Involvement of Activation of Mast Cells via IgE Signaling and Epithelial Cell–Derived Cytokines in the Pathogenesis of Pollen Food Allergy Syndrome in a Murine Model

Pollen food allergy syndrome (PFAS) is defined as an oral allergy syndrome caused by inhaled pollen Ags that commonly presents with local oral itching, numbness, and oral mucosal edema (1–3). These symptoms develop within several minutes of exposure to foods including fruits and vegetables. Serious anaphylactic symptoms such as wheezing, vomiting, or rash may also occur but rarely. Along with causative food, PFAS is caused by cross-reacting allergens found in inhaled pollen. Birch pollen is the representative inhaled pollen that induces PFAS. Studies in Western countries showed that approximately half of the patients sensitized to birch pollen developed symptoms of PFAS after consuming fresh fruits, vegetables, and nuts (4–6). Our laboratory reported that in an epidemiological survey among 6824 patients who visited the Department of Otorhinolaryngology of the University of Fukui Hospital and related hospitals, PFAS symptoms were observed in 734 individuals (10.7%), and the rate of birch pollen–specific IgE positivity was significantly higher in the PFAS group than in the healthy group (control group) without PFAS symptoms (31.7% versus 8.6%, p < 0.001) (7). The Rosaceae plant family members, particularly the apple, are representative food groups that cross-react with birch pollen. Bet v 1, the major birch pollen allergen, is a member of the pathogenesis-related protein 10 that exhibits high-grade amino acid sequence identity with the pathogenesis-related protein 10 found in apple (8, 9). Therefore, the Bet v 1–specific IgE cross-reacts with the apple allergens to induce PFAS symptoms (10, 11).

Allergic diseases, such as asthma, allergic rhinitis, atopic dermatitis, and food allergy, have been well analyzed using murine models (12–15). The analysis of changes in responses to various stimuli, histologic analyses using murine models, and outcome evaluations in knockout mice have greatly contributed to the elucidation of disease pathogenesis. However, compared with other allergic diseases, PFAS pathogenesis has not been studied in murine models. Most studies on PFAS reported on epidemiological surveys, which have not been investigated in basic research studies. Therefore, there are currently no useful therapies for PFAS, and patients have to avoid eating food containing the allergens to prevent onset. Because inhaled pollens show cross-reactivity to Ags contained in many types of food, patients with PFAS cannot avoid all causative food. Therefore, PFAS continues to be an important health issue due to its increasing incidence and adverse impact on the quality of life related to extensive food restriction (16). As a result, murine PFAS models are critical to clarify PFAS pathogenesis and discover useful therapeutic strategies that allow patients to freely consume food. Therefore, in this study, we established a novel, to our knowledge, murine PFAS model to examine the correlation between PFAS and several factors involved in the Th2 immune response.

Wild-type BALB/c mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). Fcer1a^−/−, Il-33^−/−, and thymic stromal lymphopoietin (TSLP) receptor–deficient (Crlf2^−/−) mice, all with the BALB/c background, were provided by the Department of Immunology of the Hyogo College of Medicine in Hyogo, Japan. Mast cell–deficient WBB6F1/Kit-Kit^W/Kit^W-v mice and littermate control WBB6F1/Kit mice were purchased from Sankyo Labo Service Corporation Japan (Hamamatsu, Japan). Bas-toxin receptor–mediated conditional cell knockout (TRECK) mice and Mas-TRECK mice were provided by Dr. Masato Kubo (Division of Molecular Pathology, Research Institute for Biological Sciences, Tokyo University of Science, Tokyo, Japan). All animal experiments were performed with the approval of and in accordance with the guidelines of Animal Research Committee at University of Fukui (No. 29005, No. 29040, No. 30085, No. R01036).

