Food allergy enhances allergic asthma in mice

6 weeks old female Balb/c mice were sensitized for the food allergy, respiratory allergy or both. The induction of the food allergy was carried out on days 0, 14 and 21 by intraperitoneal (i.p.) injection of 10 μg of OVA (Sigma-Aldrich, L'Isle d'Abeau Chesnes, France) diluted in 100 μl of Imject® Alum (Pierce, Rockford, USA) followed by intra-gastric (i.g.) administration of 20 mg of OVA solubilized in water on D27 to D29 (Figure 1A). Control mice were i.p.-sensitized with alum and challenged with water. Respiratory allergy was obtained as follows: mice were sensitized on days 0, 7, 14 and 21 by cutaneous application (p.c.) of 500 μg of total extract of Der f (Stallergenes, Antony, France) diluted in 20 μL of dimethylsulfoxyde (DMSO, Sigma-Aldrich, L'Isle d'Abeau Chesnes, France). Mice were challenged intranasally (i.n.) with 250 μg of Der f on D27 and D34 (Figure 2A) diluted in PBS. Control mice were sensitized with DMSO alone and challenged with PBS. Combined food and respiratory allergy was obtained by applying the two protocols successively (Figure 3A). For respiratory sensitization and challenges animals were anesthetized with 100 μl of i.p. xylazine (15 mg/kg) and 100 μl of i.m. ketamine (80 mg/kg). Mice were sacrificed using dolethal (Vetoquinol). Analyses were performed three days after the food challenge (day 32), after the 1^st respiratory challenge (day 28 and 30) and after the 2^nd respiratory challenge (day 35 and 37).

After 2 hours fast, fecal pellets were collected, counted and weighed. For permeability measurements, the jejunum and proximal colon were removed, washed in cold Krebs’s solution and sections were mounted in Ussing chambers (Physiological instruments, San Diego, CA). Paracellular permeability was assayed by fluorescein–5.6 sulfonic acid (1 mg/mL, Invitrogen) gradient over time by fluorimetry (Varioskan, Thermo SA, France). Transcellular permeability was assayed by HRP (10 mg/ml, Sigma-Aldrich) activity over the time using a Varioskan spectrofluorimeter (Thermo SA, Saint Herblain, France).

Unrestrained mice were nebulized in a plethysmography chamber with increasing doses of methacholin (0-40 mg/mL). Airway function was measured and expressed as enhanced pause (Penh) (Emka, Paris, France). Dynamic lung resistances were measured using the flexivent® (SCIREQ, Emka) system. Mice were anesthetized with a mix of xylazine (0.05 mg/kg), ventilated, paralyzed with rocuronium bromide (Organon, 10 mg/ml) and nebulized with methacholin (0-20 mg/mL). Airway resistances were continuously monitored and recorded according to manufacturer’s instructions.

1 mL of paraformaldehyde (PFA) 4% was administered intra-tracheally. Excised lungs were fixed in PFA 4%, embedded in paraffin, cut and stained with hematoxylin and eosin for morphological study and inflammation scoring from 0 to 7 representing bronchial epithelial cell dystrophy grades from 0 to 3 and peribronchial/perivascular inflammatory infiltrate abundance grades from 0 to 4.

Total IgE and IgG1 were assayed by ELISA in serum. Specific anti-Der f 1 IgE and anti-OVA IgE were assessed by indirect ELISA. 96 wells plates were coated with sodium bicarbonate 50 μM and 0.25 μg of purified Der f 1 (Indoor Biotechnologies, Wiltshire, UK) or 5 μg OVA (Sigma-Aldrich, L'Isle d'Abeau Chesnes, France) and left overnight at 4°C. Wells were then incubated with bovine serum albumine 1% for 12 h at 4°C, washed, filled with sera diluted in CGS1 (Cosmo Bio, California, USA) and incubated overnight at 4°C. Specific Ig was revealed with anti-mouse IgE (AbD serotec, Colmar, France) coupled to HRP. Substrate ABTS (Roche, Boulogne-Billancourt, France) was added and absorbance measured at 405 nm with VictorTMX3 (PerkinElmer, Courtaboeuf, France).

