The house dust mite is the most common cause of allergic diseases, and TLR4 acts as an overarching receptor for allergic responses. This study aimed to identify novel allergen binding to TLR4 in house dust mites and unveil its unique role in allergic responses. Der p 38 was purified and characterized by liquid chromatography tandem mass spectrometry–based peptide mapping. Biolayer interferometry and structure modeling unveiled TLR4-binding activity and the structure of recombinant Der p 38. The allergenicity of Der p 38 was confirmed by a skin prick test, and basophil activation and dot blot assays. The skin prick test identified 24 out of 45 allergic subjects (53.3%) as Der p 38+ subjects. Der p 38–augmented CD203c expression was noted in the basophils of Der p 38+ allergic subjects. In animal experiments with wild-type and TLR4 knockout BALB/c mice, Der p 38 administration induced the infiltration of neutrophils as well as eosinophils and exhibited clinical features similar to asthma via TLR4 activation. Persistent Der p 38 administration induced severe neutrophil inflammation. Der p 38 directly suppressed the apoptosis of allergic neutrophils and eosinophils, and enhanced cytokine production in human bronchial epithelial cells, inhibiting neutrophil apoptosis. The mechanisms involved TLR4, LYN, PI3K, AKT, ERK, and NF-κB. These findings may contribute to a deep understanding of Der p 38 as a bridge allergen between eosinophilic and neutrophilic inflammation in the pathogenic mechanisms of allergy.

Visual Abstract

Allergy is a hypersensitivity disease, which includes asthma, allergic rhinitis, and atopic dermatitis, induced by immune activation and deviation (13). House dust mites (HDMs) act as a key allergen in allergic diseases and induce IgE reactivity against their allergen proteins, demonstrating an atopy type of allergic response compared with nonatopic allergy (4, 5). Dermatophagoides pteronyssinus and Dermatophagoides farinae are considered dominant HDM species. D. pteronyssinus includes various allergenic proteins, such as cysteine and serine protease, fatty acid–binding protein, chitinase, arginine kinase, and peritrophin-like protein (6, 7). These active components induce their effects via pattern recognition receptors and protease-activated receptors (8).

TLR4 is a member of the pattern recognition receptors and TLR4-mediated signaling is a potent pathogenic pathway in HDM-induced allergic diseases (912). The inhibition of TLR4 was shown to suppress allergic inflammation and alleviate allergic responses (13, 14). In contrast, TIR-8, a TLR4-negative regulator, induced Th2 immune responses to HDMs (15). Information supporting the relationship between allergies and TLR4 indicates that the TLR4-mediated pathway is a complex mechanism that includes different and integrated activation of immune and structural cells depending on a variety of allergens. Although Der p 2 triggers allergic responses through TLR4, and Der f 35 was recently identified as an MD2-like protein, their specific mechanisms have not been clearly established (7, 16).

Several essential HDM allergens have been identified and characterized by classical tools such as screening with mite cDNA libraries (1719). A draft HDM genome produced by genomic and transcriptomic tools was recently used for identifying novel allergens (20, 21). Finding new allergens and producing their recombinant proteins can help clinicians and researchers develop clearer diagnoses and allergen immunotherapy. Nevertheless, we believe that several unrecognized, important allergens are hidden in HDMs waiting to be discovered. In this study, we investigated the unknown allergens in D. pteronyssinus, focusing on a direct TLR4-binding allergen and, to our knowledge identified a novel allergen in an allergen database that was approved by the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Subcommittee to be designated Der p 38. The allergenicity of Der p 38 and its extraordinary allergic mechanism in eosinophils, neutrophils, and bronchial cells were characterized.

RPMI 1640 and FBS were purchased from Life Technologies (Gaithersburg, MD). Adult D. pteronyssinus HDMs were obtained from the Korea National Arthropods of Medical Importance Resource Bank. D. pteronyssinus extract was obtained from Cosmo Bio (Tokyo, Japan), and recombinant Der p 1 and D. pteronyssinus feces were purchased from INDOOR Biotechnologies. Seven peptides were synthesized by Peptron (Daejeon, Korea). OVA was purchased from Sigma-Aldrich Korea (Seoul, Korea). Recombinant MD2 protein was obtained from R&D Systems (Minneapolis, MN). Peptides were purchased from Peptron. All inhibitors were purchased from Calbiochem (San Diego, CA). Abs against phospho-LYN, LYN, and phospho-ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA). Abs against phospho-AKT, AKT, ERK2, cleaved caspase 3, and cleaved caspase 9 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Recombinant TLR4 protein (R&D Systems, Minneapolis, MN) was bound to a column packed with DEAE Sepharose Fast Flow resin. D. pteronyssinus extract (Cosmo Bio) was then added to the column. The columns were washed and eluted with 1M NaCl. The eluted protein was separated by SDS–PAGE and stained with Coomassie Blue. The bands were analyzed by MALDI–time of flight/time of flight. Abs against recombinant Der p 38 protein were produced by a primary injection and a second boosting, and attached to the column following the above process. The native Der p 38 protein was excised from the gel with a sterile scalpel and placed into an Eppendorf tube and its N terminus sequence was identified by N-terminal sequencing.

Briefly, total RNA from D. pteronyssinus was extracted using TRIzol reagent (Life Technologies) and purified by an RNeasy Mini Kit (QIAGEN, Hilden, Germany). The purified RNAs were treated with DNase I (New England Biolabs, Ipswich, MA), followed by cDNA synthesis using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Mature Der p 38 (aa 21–150) was amplified by PCR. The PCR product was cut with restriction enzymes and cloned into a pETDuet-1 expression vector (Merck Millipore, Darmstadt, Germany). Recombinant His-tagged Der p 38 was expressed and isolated using a nickel column (Merck Millipore). The protein was concentrated using an Amicon Ultra concentrator (Merck Millipore) with a 10,000 Da molecular mass cutoff and purified on a Superdex 200 column attached to an ÄKTA fast protein liquid chromatography system (GE Healthcare, Chicago, IL). The endotoxin level was measured using a ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ). Endotoxin was removed by a ToxinEraser Endotoxin Removal Kit (GenScript) when this step was needed. The quadrupole time of flight method (AB Sciex TripleTOF 5600+) was performed and applied to identify the peptides of Der p 38 treated with trypsin or chymotrypsin lysate for peptide-mapping analysis. Mutations in Der p 38 were introduced using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Cedar Creek, TX) according to the manufacturer's instructions. Mutant proteins (H75A, K76A, R80A, D88A, H99A, H112A, G139A, and G139W) were generated using the wild-type (WT) Der p 38 gene as a template. The pETDuet-1 plasmids with the desired mutation were confirmed by DNA sequencing, after which the mutant gene was transformed into Escherichia coli Origami B (DE3) cells for the subsequent expression of the mutant protein.