Birch, ragweed, and cedar pollen were purchased from Institute of Tokyo Environmental Allergy (Tokyo, Japan). Bet v 1 was provided by Professor Kenji Miura at the Faculty of Life and Environmental Sciences of the University of Tsukuba in Tsukuba, Japan. The protease inhibitor 5892970001 and diphtheria toxin (DT) were purchased from Sigma-Aldrich (St. Louis, MO). Purified anti-mouse CD3e (145-2C11), purified anti-mouse CD28 (37.51) Abs, purified anti-mouse CD16/32 (93), Pacific Blue–conjugated anti-mouse CD45 (30-F11), FITC-conjugated anti-mouse CD49b (pan-NK cells) (DX5), and PE-conjugated anti-mouse Fc ε receptor 1 α (MAR-1) Abs were purchased from BioLegend (San Diego, CA). Biotin-conjugated anti-mouse FceR1 (MAR-1) Ab was purchased from eBioscience (San Diego, CA). FITC-conjugated Abs against mouse CD3 (145-2C11), CD4 (RM4-5), CD8a (53-6.7), CD11b (M1/70), CD11c (N418), B220 (RA3-6B2), and Ly-6G/Ly-6c (Gr-1) (RB6-8C5); APC-conjugated Ab against mouse streptavidin; and PE-conjugated Abs against mouse CD90.2 (Thy1.2) (53-2.1) were purchased from BioLegend. FITC-conjugated anti-mouse FceR1 (MAR-1) Ab was purchased from eBioscience. Biotinylated anti-T1/ST2 (DJ8) Ab was purchased from MD Bioproducts (St. Paul, MN).

Mice were immunized against birch pollen by i.p. injection of birch pollen (100 μg) and aluminum hydroxide hydrate gel (1 mg) in 200 μl of PBS on days 0, 7, and 14. For the experiment presented in Supplemental Fig. 5, mice were immunized against Bet v 1 by i.p. injection of Bet v 1 (1 μg) and aluminum hydroxide hydrate gel (1 mg) in 200 μl of PBS on days 0, 7, and 14. The mice were then administered intranasal (i.n.) birch pollen (1 mg in 20 μl of PBS) for four consecutive days (days 21–24). The frequency of sneezing was determined for 10 min immediately after the nasal challenge on day 24. Next, the mice were challenged orally with 30 μl of apple extract on day 25. The apple extract was used immediately after a raw apple was grated. The frequency of oral rubbing was counted for 10 min immediately after the oral challenge on day 25. The frequency of nasal sneezing and oral rubbing was determined by a blinded observer. The mice were sacrificed, and serum, nose, oral mucosa, and cervical lymph nodes (CLNs) were collected for further analysis.

For the experiment presented in Supplemental Fig. 3, the mice were immunized against birch pollen by i.p. injection of birch pollen (100 μg) and aluminum hydroxide hydrate gel (1 mg) in 200 μl of PBS on days 0, 7, and 14. The mice were then given intratracheal birch pollen (100 μg in 50 μl of PBS) for four consecutive days (days 21–24). The frequency of oral rubbing over 10 min was counted immediately after the oral challenge on day 25. The mice were sacrificed, and serum and lung samples were collected for further analysis.

Basophil-depleted mice were established by the administration of MAR-1 Ab (13). The mice were immunized against birch pollen by i.p. injection of birch pollen (100 μg) and aluminum hydroxide hydrate gel (1 mg) in 200 μl of PBS on days 0, 7, and 14. The immunized mice were administered i.p. MAR-1 Ab (5 μg) in 200 μl of PBS every day on days 20–24. The mice were also administered i.n. birch pollen (1 mg) in 20 μl of PBS for four consecutive days (days 21–24) and orally challenged with 30 μl of apple extract on day 25 (Supplemental Fig. 6A). We confirmed that the CD45⁺/FceR1⁺/DX-5⁺ basophils in serum were depleted by the administration of MAR-1 Ab using flow cytometry (Supplemental Fig. 6B).

The Bas-TRECK and Mas-TRECK mice were examined as previously described (17). For DT treatment, the mice were injected i.p. with 500 ng of DT in 250 μl of PBS per mouse for the Bas-TRECK mice or 250 ng of DT in 250 μl of PBS per mouse for the Mas-TRECK mice. The Bas-TRECK or Mas-TRECK mice were immunized against birch pollen by i.p. injection of birch pollen (100 μg) and aluminum hydroxide hydrate gel (1 mg) in 200 μl of PBS on days 0, 7, and 14. Then, the immunized Bas-TRECK mice were injected i.p. with 500 ng of DT in 250 μl of PBS on days 15, 18, 21, and 24. The immunized Mas-TRECK mice were injected i.p. with 250 ng of DT in 250 μl of PBS on days 15 − 19, 22, and 24. The mice were given i.n. birch pollen (1 mg) in 20 μl of PBS for four consecutive days (days 21–24) and orally challenged with 30 μl of apple extract on day 25. The frequency of oral rubbing over 10 min was counted immediately after the oral challenge on day 25. The mice were sacrificed, and serum samples were collected for IgE analysis (Fig. 4A, 4D).