Broncho-alveolar lavage (BAL) fluid was recovered by intra-tracheal administration and aspiration of 1 mL of PBS. To obtain total lung immune cells, the lungs were disrupted and digested at 37°C for 1H in a solution containing Dnase I (Roche) and collagenase II (Invitrogen). To determine the immune profile, lung and BAL cells were counted and stained by flow cytometry as previously described [16] with specific anti-mouse antibodies: CD3-APC, CD8-APC-H7, CD19 PE-Cy7, F4/80-FITC, Ly6G-PerCP-Cy5.5, and CCR3-PE (R&D, Lille, France). Analyses were performed on a BD LSR™ II with BD FACSDiva™ software (BD Biosciences).

Cytokines (IL-4, IL-5, IL-10, IFN-γ, IL-17, chemoattractant keratinocyte (KC) and CCL5/RANTES (Regulation on Activation, Normal T cell Expressed and Secreted) were quantified by Luminex (BioPlex 200 system, Bio-Rad, Marnes-La-Coquette, France) according to manufacturer’s instructions (Bio-Rad).

GraphPad Prism 4.0b (Graphpad Software Inc., La Jolla, CA USA) was used. Data are expressed as mean ± standard error to the mean (SEM). Means were compared among groups using ANOVA. In case of positive ANOVA means were compared by Mann-Whitney test. Results were corrected by a Bonferroni multiple comparisons correction test. A p < 0.05 was considered statistically significant.

To characterize the food allergy part of the model, serum-allergen specificity and intestinal function of OVA and control mice were evaluated on day 32, 3 days after final challenge (Figure 1A). Type I allergy was confirmed by a significant increase of total (697 vs 5603 ng/ml) and OVA-specific IgE levels (2680 vs 15913 fluorescence units) in OVA mice compared to controls (Figure 1B). Th2-supported IgG1, and Th1-supported IgG2a proteoglycan-specific antibody levels in serum were determined. The IgG2a predominance (1.33.10⁷ vs 1,399.10⁶ ng/ml) in allergic mice suggests a bias towards a Th2-type response (Figure 1C). Intestinal parameters were assessed 30 minutes after the final intra-gastric challenge (day 29) (Table 1). Transit time, fresh faecal pellet weight and humidity were similar in both groups. Contrastingly, a significant increase in paracellular permeability was found in OVA mice, whereas there was no difference is transcellular permeability between the groups. OVA sensitization did not trigger any change in respiratory function or airway inflammation (not shown). Taken together these results show that OVA i.p. sensitization and intra-gastric challenge induces a systemic type I allergy and an increase in intestinal permeability.

In order to follow as closely as possible the atopic march, asthma was induced by percutaneous sensitization and intranasal challenge (Figure 2A). Total and specific serum IgE levels significantly increased in Der f-allergic mice on day 35, one day after the 2^nd challenge (0.36 vs 1.14 μg/ml and 0.44 vs 1.6 A.U respectively) (Figure 2B), but not on day 26, before challenges (not shown), suggesting that skin exposure is not sufficient for inducing a full allergic response and that bronchial exposure is essential. Regarding respiratory function, Der f-allergic mice displayed airway hyperresponsiveness (AHR) one and three days (day 28 and day 30) after the first challenge (Figure 2C). A second challenge on day 35 increased AHR dramatically, a result confirmed using flexivent® on day 37 (Figure 2D). Overall tissue inflammation was assessed by histological evaluation of lung sections at the same time points. A clear increase in inflammation between the 1^st (day 28) and the 2^nd challenge (day 35) (Figure 2E) was observed, concordant with respiratory function measurements. Figure 2F shows the extensive perivascular and peribronchial cell infiltration on day 35 (Figure 2F).