Adult D. pteronyssinus mites were obtained from the Korea National Arthropods of Medical Importance Resource Bank and immediately fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate solution (pH 7.0) for 3 d at 4°C, followed by postfixation in 2% osmium tetroxide for 48 h at 4°C. The block was stained using 0.1 mg thiocarbohydrazide in 10 ml distilled water and 2% uranyl acetate, followed by dehydration in a graded ethanol series. The block was also stained using the National Center for Microscopy and Imaging Research method. The stained samples were then embedded into an epoxy medium (EMS, Delaware, OH). The block sections were incubated with primary anti–Der p 38 rabbit Ab (1:30, diluted with 0.1% blocking solution). The sections were then rinsed several times and incubated for 1 h in secondary anti-rabbit IgG Ab conjugated to 10 nm gold particles (Sigma-Aldrich Korea). The gold particles were observed at 200 kV using the Tecnai G2 (FEI, Hillsboro, OR).

The interaction of Der p 38, MD2 (R&D Systems), Der p 2 (INDOOR Biotechnologies, Charlottesville, VA) and mutant Der p 38 proteins with TLR4 were evaluated by biolayer interferometry (BLI) using an Octet QKe machine (ForteBio, Fremont, CA). Recombinant TLR4 (1 μg/ml) was immobilized in PBS by amine coupling on an AR2G sensor chip. A blocking step was conducted to prevent nonspecific binding using 1 M ethanolamine (pH 8.5), and the biosensors were equilibrated in assay buffer. The sensors were dipped in 2-fold serial dilutions of Der p 38 (6400 nM, 3200 nM, 1600 nM, 800 nM, 400 nM, and blank control) for 300 s during the association phase, and thereafter, a 1200 s dissociation phase proceeded in assay buffer. The BLI Data Acquisition Software 9.0.0.26 and Data Analysis Software 9.0.0.10 (ForteBio) were used for the production and analysis of the data.

A total of 41 allergic subjects with allergic rhinitis (n = 34), allergic rhinitis/asthma (n = 3), or asthma (n = 4) were recruited from Eulji University and Eulji University Hospital. The allergic status was based on history and the presence of HDM-positive skin prick test results. Additionally, 45 healthy subjects were recruited as controls. The normal subjects had no prior history of allergy and other diseases. The normal and allergic subjects were currently not on any medication. This study was approved by the Institutional Review Board of Eulji University. All participants in this study provided written informed consent (Table I).

Table I.

The results of skin prick test

Normal (n = 45)Allergy (n = 41)
Number of subjects (female/male) 45 (34/11) 41 (24/17) 
Age (y) 22.5 ± 4.9 (20∼41) 23.4 ± 4.4 (20∼48) 
D. pteronyssinus+, D. farinae16 (35.6%) 37 (90.2%) 
D. pteronyssinus+, D. farinae 2 (4.4%) 0 (0.0%) 
D. pteronyssinus, D. farinae1 (2.2%) 4 (9.8%) 
D. pteronyssinus, D. farinae 26 (57.8%) 0 (0.0%) 
Der p 1 6 (13.3%) 30 (73.2%) 
Der p 2 11 (24.4%) 31 (75.6%) 
Der p 38 3 (6.7%) 24 (58.5%) 
Normal (n = 45)Allergy (n = 41)
Number of subjects (female/male) 45 (34/11) 41 (24/17) 
Age (y) 22.5 ± 4.9 (20∼41) 23.4 ± 4.4 (20∼48) 
D. pteronyssinus+, D. farinae16 (35.6%) 37 (90.2%) 
D. pteronyssinus+, D. farinae 2 (4.4%) 0 (0.0%) 
D. pteronyssinus, D. farinae1 (2.2%) 4 (9.8%) 
D. pteronyssinus, D. farinae 26 (57.8%) 0 (0.0%) 
Der p 1 6 (13.3%) 30 (73.2%) 
Der p 2 11 (24.4%) 31 (75.6%) 
Der p 38 3 (6.7%) 24 (58.5%) 

Conventional skin prick tests were conducted according to the instructions of the SoluprickR test. The test reagents included histamine (positive control), D. pteronyssinus, and D. farinae (ALK-Abello A/S, Horsholm, Denmark). Additionally, recombinant Der p 1, Der p 2, Der p 38, and mutant Der p 38 proteins (10 μg/ml) were dissolved in PBS (pH 7.4) and used for the skin prick test. Test interpretations were conducted 15–20 min after the application, and a positive result was defined as a wheal ≥ 3 mm in diameter.

For the nondenaturing dot blot assay, the proteins or peptides were dotted on nitrocellulose membranes and blocked with 3% BSA solution. After adding 1:10 diluted normal or patient sera, the membrane was washed and incubated with a secondary mouse anti-human IgE Ab. Finally, it was developed using the ECL detection system (Thermo Fisher Scientific, Waltham, MA).

The heparinized peripheral blood of healthy persons and allergic subjects was subjected to Ficoll-Hypaque gradient centrifugation for the isolation of granulocytes. A CD16 MicroBead Magnetic Cell Sorting Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was applied to collect the neutrophils, and erythrocyte removal was achieved by hypotonic lysis. The isolated cells were collected and washed with PBS buffer. The cells, including basophils and eosinophils, were incubated with PBS or Der p 38 for 30 min at 37°C. The cells were incubated on ice for 5 min and washed with PBS. After incubation, the cells were incubated with PE-conjugated anti-human IgE and allophycocyanin-conjugated CD203c (BioLegend, San Diego, CA) for 20 min on ice. Following washing, the stained cells were analyzed on a FACSort cytofluorimeter (Becton Dickinson, Franklin Lakes, NJ). Flow cytometric analysis of the cells was performed with a FACScan flow cytometer (BD Biosciences, Heidelberg, Germany), using CellQuest and FlowJo V10 software. The basophils were identified as IgE-positive cells, and activated basophils were additionally detected by the presence of CD203c.