An investigator who was unaware of the clinical data chose a mouse randomly and blindly. Awake mice were given i.n. birch pollen (1 mg) in 20 μl of PBS for four consecutive days (days 21–24) and orally challenged with 30 μl of apple extract on day 25. Immediately after the challenge, each mouse was put in a new standard breeding cage but without bedding (1 mouse per cage). An investigator directly monitored the behavior of each mouse for 10 min, and each sneezing and oral rubbing behavior was counted.

Histological examinations of nasal and oral mucosa specimens from mice were performed as previously described (18). Briefly, facial skin was stripped. The noses and oral mucosa were removed, fixed in 4% paraformaldehyde overnight, and decalcified in 0.12 mol/l EDTA solution (pH 6.5) at room temperature for 10 d. Nasal and oral mucosal specimens were embedded in paraffin, and 4-μm-thick coronal sections were prepared and stained with H&E and toluidine blue for the analysis of eosinophils and mast cells, respectively. The total number of eosinophils in nasal specimens was calculated.

For immunohistochemistry of mast cell tryptase, the sections were deparaffinized and heated in a microwave oven using target retrieval solution (Dako, Tokyo, Japan) for Ag retrieval. After blocking, the sections were incubated in rabbit anti-mouse monoclonal TPSAB1 Ab (10 μg/ml; SC68-07; Thermo Fisher Scientific, Waltham, MA) overnight at 4°C. The sections were rinsed and incubated in anti-rabbit HRP-conjugated Ab (Envision dual-link system; Dako) for 1 h at room temperature. The sections were rinsed and incubated in a DAB/Tris solution (Muto Pure Chemicals, Tokyo, Japan) for 10 min at room temperature and counterstained with hematoxylin. The number of mast cells in the oral mucosa specimens was counted in the three densest areas with cellular infiltration at high-power field (original magnification ×400), which were chosen by a blinded observer who was unaware of the clinical data, and the mean number of mast cells was calculated.

Serum total IgE concentrations were measured using a specific ELISA kit (YAMASA, Tokyo, Japan). To measure specific IgE levels in serum, 96-well plates were coated with the birch pollen extract (30 μg/ml; Institute of Tokyo Environmental Allergy), Bet v 1 (10 μg/ml), or apple extract (10-fold dilution) and incubated overnight at 4°C. After blocking, the samples were added to the plates and incubated overnight at 4°C. After washing, the biotinylated anti-mouse IgE Ab (BioLegend) was added to the plates and incubated for 1 h at room temperature. After washing, avidin-HRP (BioLegend) was added to the plates and incubated for 30 min at room temperature. After washing, the 3,3′,5,5′-tetramethylbenzidine substrate solution was added to the plates and incubated in the dark for 20 min. The stop solution was added, and the OD was measured at 450 nM. IL-4, IL-5, and IL-13 concentrations were measured using specific ELISA kits (Thermo Fisher Scientific).

Cell cultures were performed as previously described (18). Briefly, mice were sacrificed and noses, oral mucosa, and CLNs were dissected for further analysis. The nasal tissue and oral mucosa were cut into small fragments with scissors and digested for 50 min at 37°C with collagenase (150 U/ml) and DNase I (10 mg/ml). The cell suspensions were filtered using a cell strainer, and RBCs were lysed. The CLNs were dissected from mice, and single cell suspensions were prepared by sieving and gentle pipetting. The nasal, oral mucosal, and CLN cells were cultured for 2 d in 96-well plates at concentrations of 2 × 10⁴, 2 × 10⁴, and 2 × 10⁵ per 200 μl per well, respectively, in the presence of purified anti-mouse CD3e (1 μg/ml) and purified anti-mouse CD28 (1 μg/ml) Abs with RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The culture supernatants were collected, and cytokine concentrations were assessed by ELISA.

Oral mucosa cells were isolated from mice. For surface staining, the cells were washed in ice-cold staining buffer (1% BSA in PBS), incubated with each Ab for 20 min, and washed twice with the staining buffer. Group 2 innate lymphoid cells (ILC2s) in the oral mucosa were stained with Abs for CD45, Thy1.2, and ST2 as well as against the following lineage markers: CD3, CD4, CD8, CD11b, CD11c, B220, CD49b, Gr-1, Siglec F, and FceR1. The stained cells were analyzed by FACS Canto II flow cytometer and the FlowJo software. Lineage⁻/CD45⁺/ST2⁺/Thy1.2⁺ cells were defined as ILC2s.