In order to characterize the inflammatory profile induced by Der f in the model, the proportions of eosinophils, neutrophils and lymphocytes in lungs and BAL were analyzed after the 1^st (day 28) and the 2^nd challenge (day 35) (Figure 3). On day 28, a significant increase in neutrophils (16.10³versus 130.10³ cells), but not in eosinophils and lymphocytes was identified in BAL (Figure 3A). These results were confirmed in lungs, with a significant increase of total cell concentration, due to a major influx of neutrophils (287.10³ vs 1275.10³ cells) and to a lesser extent of eosinophils and lymphocytes (Figure 3A). After the second challenge (day 35) proportions of eosinophils, neutrophils and lymphocytes were strongly elevated in BAL and lungs from allergic mice compared to control mice and mice which had only undergone one challenge (Figure 3A).

In order to assess the role of skin sensitization in the asthma process, experiments were also performed in challenged, unsensitized mice and in sensitized, unchallenged mice. In neither group difference in AHR was found compared to control mice (Figure 3B). In addition, sensitization or challenge alone did not induce inflammation, detected by cell infiltration in the lungs (Figure 3C). These results show that skin sensitization to HDM is necessary, but not sufficient, to induce asthma. In order to further define the mechanisms of allergen-induced inflammation, cytokines specific to Th1 (IFN-γ), Th2 (IL-5), Th17 (IL-17) and Treg (IL-10) activation were assayed in BAL. IFN-γ, IL-17 and IL-10 significantly increased in BAL after the 1^st challenge, whereas IL-5 significantly increased only after the 2^nd (Figure 3D). Interestingly, secretion of IL-5, IL-10 and IL-17 increased greatly after the 2^nd challenge (Day 35). Similar results were observed in lungs (data not shown). These data highlight a Th1/Th17-oriented inflammation after the first challenge and a mixed Th2/Th17 inflammation after the second challenge.

Overall, these results show that cutaneous sensitization with Der f followed by Der f respiratory challenges induces a systemic type one allergy to Der f. Skin sensitization primes the immune system to respond to a pulmonary challenge with a biphasic lung inflammation and hugely increased AHR after a second challenge.

In order to assess whether a synergy between OVA and HDM allergies could aggravate asthma in food allergic mice, a number of parameters were compared between respiratory allergy, food allergy and dual (food plus respiratory) allergy. Immunoglobulin levels were assayed in serum obtained 3 days after the last intranasal HDM challenge. Total IgG1 and IgE (Figure 4B) levels were significantly higher in dual allergy mice compared to control and mice with asthma only. However total IgG1 levels were not higher in dual allergy mice than in mice allergic to OVA only. As expected, OVA-specific IgE levels were significantly higher in food allergic mice. In dual allergy mice, no further increase in OVA-specific IgE was observed (Figure 4C, upper panel).

In contrast, Der f-specific IgE levels were higher in asthmatic mice and significantly higher in dual allergy mice compared to control and mice allergic to OVA only (Figure 4C, lower panel). Dual allergy mice displayed significantly greater AHR than mice sensitized to one allergen only (Figure 4D). Inflammation was also increased in BAL and lungs of mice with two allergies (Figure 5A and B, respectively). Neutrophils, eosinophils and lymphocytes numbers were elevated in dual allergy mice compared to all other groups, with a predominant eosinophil infiltrate. Cytokine production was analysed in BAL supernatants (Figure 5C). As expected from the inflammatory profile, BAL IL-4 and IL-5 levels were increased in asthmatic mice compared to controls and mice allergic to OVA only. BAL IL-4 and IL-5 were also found increased in dual allergy mice compared to asthmatic, OVA allergic and controls mice. In addition RANTES, a chemotactic agent for T cells, eosinophils and basophils, was significantly higher in dual allergy mice than in others groups. However, KC, a chemotactic agent for neutrophils, was found at significantly higher levels in asthmatic mice, whether they were also OVA-allergic or not, than in controls and mice only allergic to OVA. Similarly, IL-17 was significantly increased in dual allergy mice compared to controls, but not compared to mice allergic to a single agent. IL-10 was found at the same level in all groups (Figure 5C).

These results show that an OVA food allergy primes the immune system to increase its response to Der f respiratory challenges in mice percutaneously sensitized to Der f.