The ClustalW2 program was used for sequence alignment, and PyMOL software was used to predict the structure of Der p 38.

Six-week-old female WT and TLR4 knockout (KO) BALB/c mice were used in the animal experiments. The mice were maintained in a specific pathogen–free facility. In the first experiment, each group (n = 5) was administered intranasal (IN) injections of PBS, Der p 38 (10, 25, or 50 μg/50 μl) or/and LPS (20 μg/50 μl) for 1 wk. In the second experiment, each group was immunized by the i.p. injection of PBS, Der p 38 (20, 50 or 100 μg/50 μl), D. pteronyssinus extract (100 μg/50 μl), OVA (20 μg/50 μl), or LPS (2 or 40 μg/50 μl) on days 1 and 14. The mice were IN administered PBS, Der p 38 (10, 25, or 50 μg/50 μl), D. pteronyssinus extract (50 μg/50 μl), OVA (10 μg/50 μl), or LPS (1 or 20 μg/50 μl) from days 21 to 27 after the second sensitization. In the chronic model experiment, the first administrations to the mice were the same as those in the second experiment. After the first IN injection, the process was carried out two times at 1-wk intervals. The PBS-treated group was considered as the control. The groups were named according to the stimulators administered i.p./IN. All animal experiments used in this study were approved by the Institutional Animal Care and Use Committee of Eulji University.

Bronchoalveolar lavage fluid (BALF) (∼0.7 ml) was collected five times by lung lavage via the trachea with 1 ml of PBS. Blood was collected by heart puncture. The BALF and blood were centrifuged, and the supernatants were stored at −70°C. The cells in the BALF were resuspended in 100 μl of PBS for total cell and differential counts. Total cell numbers were counted using a Neubauer hemocytometer. The cells suspended in PBS were attached to a slide by cytospinning and stained with a Diff-Quick Kit (Sysmex Corporation, Kobe, Japan). The ratio of each leukocyte was represented as a percentage.

At the end of the study period, all animals were euthanized, and lung tissues were harvested and fixed in formalin solution. The fixed tissues were embedded in paraffin and cut into 5-μm sections using a Microtome (Leica Microsystems, Wetzlar, Germany). The sections were deparaffinized and stained with H&E (Sigma-Aldrich Korea) or with periodic acid–Schiff (PAS) stain (Sigma-Aldrich Korea) to detect mucus production. Immunohistochemical staining was performed on 3 μm-thick paraffin-embedded sections. Briefly, the sections were placed on Superfrost Plus Microscope Slides (Thermo Fisher Scientific). A VECTASTAIN Elite ABC HRP Kit (Vector Laboratories, Burlingame, CA) was used as a 3,3′-diaminobenzidine chromogen for detecting Abs against Ly-6G, tryptase (Abcam, Cambridge, UK), and eosinophil peroxidase (Bioss Antibodies, Woburn, MA). Sections were deparaffinized by xylene. A solution of proteinase K was applied for 30 min for Ag retrieval, followed by treatment with 0.3% H2O2 in methanol for 30–40 min. Slides were blocked and subsequently incubated with primary Abs and secondary Abs. After incubating with Alcian Blue and 3,3′-diaminobenzidine reagents, the specimens were counterstained with H&E and examined under light microscopy (Leica Microsystems, Wetzlar, Germany) for histological evaluation.

Airway hyperresponsiveness to methacholine was assessed by whole-body plethysmography (Buxco Electronics, Wilmington, NC). The mice were placed in a chamber for adaptation, and the basal enhanced pause value was measured in the absence of methacholine treatment. The basal enhanced pause values were next determined following the exposure of mice to nebulized PBS and methacholine (6.25, 12.5, 25, and 50 mg/ml) (Sigma-Aldrich Korea) at 5 min intervals.

To evaluate IgE binding activity to recombinant Der p 38 protein, ELISA plates were coated overnight at 4°C with Der p 38 at a concentration of 1 μg/ml Der p 38 or peptides per well prepared in carbonate-buffered solution (15 mM Na2CO3 and 35 mM NaHCO3 [pH 9.5]). After blocking with 3% BSA in PBS at 37°C for 1 h, the plates were incubated with 1:5 diluted serum for 1 h at 37°C, followed by incubation with biotin-conjugated goat anti-mouse IgE (1:2000) and streptavidin-HRP reagent for 1 h at 37°C. The reaction was stopped by adding 2 M H2SO4 solution. A Mouse IgE ELISA Kit (BD Biosciences) was used to measure total IgE in the mice sera. The OD values were measured by an ELx808 Absorbance Microplate Reader (BioTek Instruments, Winooski, VT) at 450 nm. Cytokine ELISA was performed as previously described (22).

Isolation of neutrophils and eosinophils was performed as previously described (10, 22). An annexin V–FITC Apoptosis Detection Kit (BD Biosciences) was used for apoptosis evaluation. Apoptotic cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and reported as the percentage of cells showing annexin V+/propidium iodide (PI) and annexin V+/PI+.

The human bronchial epithelial cell line, BEAS-2B (CRL-9609; American Type Culture Collection, Manassas, VA), was cultured in DMEM/F12 medium supplemented with 10% FBS. Western blotting was performed as previously described (23).

Data were expressed as the means ± SD. Statistical differences were analyzed using a paired t test for two-group comparisons and one-way ANOVA for comparison of more than two groups. All analyses were conducted using the SPSS statistical software package (version 10.0; IBM, Chicago, IL), and a p value < 0.05 was considered to indicate statistical significance.

We identified Der p 38 by MALDI–time of flight/time of flight after separating TLR4-binding proteins in the D. pteronyssinus extract using a TLR4-bound column (Fig. 1A). Although the protein has been reported to be a bacteriolytic enzyme or LytFM variant (LytFM1), which is a possible allergen, it has never been clearly identified as an allergen (20, 24, 25). Abs against Der p 38 were used for isolating native Der p 38 from the D. pteronyssinus extract. We found that the N terminus of mature native Der p 38 was NGAAIVSAAR by N-terminal sequencing, indicating that it has a signaling peptide (20 aa) and a mature form (130 aa) (Supplemental Fig. 1A). The recombinant Der p 38 protein was produced, purified by fast protein liquid chromatography and confirmed by peptide-mapping analysis (sequence coverage: 100%) (Fig. 1B, Supplemental Fig. 1B). The interaction of recombinant Der p 38 with TLR4 was investigated using a BLI analysis (KD = 97.9 nM) (Fig. 1C). TLR4-binding affinity was not detected in Der p 2, known as an MD-like allergen. Der p 38 was present in the body and feces of D. pteronyssinus (Fig. 1D). The posterior midgut of D. pteronyssinus showed the widespread presence of Der p 38 by electron microscopy, and the lumen showed relatively more Der p 38 particles than the epithelium (Fig. 1E).