GraphPad Prism software (San Diego, CA) was used for all statistical analyses. All data were reported as mean ± SEM unless otherwise noted. Differences between groups were analyzed by one-way ANOVA with Dunnett post hoc test or t test. A p value <0.05 was considered statistically significant.

To establish the murine PFAS model, we first developed a murine model of birch pollen–sensitized allergic rhinitis. The sneezing frequency after nasal challenge on day 24 was significantly higher in the mice immunized with i.p. injection of birch pollen (100 μg) and aluminum hydroxide hydrate gel (1 mg) in 200 μl of PBS on days 0, 7, and 14 and administered i.n. birch pollen (1 mg in 20 μl of PBS) on days 21–24 as compared with the naive mice (Fig. 1A). The birch pollen–immunized mice exhibited significantly higher serum levels of total and birch pollen–specific IgE (Fig. 1B). To investigate the ability of activated Th2 cells in regional lymph nodes to produce Th2 cytokines, CLN cells were stimulated with aCD3/aCD28 to assess the production of IL-4, IL-5, and IL-13. The birch pollen–sensitized mice showed significant increases in the production of all three Th2 cytokines (Fig. 1C). Furthermore, the birch pollen–sensitized mice showed stronger eosinophilic infiltration in the nasal mucosa than the naive mice (Fig. 1D–F).

The birch pollen–immunized mice that were administered apple extract orally on day 25 (Fig. 2A) exhibited oral rubbing action, which was clearly different from the nasal rubbing action observed after the nasal administration of birch pollen in the murine model of birch pollen–sensitized allergic rhinitis (Supplemental Fig. 1A, 1B). This oral rubbing action is the exact allergic response in PFAS. The nasal apple-extract administration increased sneezing, which was absent in the mice administered apple extract orally, indicating that the response elicited by the oral apple-extract challenge did not play a role in the nasal response (Supplemental Fig. 1C, 1D). The oral rubbing frequency after the oral challenge was significantly increased in the birch pollen–immunized mice compared with the naive mice (Fig. 2B). The oral apple-extract challenge 1 h after the nasal birch pollen administration did not increase the oral rubbing frequency as compared with the oral apple-extract challenge on the day after the nasal birch pollen administration (Supplemental Fig. 1E). Of note, the rectal temperature was comparable between the birch pollen–immunized mice after the oral challenge and the naive mice (Fig. 2C). A positive reaction in the prick test with food is an effective diagnostic method for PFAS. The birch pollen–immunized mice were administered birch pollen or the apple extract in s.c. ear tissue, and the ear thickness was examined. The birch pollen–immunized mice exhibited significantly more ear swelling after the administration of birch pollen or the apple extract compared with the naive mice (Fig. 2D, 2E), suggesting that the oral challenge with the apple extract in birch pollen–immunized mice led to the development of PFAS-like symptoms, including oral rubbing action without systemic symptoms and a positive reaction by the prick test. Mice sensitized with birch pollen peritoneally and nasal administration had significantly higher frequencies of oral itching after oral challenge with apple extract and total and birch pollen–specific IgE levels in serum than the mice sensitized with birch pollen peritoneally without nasal administration (Supplemental Fig. 2A, 2B). To induce asthma-like lung inflammation, the mice were injected i.p. with birch pollen on days 0, 7, and 14 and intratracheal with birch pollen on days 21–24. We confirmed that the mice sensitized with birch pollen peritoneally and lung administration induced lung inflammation (Supplemental Fig. 3A). The frequency of oral itching after oral challenge with apple extract and the total and birch pollen–specific IgE in serum were significantly increased in the mice with induced asthma-like lung inflammation (Supplemental Fig. 3B, 3C). The oral rubbing frequency was higher with the oral apple-extract administration than with the oral birch pollen administration in the birch pollen–immunized mice (Fig. 2F). The challenge with apple extract mixed with a protease inhibitor significantly reduced the oral rubbing frequency after the oral apple-extract administration (Fig. 2G), which suggests that the protease activity included in food Ag stimulation might be closely associated with the exacerbation of oral symptoms in PFAS.