FIGURE 1.

Der p 38 is identified as a TLR4-binding protein. (A) D. pteronyssinus extract was added to a TLR4-bound column and eluted. The eluates, including native Der p 38, were loaded onto gels. (B) Recombinant Der p 38 was produced and loaded onto the gel. The proteins were separated by SDS–PAGE and stained with Coomassie Brilliant Blue (A and B). (C) TLR4 was coated on an AR2G sensor chip. The binding affinity (KD) of Der p 38, Der p 2, and MD2 to TLR4 was measured by BLI. (D) Recombinant Der p 38, D. pteronyssinus body, and D. pteronyssinus feces at the indicated concentrations were analyzed by SDS–PAGE, and anti–Der p 38 polyclonal Abs are used for the detection of Der p 38 by Western blotting. (E) Electron micrograph showing the presence of Der p 38 in the posterior midgut of D. pteronyssinus. White arrows indicate gold particles (Der p 38).

FIGURE 1.

Der p 38 is identified as a TLR4-binding protein. (A) D. pteronyssinus extract was added to a TLR4-bound column and eluted. The eluates, including native Der p 38, were loaded onto gels. (B) Recombinant Der p 38 was produced and loaded onto the gel. The proteins were separated by SDS–PAGE and stained with Coomassie Brilliant Blue (A and B). (C) TLR4 was coated on an AR2G sensor chip. The binding affinity (KD) of Der p 38, Der p 2, and MD2 to TLR4 was measured by BLI. (D) Recombinant Der p 38, D. pteronyssinus body, and D. pteronyssinus feces at the indicated concentrations were analyzed by SDS–PAGE, and anti–Der p 38 polyclonal Abs are used for the detection of Der p 38 by Western blotting. (E) Electron micrograph showing the presence of Der p 38 in the posterior midgut of D. pteronyssinus. White arrows indicate gold particles (Der p 38).

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To examine the IgE reactivity of Der p 38, we conducted skin prick tests and dot blot assays in healthy persons and subjects with asthma or/and allergic rhinitis. In the skin prick tests, 58.5% of the allergic subjects (24/41) showed positive responses to Der p 38 and 73.2% (30/41) and 75.6% (31/41) of the same group exhibited positive reactions to Der p 1 and Der p 2, respectively (Table I). According to the dot blot assay results, the frequency of IgE reactivity to Der p 38 was 60% (27/45) in the allergic subjects (Fig. 2A). The Der p 38+ subjects in the skin prick test showed positive reactions to Der p 38 in the dot blot assay. After considering the epitopes predicted by the BCPreds, ABCpred, and BepiPred programs, we synthesized peptides to find the epitope region of Der p 38 involved in allergenicity, and peptides P5 and P6 weakly reacted to Der p 38+ serum (Fig. 2B, 2C). Based on the peptide results and expected sites (26), we produced several mutant proteins. After conducting the skin prick test using mutant proteins in Der p 38+ allergic subjects, we evaluated the percentage of the population that showed more than a 70% swelling decrease compared with WT Der p 38. H75A, K76A, R80A, D88A, K90A, K99A, and H112A proteins exhibited more than the 70% swelling decrease in 20%, 70%, 60%, 62.5%, 37.5%, 40%, and 30% of Der p 38+ allergic subjects, respectively (Fig. 2D). By analysis of the model structure of Der p 38, the expected epitope sites are exposed on the exterior side (Fig. 2E; data not shown). In addition, we evaluated the allergenic activity of Der p 38, which enhanced the expression of the basophil activation marker CD203c (Fig. 2F).

FIGURE 2.

Der p 38 has IgE reactivity and induces basophil activation. (A) The IgE reactivity of Der p 38 was evaluated by dot blot assay using the sera of normal and allergic subjects. (B) Schematic representation of seven peptides derived from Der p 38. (C) The dot-blotted peptides were evaluated for IgE reactivity with the sera of Der p 38+ subjects. (D) Skin prick tests were performed using WT and mutant Der p 38 proteins. The y-axis represents the percentage of Der p 38+ allergic subjects (8 < n < 10) exhibiting more than a 70% swelling decrease when the skin wheal to WT Der p 38 protein was set at 100%. (E) The three-dimensional structure of Der p 38 predicted by the ClustalW2 program and PyMOL software using RipA protein as a template. (F) Granulocytes were isolated from normal and allergic subjects and treated with PBS or Der p 38 at the indicated concentrations (μg/ml). The CD203c expression of basophils was evaluated using flow cytometry. Allergic subjects were divided into Der p 38 and Der p 38+ groups based on the skin prick test. The data are presented as the mean ± S.D. *p < 0.05 indicates significant differences between the PBS-treated group and Der p 38–treated groups.

FIGURE 2.

Der p 38 has IgE reactivity and induces basophil activation. (A) The IgE reactivity of Der p 38 was evaluated by dot blot assay using the sera of normal and allergic subjects. (B) Schematic representation of seven peptides derived from Der p 38. (C) The dot-blotted peptides were evaluated for IgE reactivity with the sera of Der p 38+ subjects. (D) Skin prick tests were performed using WT and mutant Der p 38 proteins. The y-axis represents the percentage of Der p 38+ allergic subjects (8 < n < 10) exhibiting more than a 70% swelling decrease when the skin wheal to WT Der p 38 protein was set at 100%. (E) The three-dimensional structure of Der p 38 predicted by the ClustalW2 program and PyMOL software using RipA protein as a template. (F) Granulocytes were isolated from normal and allergic subjects and treated with PBS or Der p 38 at the indicated concentrations (μg/ml). The CD203c expression of basophils was evaluated using flow cytometry. Allergic subjects were divided into Der p 38 and Der p 38+ groups based on the skin prick test. The data are presented as the mean ± S.D. *p < 0.05 indicates significant differences between the PBS-treated group and Der p 38–treated groups.