We compared the oral rubbing reaction with the various oral food-extract administrations in the mice immunized with birch pollen. The oral rubbing frequency after the oral peach-extract challenge was significantly higher in the birch pollen–immunized mice than in the naive mice. In contrast, the oral rubbing frequency after the oral tomato-extract challenge in the birch pollen–immunized mice was comparable with that in the naive mice (Supplemental Fig. 4A). We compared the oral rubbing reaction to the oral apple-extract administration in mice immunized with various inhaled Ags. The serum IgE levels were comparable among the mice immunized by the i.p. injection and subsequently nasally administered ragweed pollen, cedar pollen, or birch pollen (Supplemental Fig. 4B). However, the oral rubbing frequency after the oral apple-extract challenge was significantly higher in the birch pollen–immunized mice compared with the ragweed pollen– and cedar pollen–immunized mice (Supplemental Fig. 4C). Furthermore, the apple extract that was heated and denatured for 30 min at 100°C failed to induce an increase in the oral rubbing frequency (Supplemental Fig. 4D). Apple-specific IgE levels were increased in birch-immunized mice (Supplemental Fig. 4E). These results suggest that the Ag-specific cross-reaction between the birch pollen and the apple extract induced the oral symptoms in the murine PFAS model.

Bet v 1 is the major protein component of birch pollen. The birch pollen–immunized mice exhibited significantly increased serum levels of Bet v 1–specific IgE (Supplemental Fig. 5A). To examine the role of Bet v 1 in the murine PFAS model, mice were immunized with Bet v 1 and orally challenged with the apple extract. The serum levels of total and Bet v 1–specific IgE, oral rubbing frequency after the oral challenge, and the Th2-cytokine production by the CLN cells following aCD3/aCD28 stimulation were significantly higher in the Bet v 1–immunized mice compared with the naive mice (Supplemental Fig. 5B–D). These results indicated that oral rubbing in the murine PFAS model was due to the cross-reaction between a Bet v 1–specific Ab and the allergens in the apple extract.

Allergen-specific reactions via IgE signaling have major roles in allergic diseases. We therefore used Fcer1a^−/− mice deficient for the high-affinity receptor for IgE (Fcer1a) to examine the role of IgE signaling in the murine PFAS model. The oral rubbing frequency after the oral challenge with apple extract was significantly reduced in the birch pollen–immunized Fcer1a^−/− mice compared with the immunized wild-type mice (Fig. 3A). The serum levels of total IgE and birch pollen–specific IgE as well as the Th2-cytokine production by the CLN cells following aCD3/aCD28 stimulation were comparable between the immunized Fcer1a^−/− mice and the immunized wild-type mice (Fig. 3B, C), demonstrating that IgE signaling was necessary to induce oral rubbing in the murine PFAS model.

Next, we examined the role of mast cells and basophils in IgE signaling. We used basophil-depleted mice that were administered the MAR-1 Ab as well as the mast cell–deficient WBB6F1/Kit-Kit^W/Kit^W-v mice. The serum levels of total and birch pollen–specific IgE in the immunized basophil-depleted mice and the immunized mast cell–deficient WBB6F1/Kit-Kit^W/Kit^W-v mice were comparable with the respective immunized control mice (Supplemental Fig. 6C, 6E). The oral rubbing frequency after the oral challenge with apple extract in basophil-depleted, birch pollen–immunized mice was comparable with that in the immunized control mice (Supplemental Fig. 6D). In contrast, the oral rubbing frequency after the oral challenge with apple extract in birch pollen–immunized WBB6F1/Kit-Kit^W/Kit^W-v mice was significantly decreased compared with that in the immunized control mice (Supplemental Fig. 6F). In addition, we used mice with Mas-TRECK and Bas-TRECK, which are DT-based conditional mast cell and basophil deletion systems, respectively (TRECK). The DT treatment of the Bas-TRECK mice resulted in a specific deletion of basophils, whereas both mast cells and basophils were deleted in the Mas-TRECK mice by DT because of the expression of the DT receptor on their cell surfaces. The serum total and birch pollen–specific IgE levels in the immunized DT-treated Bas-TRECK mice and immunized DT-treated Mas-TRECK mice were comparable with those in the immunized control mice (Fig. 4B, 4E). The oral rubbing frequency after the oral challenge with apple extract in the immunized DT-treated Bas-TRECK mice was comparable with that in the immunized control mice (Fig. 4C). By contrast, the oral rubbing frequency after the oral challenge with apple extract in the immunized DT-treated Mas-TRECK mice was significantly decreased as compared with that in the immunized control mice (Fig. 4F). Indeed, no abundant mast cells were found in the oral mucosa of both the naive and immunized mice (Fig. 4G, 4H). The numbers of mast cells in the oral mucosa did not increase in the immunized mice as compared with the naive mice, which suggests that the developed mast cells degranulation via IgE signaling played a central role in oral rubbing in the murine PFAS model.

We examined the relationship between the symptoms of PFAS and epithelial-derived cytokines using Il-33^−/− mice and Crlf2^−/− mice. The wild-type and Il-33^−/− mice were immunized by i.p. injection and nasal administration of birch pollen, followed by the oral apple-extract challenge. The birch pollen–immunized Il-33^−/− mice exhibited a lower frequency of oral rubbing after the oral challenge with apple extract as compared with the immunized wild-type mice (Fig. 5A). The serum levels of total and birch pollen–specific IgE and the Th2-cytokine production by the CLN cells following aCD3/aCD28 stimulation were significantly reduced in the immunized Il-33^−/− mice compared with the immunized wild-type mice (Fig. 5B, 5C). Similarly, the wild-type and Crlf2^−/− mice were immunized by i.p. injection and nasal administration of birch pollen, followed by oral apple-extract challenge. The oral rubbing frequency following the oral apple-extract challenge was significantly decreased in the birch pollen–immunized Crlf2^−/− mice compared with the immunized wild-type mice (Fig. 5D). The serum levels of total and birch pollen–specific IgE and the Th2-cytokine production by the CLN cells following aCD3/aCD28 stimulation were also significantly decreased in the immunized Crlf2^−/− mice compared with the immunized wild-type mice (Fig. 5E, 5F), suggesting that the epithelial-derived cytokines played an important role in the PFAS pathogenesis in our murine model.

Chronic nasal inflammation caused by the activation of Th2 cells and eosinophils following Ag stimulation of the nasal mucosa is an important pathogenic feature of allergic rhinitis. The birch pollen–sensitized mice showed significant increases in the production of Th2 cytokines and strong eosinophilic infiltration in the nasal mucosa (Fig. 1). Conversely, whether Ag stimulation of the oral mucosa in PFAS induces a local Th2/eosinophilic chronic inflammation remains unclear. Therefore, we investigated local inflammatory response in our murine PFAS model. The mice were orally challenged with apple extract on day 25 (immune per apple 1 d) or for 14 consecutive days (days 25–38; immune per apple 14 d) after i.p. and i.n. immunization with birch pollen. We confirmed that the oral rubbing frequency after the final oral challenge in the birch pollen–immunized mice orally challenged with apple extract for 14 consecutive days was comparable with that in the birch pollen–immunized mice orally challenged with apple extract on day 25, and the exposure to the apple extract for 14 consecutive days did not induce oral desensitization (Fig. 6A). To examine the production of Th2 cytokines by Th2 cells in the local mucosa, cells derived from the nasal and oral mucosa were stimulated with aCD3/aCD28 to measure IL-4, IL-5, and IL-13 levels. The aCD3/aCD28 stimulation led to a significantly higher production of Th2 cytokines by the nasal mucosa–derived cells from the birch pollen–immunized mice compared with the nasal mucosa–derived cells from the control mice. In contrast, the aCD3/aCD28 stimulation of the cells derived from the oral mucosa did not lead to an increased production of Th2 cytokines in the birch pollen–immunized mice or the birch pollen–immunized mice orally challenged with the apple extract for 14 consecutive days (Fig. 6B). Next, we evaluated the eosinophilic infiltration of the oral mucosa in the murine PFAS model by H&E staining and found that, consistent with the poor production of Th2 cytokines in the oral mucosa, the oral mucosa of the birch pollen–immunized mice did not exhibit eosinophilic infiltration 24 h after the oral apple-extract challenge, even after exposure to the extract for 14 consecutive days (Fig. 6C). Group ILC2s were present in the oral mucosa (Fig. 6D); however, consecutive Ag stimulation of the oral mucosa in the immunized mice did not increase the number of ILC2s in the oral mucosa (Fig. 6E), demonstrating that the pathogenic course of local Th2/eosinophilic inflammation differed between allergic rhinitis and PFAS.

We established a novel, to our knowledge, PFAS murine model to analyze the underlying pathogenesis and showed that allergen stimulation of the oral mucosa induced an early-phase response involving mast cells and epithelial cell–derived cytokines but not a late-phase response involving local activated Th2 cells and eosinophils, a finding that is distinct from other allergic inflammatory processes. The birch pollen–sensitized mice exhibited oral rubbing action without a change in rectal temperature after the oral apple-extract challenge and increased ear thickness following apple-extract injection to the s.c. ear tissue. The diagnosis of PFAS is based on a positive history and a positive skin-prick test triggered by a fresh extract of the specific food (18). Our novel, to our knowledge, murine model focusing on oral rubbing action as a closely representative characteristic of PFAS can be a useful tool to study the pathophysiology and clinical course of PFAS.