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To better define the role of Der p 38 in allergies, we tested the inflammatory effects and mechanism of Der p 38 in a mouse model. Because the clinical features of asthma appear differently depending upon the type of allergen, exposure time, administration route, and binding receptor, we designed experimental groups based on the IN or/and i.p. exposure of Der p 38 for short- and long-terms. IN injections of Der p 38 for 1 wk induced the recruitment of neutrophils into the lungs compared with the PBS-treated group (Fig. 3; Supplemental Fig. 2A, 2B) and had little effect on mucin secretion by goblet cells and total and Der p 38–specific IgE (Supplemental Fig. 2B, 2C). The addition of an i.p. administration of Der p 38 before the IN injection strongly induced peribronchial inflammation by the infiltration of immune cells and mucus hypersecretion by goblet cell hyperplasia (Fig. 3B, Supplemental Fig. 2D). Notably, Der p 38 triggered the movement of eosinophils and neutrophils compared with stimulation by D. pteronyssinus extract, although i.p. and IN injections of general allergens induced eosinophil recruitment (Fig. 3C). Der p 38 induced hyperreactivity to methacholine and increased total IgE compared with the effect of D. pteronyssinus extract and increased Der p 38–specific IgE (Fig. 3C). Because Der p 38 binds to TLR4 (Fig. 1C), we next investigated whether the inflammatory effects of Der p 38 were related to TLR4. After the IN or IN/i.p. injection of Der p 38 to TLR4 KO mice, leukocyte infiltration and IgE increases were not seen compared with the WT group (Fig. 3F–H). C57BL/10ScNJ WT and C57BL/10ScNJ TLR4 KO mice showed findings similar to those in the WT and TLR4 KO BALB/c mice (data not shown).

FIGURE 3.

Der p 38 induces the infiltration of eosinophils and neutrophils into airways and mucin hypersecretion in mice via TLR4. (A) Differential cell counts in BALF of BALB/c mice after the IN administration of PBS and the indicated concentration (μg) of Der p 38 for 1 wk. (BE) Lung tissues from the mice after i.p. and IN injections of Der p 38 and D. pteronyssinus extract were stained with H&E and PAS (B), and the cells of BALF were differentially counted (C). Hyperresponsiveness to methacholine at the indicated concentrations was measured by whole-body plethysmography (D). Total IgE and Der p 38–specific IgE were evaluated in the mice sera (E). (FH) Mice with or without i.p. injections of Der p 38 (100 μg/50 μl) were IN administered Der p 38 (50 μg/50 μl). Differential cell counts (F), histopathological features (G), and the levels of total IgE and Der p 38–specific IgE (H) in BALF, lung tissues, and sera of WT and TLR4 KO BALB/c mice. Magnification, ×200 or ×400 (B and G). The data are presented as the mean ± S.D. *p < 0.05 and **p < 0.01 indicate significant differences between the PBS-treated group and the stimulator-treated groups or between the WT and TLR4 KO mice. ##p < 0.01 indicates a significant difference between the two groups.

FIGURE 3.

Der p 38 induces the infiltration of eosinophils and neutrophils into airways and mucin hypersecretion in mice via TLR4. (A) Differential cell counts in BALF of BALB/c mice after the IN administration of PBS and the indicated concentration (μg) of Der p 38 for 1 wk. (BE) Lung tissues from the mice after i.p. and IN injections of Der p 38 and D. pteronyssinus extract were stained with H&E and PAS (B), and the cells of BALF were differentially counted (C). Hyperresponsiveness to methacholine at the indicated concentrations was measured by whole-body plethysmography (D). Total IgE and Der p 38–specific IgE were evaluated in the mice sera (E). (FH) Mice with or without i.p. injections of Der p 38 (100 μg/50 μl) were IN administered Der p 38 (50 μg/50 μl). Differential cell counts (F), histopathological features (G), and the levels of total IgE and Der p 38–specific IgE (H) in BALF, lung tissues, and sera of WT and TLR4 KO BALB/c mice. Magnification, ×200 or ×400 (B and G). The data are presented as the mean ± S.D. *p < 0.05 and **p < 0.01 indicate significant differences between the PBS-treated group and the stimulator-treated groups or between the WT and TLR4 KO mice. ##p < 0.01 indicates a significant difference between the two groups.

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Because LPS IN treatment showed a synergic effect on neutrophil movement when injected with Der p 38 (Fig. 3A), we wondered whether the effects of Der p 38 were altered in the presence of LPS when it induced the movement of both eosinophils and neutrophils. We divided the LPS treatments into low- (1 μg per a dose) and high-dose (20 μg per a dose) stimulations because Th1 or Th2 responses are elicited depending upon the treatment concentration (27, 28). After the i.p. administration of Der p 38, the IN injection of LPS induced neutrophil infiltration similar to LPS in treatment alone, regardless of the i.p. injection of Der p 38 (Fig. 4A, 4B). The Der p 38/Der p 38 plus LPS20 group showed decreased numbers of eosinophils and increased numbers of neutrophils with no significance compared with the Der p 38/Der p 38 group. In other words, Der p 38 suppressed the neutrophil numbers increased by LPS. Low-dose LPS did not affect mucus production in the lung or IgE in the serum increased by Der p 38, but high-dose LPS decreased the production (Fig. 4B, 4C). These results indicate that Der p 38 showed different effects, although Der p 38 and LPS have a common receptor, TLR4. The results on the relationship between LPS and Der p 38 led us to investigate whether Der p 38 had interactive effects with OVA, which induces eosinophil infiltration and is not related to TLR4. IN treatment with OVA instead of Der p 38 (Der p 38/OVA) did not trigger the recruitment of eosinophils and neutrophils (Supplemental Fig. 3A, 3B), and IN treatment with OVA in the absence of OVA i.p. injection had no meaningful effect on the lungs. OVA treatment promoted the production of total IgE Igs and Der p 38–specific IgE in the Der p 38/OVA groups compared with the Der p 38/Der 38 group (Supplemental Fig. 3C). Next, we investigated the effects of an IN injection of Der p 38 or LPS after an i.p. injection of OVA to determine whether Der p 38 enhanced the allergic effects of OVA. Both i.p. and IN injections with OVA strongly increased the numbers of eosinophils in BALF (Fig. 4D). The number of eosinophils in the Der p 38/Der p 38 group was lower but the number of neutrophils was greater than that in the OVA/OVA group (Fig. 4D). An additional IN injection of Der p 38 to the OVA/OVA plus Der p 38 group produced a slight decrease in the number of eosinophils and an increase in neutrophils compared with the OVA/OVA group. An additional IN injection of high-dose LPS (OVA/OVA plus LPS20) markedly increased the neutrophils and markedly decreased the eosinophils compared with the OVA/OVA and OVA/OVA plus Der p 38 groups. The OVA/OVA plus Der p 38 group showed the most severe histological changes, including the infiltration of inflammatory cells, mucus hypersecretion resulting in narrowing of the bronchi, and thickened alveolar walls, between the groups (Fig. 4E). Although different stimulators were used for the IN and i.p. injections, total IgE and OVA-specific IgE increased in the OVA/Der p 38 group similar to the Der p 38/OVA group (Fig. 4F, Supplemental Fig. 3C). These results indicate that Der p 38 may modulate the number of eosinophils and neutrophils altered by OVA in contrast to LPS and aggravate allergic responses induced by other allergens.