The cross-reactive epitopes that induce PFAS are readily broken down and denatured by digestive proteases and the body temperature. As a result, a stimulation by food Ags in PFAS would rarely induce anaphylaxis and heat treatment can reduce oral symptoms (19). Indeed, oral challenge of apple extract did not exhibit systemic anaphylaxis and the heat-denatured apple extract failed to induce oral rubbing in the murine PFAS model. In contrast, studies on conventional food allergy induced by foods, including wheat, peanut, egg, and milk, have shown that oral challenge with a food Ag in murine food allergy models can induce systemic symptoms and heat treatment is often not useful in suppressing these symptoms (20–22). Therefore, our murine PFAS model is novel, to our knowledge, and distinct from conventional murine models of food allergy. The oral symptom in PFAS may not be necessarily exacerbated during the pollen dispersed time because in our study, the oral apple-extract challenge 1 h after the nasal birch pollen administration did not increase the oral rubbing frequency as compared with the oral apple-extract challenge on the next day after the nasal birch pollen administration. The frequency of oral rubbing and serum birch-specific IgE level were not increased only in the i.p. immunized mice. The mice with induced asthma-like lung inflammation showed high frequency of oral rubbing and serum birch-specific IgE level. The onset of PFAS was higher in the patients with than in the patients without bronchial asthma (7). Sufficient increase in serum birch-specific IgE level is essential for the establishment of the PFAS murine model. The oral apple-extract challenge in our PFAS model led to significantly more oral symptoms than that induced by birch pollen. Most fruits and vegetables are capable of exhibiting strong protease activity (23). Proteases can promote inflammation and exacerbate allergic symptoms in Th2 environment by inducing IL-33 and TSLP release (24). The apple extract mixed with a protease inhibitor suppressed the oral symptoms in our murine PFAS model. This protease inhibitor tablet is used for the inhibition of an extended range of proteases and inhibits aspartic proteases, as well as serine, cysteine, and metalloproteases, so the protease in apple that affects them must be further verified. Some protease activity in the apple extract might enhance the histamine release from mast cells activated by the cross-reactivity between birch pollen and apple.

Ag-specific cross-reactivity is essential for oral symptom development in PFAS (25–27). The oral rubbing frequency after peach-extract oral administration was significantly increased in the birch pollen–immunized mice, but not in those that received oral administration of tomato extract. Apple and peach are Rosaceae plant family members that cross-react with birch pollen, but tomato does not show a cross-reaction with birch pollen. The oral rubbing frequency after apple extract oral administration was significantly increased in the birch pollen–immunized mice but not in the ragweed pollen– or cedar pollen–immunized mice. Furthermore, apple-specific IgE levels were increased in birch-immunized mice. Patients with PFAS sensitized with specific allergens develop PFAS symptoms against specific food Ags (e.g., sensitization with birch, ragweed, and Japanese cedar leading to PFAS symptoms against apple, melon, and tomato, respectively). These responses depend on the high similarity of the allergenic protein components. Most patients with PFAS sensitized to birch pollen are positive for Bet v 1–specific IgE, which can cross-react with fruits belonging to the Rosaceae plant family, such as apple, peach, cherry, and hazel nuts (28). In the current study, Bet v 1–specific IgE in birch pollen was demonstrated to play an important role in oral symptom development following oral apple-extract challenge. Our murine PFAS model can be used to test specific combinations of various inhaled allergens and foods because the oral rubbing reaction is an Ag-specific response that can effectively help identifying unknown cross-reactivity and correlation among specific pollens and foods.

Mast cells and basophils play an important role in IgE signaling. The symptoms of both allergic rhinitis and food allergy improve in mast cell– and basophil-depleted mice (13, 21). In the murine PFAS model, the significant reduction in the frequency of oral rubbing following oral apple-extract challenge observed in the immunized, mast cell–deficient WBB6F1/Kit-Kit^W/Kit^W-v and DT-treated Mas-TREC mice, which was not observed in the basophil-depleted MAR-1– and DT-treated Bas-TREC mice, suggests that mast cells play a more important role in the oral symptoms of PFAS compared with basophils. Nevertheless, basophils play an essential role in the pathogenesis of PFAS by contributing to the Th2–IgE responses via IL-4 production and the presentation of peptide–MHC class II complexes to CD4⁺ T cells (29–31). In our murine PFAS model, we administered MAR-1 Ab to wild-type mice or DT to the Bas-TRECK mice after the i.p. immunization with birch pollen and examined responses to the oral administration of the cognate Ag. Therefore, the Th2-IgE responses via basophils were established by the i.p. immunization; basophils play an important role in Th2 cell differentiation, and histamine released by mast cells induced the oral symptoms in PFAS.

The IL-33/ST2 pathway and TSLP signaling play important roles in innate and adaptive immune responses and Th2-type inflammation of the airway (24, 32). These cytokines are induced by several factors including allergens, proteases, bacteria, and viruses and act on various cells such as Th2 cells, mast cells, eosinophils, and ILC2s (33). Many reports demonstrated that IL-33 and TSLP drive Th2 differentiation and Ab production by B cells and enhance IgE-mediated mast cell degranulation (20, 34–36). However, the role of epithelial-derived cytokines in oral mucosa remains unclear. We found that serum IgE levels, Th2-cytokine production by the CLNs, and oral rubbing frequency were significantly reduced in the birch pollen–immunized Il-33^−/− and Crlf2^−/− mice, suggesting that IL-33 and TSLP might be involved in PFAS pathogenesis by inducing the differentiation and activation of Th2 cells in CLNs, with subsequent IgE production. Furthermore, IL-33 and TSLP are released following exposure to the allergen and protease and induce mast cell degranulation (33, 37). Because the allergen and protease included in evoked food can exacerbate the PFAS symptoms, IL-33 and TSLP might be closely associated with the PFAS pathogenesis by acting on the degranulation response of the mast cells in the oral mucosa and on the innate immunity, in addition to the adaptive immunity such as Th2 cell differentiation in the CLNs and IgE production.

Allergen administration in allergic murine models usually induces chronic eosinophilic inflammation (38). However, consecutive oral apple-extract administration did not induce eosinophilic infiltration of the oral mucosa in our murine PFAS model. The allergic response in allergic rhinitis is divided into the early and late phases (39). The early-phase response occurs within minutes after allergen exposure by the release of histamine from mast cells, whereas the late-phase response develops 6–10 h later by the eosinophil-derived mediators. In contrast, the allergic response in PFAS depends on the early phase and does not include a late phase; therefore, PFAS symptoms are short-lived. Various cells associated with type II inflammation can induce eosinophilic infiltration during allergic diseases (40). Activated Th2 cells are the most important players in eosinophilic infiltration as they release IL-5. Unlike the CLN and nasal Th2 cells, the oral Th2 cells did not lead to an increase in Th2 cytokines in the current murine PFAS model, indicating that the activated Th2 cells were located in the regional CLNs and the nose rather than the oral mucosa, which might explain the relatively less local eosinophilic infiltration in PFAS compared with other allergic diseases. Furthermore, the number of ILC2s did not increase upon allergen stimulation in oral mucosa. IL-33 and TSLP might be more strongly associated with mast cell activation than with Th2-cytokine production in the oral mucosa. As the epitope recognized as the common Ag between Bet v 1 and the apple Ag is limited, it might be degraded by salivary protease, and the organizational structure of the epithelium and certain factors in the oral mucosa might underlie the differences between the nasal and oral allergic inflammatory processes, which should be elucidated in future studies.

In summary, the activation of mast cells via IgE signaling following cross-reaction between Bet v 1–specific IgE and the allergens derived from apple induced the oral symptoms during the early phase of PFAS. However, local Th2/eosinophilic inflammation in the oral mucosa as a late phase reaction was not induced by the allergen stimulation. IL-33 and TSLP released induced by the allergens and proteases in apple might be exacerbating the PFAS symptoms via mast cell degranulation. Appropriate control of these factors might be useful as a therapeutic strategy in PFAS. The pathogenesis of allergic diseases includes various factors such as Th2 cells, B cells, dendritic cells, and ILC2s, among others. Future studies using our novel, to our knowledge, murine model might clarify the pathogenesis of PFAS and provide a useful therapeutic platform for PFAS.

We thank all the colleagues in our laboratories, Miyuki Shirasaki, and Yumie Yasusaki for secretarial assistance and Hiroko Tsuchiya for technical assistance. We also thank Enago (www.enago.jp) for the English language review.

The authors have no financial conflicts of interest.