FIGURE 4.

Eosinophil recruitment induced by Der p38 is suppressed by LPS, and Der p 38 increases neutrophil infiltration and mucus hyperproduction in OVA-induced responses. (AF) After i.p. injection, BALB/mice received one IN injection. Differential (A and D) cell counts, histopathological features after staining with H&E (upper panel) and PAS (lower panel) (B and E), and the levels of total IgE and Der p 38–specific IgE (C and F) in BALF, lung tissues, and mice sera. Magnification, ×200 (B and E). The data are presented as the mean ± S.D. *p < 0.05 and **p < 0.01 indicate significant differences between the PBS-treated group and the stimulator-treated groups. #p < 0.05 and ##p < 0.01 indicate a significant difference between two groups.

FIGURE 4.

Eosinophil recruitment induced by Der p38 is suppressed by LPS, and Der p 38 increases neutrophil infiltration and mucus hyperproduction in OVA-induced responses. (AF) After i.p. injection, BALB/mice received one IN injection. Differential (A and D) cell counts, histopathological features after staining with H&E (upper panel) and PAS (lower panel) (B and E), and the levels of total IgE and Der p 38–specific IgE (C and F) in BALF, lung tissues, and mice sera. Magnification, ×200 (B and E). The data are presented as the mean ± S.D. *p < 0.05 and **p < 0.01 indicate significant differences between the PBS-treated group and the stimulator-treated groups. #p < 0.05 and ##p < 0.01 indicate a significant difference between two groups.

Close modal

Chronic exposure to Der p 38 strongly increased leukocyte infiltration, in particular neutrophils, and more severe histological features compared with the Der p 38/Der p 38 group (Fig. 5A–D). The PBS-treated group showed normal histological features. However, mucin secretion, the total IgE, and Der p 38–specific IgE were decreased compared with the Der p 38/Der p 38 group (Fig. 5C, 5E).

FIGURE 5.

Persistent Der p 38 stimulation induces severe neutrophil infiltration. (AE) After i.p. injection, BALB/mice received three IN injections. Total (A) and differential (B) cell counts, histopathological features after staining with H&E (upper panel) and PAS (lower panel) (C), and with Abs against Ly-6G, eosinophil peroxidase, and tryptase (D), and the levels of total IgE and Der p 38–specific IgE (E) in BALF, lung tissues, and mice sera. Magnification, ×200 (C and D). The data are presented as the mean ± S.D. **p < 0.01 indicates significant differences between the PBS-treated group and the stimulator-treated groups.

FIGURE 5.

Persistent Der p 38 stimulation induces severe neutrophil infiltration. (AE) After i.p. injection, BALB/mice received three IN injections. Total (A) and differential (B) cell counts, histopathological features after staining with H&E (upper panel) and PAS (lower panel) (C), and with Abs against Ly-6G, eosinophil peroxidase, and tryptase (D), and the levels of total IgE and Der p 38–specific IgE (E) in BALF, lung tissues, and mice sera. Magnification, ×200 (C and D). The data are presented as the mean ± S.D. **p < 0.01 indicates significant differences between the PBS-treated group and the stimulator-treated groups.

Close modal

Because Der p 38 triggered an increase in neutrophils and eosinophils in the mice lungs, we examined the precise mechanism of Der p 38 in this process. Der p 38 blocked the apoptosis of allergic neutrophils and eosinophils in a dose-dependent manner (Fig. 6A). Der p 38 also induced neutrophil apoptosis in healthy people (data not shown). However, Der p 38 inhibited eosinophil apoptosis that was not dependent upon Der p 38 sensitization in 33.3% (5/15) of the allergic subjects and 20% (2/10) of the healthy subjects. Based on our previous paper, we investigated the association of TLR4, LYN, PI3K, AKT, ERK, and NF-κB with HDMs (29). Pretreatment with TLR4i, PP2, LY294002, AKTi, PD98059, and BAY-11-7085 inhibited the antiapoptotic effects of Der p 38 on neutrophils and eosinophils (Fig. 6B). LYN, AKT, and ERK were activated by Der p 38 in a time-dependent manner (Fig. 6C). ERK phosphorylation due to Der p 38 was suppressed by TLR4i, PP2, LY294002, and AKTi treatment (Fig. 6D). Der p 38 induced NF-κB activation, which was blocked by TLR4i, PP2, LY294002, AKTi, and PD98059 (Fig. 6E). These results indicate that Der p 38 signal transduction occurred via TLR4/LYN/PI3K/AKT/ERK/NF-κB. Cleaved caspase 9 and caspase 3 were upregulated in a time-dependent fashion after initiating constitutive apoptosis, and the cleavage of both proteins was inhibited by Der p 38 (Fig. 6F). The importance of structural cells led us to examine whether cytokine secretion induced by Der p 38 in the human bronchial epithelial cell line BEAS-2B affected the survival of allergic neutrophils and eosinophils. The supernatants of BEAS-2B cells treated with Der p 38 blocked the apoptosis of allergic neutrophils but not the apoptosis of eosinophils (Fig. 6G). MCP-1, IL-6, and IL-8 related to neutrophil survival increased after treatment with Der p 38, and this increase was prevented by treatment with signal protein inhibitors (Fig. 6H). Activation of the signal molecules due to Der p 38 was confirmed by Western blotting and NF-κB assays (Fig. 6I–K).

FIGURE 6.

TLR4, LYN, PI3K, AKT, ERK, and NF-κB are involved in Der p 38–induced inflammatory effects. (A, B, and G) Allergic neutrophils and eosinophils were incubated without (Con) or with Der p 38 (10 μg/ml) for 24 h or 48 h, respectively (A), after pretreatment without (A) or with (B) the indicated inhibitors. Allergic cells were treated with the supernatant (Sup) collected from BEAS-2B cells after Der p 38 treatment or/and Derp 38 (G). Apoptosis was analyzed (C, D, F, I, and J). Allergic cells (C, D, and F), and BEAS-2B cells (I and J) were pretreated for 1 h with the indicated inhibitors and then incubated with Der p 38 for the indicated time (C, F, and I) or for 30 min (D and J). The indicated proteins in the lysates were detected by Western blotting. (E and K) NF-κB activation was detected in allergic cells (E) and BEAS-2B cells (K) after treatment with Der p 38 for 1 h in the presence of inhibitor pretreatment. (H) BEAS-2B cells were incubated with Der p 38 for 48 h after pretreatment with the inhibitors. The supernatant was analyzed by ELISA. *p < 0.05 and **p < 0.01 (between the untreated group and Der p 38–treated groups), #p < 0.05 and ##p < 0.01 (between the Der p 38–treated group and the inhibitor-treated groups).

FIGURE 6.

TLR4, LYN, PI3K, AKT, ERK, and NF-κB are involved in Der p 38–induced inflammatory effects. (A, B, and G) Allergic neutrophils and eosinophils were incubated without (Con) or with Der p 38 (10 μg/ml) for 24 h or 48 h, respectively (A), after pretreatment without (A) or with (B) the indicated inhibitors. Allergic cells were treated with the supernatant (Sup) collected from BEAS-2B cells after Der p 38 treatment or/and Derp 38 (G). Apoptosis was analyzed (C, D, F, I, and J). Allergic cells (C, D, and F), and BEAS-2B cells (I and J) were pretreated for 1 h with the indicated inhibitors and then incubated with Der p 38 for the indicated time (C, F, and I) or for 30 min (D and J). The indicated proteins in the lysates were detected by Western blotting. (E and K) NF-κB activation was detected in allergic cells (E) and BEAS-2B cells (K) after treatment with Der p 38 for 1 h in the presence of inhibitor pretreatment. (H) BEAS-2B cells were incubated with Der p 38 for 48 h after pretreatment with the inhibitors. The supernatant was analyzed by ELISA. *p < 0.05 and **p < 0.01 (between the untreated group and Der p 38–treated groups), #p < 0.05 and ##p < 0.01 (between the Der p 38–treated group and the inhibitor-treated groups).

Close modal

To precisely investigate the regulatory site of Der p 38 for binding to TLR4, we evaluated the effect of mutant proteins on neutrophil and eosinophil apoptosis and cytokine release by BEAS-2B cells. Recently, we found that the Der f 38 allergen in D. farinae was homologous to Der p 38 (data not shown). However, Der f 38 had a weak effect on neutrophil and eosinophil apoptosis compared with Der p 38 (Fig. 7A). After predicting a putative TLR4-binding regulatory site, mutant proteins G139W and G139A were produced. The mutant proteins were not effective on the apoptosis of neutrophils and eosinophils and cytokine secretion from BEAS-2B cells (Fig. 7B, 7C). Finally, we confirmed that the G139W and G139A mutants had no affinity for TLR4 because glycine mutation may alter the loop structure of that site (Fig. 7D). These results indicate that glycine at the 139 aa of Der p 38 should be considered a critical part of the regulatory site for binding to TLR4, although the exact binding site was not identified.

FIGURE 7.

Der p 38 has an important TLR4-binding regulatory site. (A and B) Allergic neutrophils (n = 8) and eosinophils (n = 5) were incubated for 24 h or 48 h in the absence (Con) and presence of Der p 38 (10 μg/ml), Der f 38 (10 μg/ml), and Der p 38 mutant proteins. Apoptosis was analyzed by measuring the binding of annexin V–FITC and PI. The data are presented as the mean ± S.D. relative to the control, which was set at 100%. (C) BEAS-2B cells (n = 3) were incubated in the absence (Con) or presence of Der p 38 (10 μg/ml) and mutant Der p 38 proteins for 48 h. Cytokines in the supernatant were analyzed by ELISA. (D) TLR4 was coated on an AR2G sensor chip. The binding affinity (KD) of Der p 38 WT and mutant proteins to TLR4 was measured by BLI. *p < 0.05 and **p < 0.01 indicate significant differences between the untreated group and Der p 38–treated groups, and #p < 0.05 indicates a significant difference between the Der p 38–treated group and the mutant protein-treated groups.

FIGURE 7.

Der p 38 has an important TLR4-binding regulatory site. (A and B) Allergic neutrophils (n = 8) and eosinophils (n = 5) were incubated for 24 h or 48 h in the absence (Con) and presence of Der p 38 (10 μg/ml), Der f 38 (10 μg/ml), and Der p 38 mutant proteins. Apoptosis was analyzed by measuring the binding of annexin V–FITC and PI. The data are presented as the mean ± S.D. relative to the control, which was set at 100%. (C) BEAS-2B cells (n = 3) were incubated in the absence (Con) or presence of Der p 38 (10 μg/ml) and mutant Der p 38 proteins for 48 h. Cytokines in the supernatant were analyzed by ELISA. (D) TLR4 was coated on an AR2G sensor chip. The binding affinity (KD) of Der p 38 WT and mutant proteins to TLR4 was measured by BLI. *p < 0.05 and **p < 0.01 indicate significant differences between the untreated group and Der p 38–treated groups, and #p < 0.05 indicates a significant difference between the Der p 38–treated group and the mutant protein-treated groups.

Close modal

HDMs have been targeted as a critical allergen in the diagnosis of and therapy for allergic diseases. Approaches to finding a clear explanation for allergic diseases has led researchers to discover novel allergenic proteins (16, 20, 21). Before beginning this research, we thought that TLR4 was a crucial receptor inducing allergic diseases. Therefore, an unknown allergen that directly binds to TLR4 and orchestrates allergic responses was pursued, based on our previous reports and those of others (9, 10, 14, 29). In this study, we applied a different principle to unveil a new, to our knowledge, allergen in HDMs and identified Der p 38, which drives mixed granulocytes asthma in the mouse model. Chronic exposure to Der p 38 may facilitate the transition from mixed granulocytic asthma to neutrophilic asthma.

The TLR4-binding capacity of allergens was not limited to Der p 38. Der p 2 is widely known as an MD2-like protein, and Der f 35 has been recently reported (16, 30, 31). It has not been completely demonstrated that TLR4-binding allergens directly bind to TLR4 using an in vitro assay such as BLI. Conversely, the action of Der p 2 was not dependent upon TLR4 but was dependent on TLR2 (31, 32). These facts led us to confirm the binding of Der p 38 and Der p 2 to TLR4. We found that Der p 38 showed TLR4-binding affinity and identified a TLR4-binding regulatory site (G139) (Fig. 7D). However, Der p 2 did not show any binding activity (Fig. 1C). The relationship between Der p 38 and TLR4 was supported by the findings that Der p 38 triggered allergic responses in the mouse model, antiapoptotic effects on neutrophils and eosinophils, and cytokine secretion (Figs. 3G, 6A, 6G). The in vivo responses due to Der p 38 were demonstrated by the infiltration of neutrophils and eosinophils in BALF and the lungs (Fig. 3A, 3C). It also induced allergic responses, such as IgE production and hyperreactivity to methacholine (Fig. 3D, 3E). Allergens, including Der p 1 and Der p 2, induced marked eosinophil recruitment without the infiltration of neutrophils (33, 34). Simpson et al. (35) suggested classifications of asthma based on eosinophilic, neutrophilic, mixed granulocytic, and paucigranulocytic subtypes. T2 type (T2 high) asthma and non-T2 type (T2 low) are used to explain the heterogeneity of asthma, including the various endotypes with phenotypes (3638). Non-T2 type (neutrophilic asthma) is seen in moderate and severe asthma and glucocorticoid-resistant asthma, although infections and cigarette smoke are considered the causative factors (36, 39, 40). Mixed granulocytes may reflect a transition stage between eosinophilic and neutrophilic endotypes. An absolute and relative increase in eosinophils and neutrophils distinguishes Der p 38 from other allergens, and it may function as a stage regulator. In a chronic model, Der p 38 exacerbated the clinical features of asthma and induced the transition from a mixed granulocyte (eosinophils and neutrophils) phenotype to a predominance of neutrophils (Fig. 5). However, this study had some limitations as a first trial. The allergic subjects, including young people with one or two allergic diseases, were recruited to investigate the pathogenesis of allergies due to Der p 38. Allergenicity may depend on different characteristics, such as race, age, the quality of the recombinant protein and mite extract, diagnostic tools, and the type of allergy (4143). In addition, innate lymphoid cells (ILCs) are closely related to allergic diseases (4446). ILC2 and ILC3 play important roles in eosinophilic asthma and noneosinophilic asthma, respectively. Thus, the precise role of Der p 38 in the clinical severity of allergic diseases and how Der p 38 induces allergenicity remain unsolved. Further studies on Der p 38 need to focus on the specific mechanisms and allergic diseases, such as asthma and atopic dermatitis, atopic/nonatopic allergy, and other populations besides Korean subjects.

TLR4 is an overarching receptor in neutrophilic and eosinophilic inflammation. Der p 38 transduced its signal via LYN, PI3K, AKT, and NF-κB, which is related to essential mechanisms in eosinophils, neutrophils, and structural cells such as bronchial epithelial cells (Fig. 6). This signal transduction increased cell survival and cytokine secretion, consistent with the recruitment of eosinophils and neutrophils in animal experiments. TLR4 shows differential action, depending upon the cell type and kind of stimulator. TLR4 in hematopoietic cells and epithelial cells is related to neutrophilic and eosinophilic airway inflammation, respectively (47), in contrast to our results that TLR4 importantly functioned as a Der p 38 counterpart in granulocytes and epithelial cells. In animal experiments, the effect of an IN injection of Der p 38 alone was similar to the action of LPS. However, the i.p. and IN injection of Der p 38 triggered eosinophil and neutrophil inflammation compared with OVA (Figs. 3A, 4A, 4D). LPS inhibited the eosinophil increase due to OVA or Der p 38. Der p 38 augmented the IgE production and mucus production induced by OVA, in contrast to a high dose of LPS (Fig. 4E, 4F) (Supplemental Fig. 4). Sensitization to Der p 38 and OVA can interact with each other and boost their actions (Fig. 4F). The allergenic effects of OVA may be modulated by TLR4 (48). These mutual effects may derive from the complex actions of allergens and cell–cell interactions, such as alterations in apoptosis due to Der p 38 associated with cytokine secretion from bronchial cells after exposure of Der p 38 (Fig. 6G). In that context, we need to broadly understand the role of TLR4 in HDM-mediated allergy from the perspective of LPS or Der p 2 to Der p 38, which will help clarify its involvement in HDM allergies. In addition, we are trying to uncover the systemic mechanisms involved.

In summary, Der p 38 is a key protein in HDM-mediated TLR4 mechanisms, which induces the infiltration of eosinophils and neutrophils as well as allergenicity. The present and further studies on Der p 38 may open up approach opportunities for deeper understanding of HDM-mediated allergies in the pathogenic, diagnostic, and therapeutic fields.

We thank normal volunteers and allergic subjects who participated in this study.

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea, which is funded by the Ministry of Education (NRF-2017R1D1A1B03034630) and the Korea Brain Research Institute (KBRI) Basic Research Program through the KBRI, funded by the Ministry of Science and ICT (21-BR-01-11).

The online version of this article contains supplemental material.

Abbreviations used in this article

BALF

bronchoalveolar lavage fluid

BLI

biolayer interferometry

HDM

house dust mite

ILC

innate lymphoid cell

IN

intranasal

KO

knockout

PAS

periodic acid–Schiff

PI

propidium iodide

WT

wild-type

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The authors have no financial conflicts of interest.

Supplementary data