A Murine Model of Food Allergy by Epicutaneous Adjuvant-Free Allergen Sensitization Followed by Oral Allergen Challenge Combined with Aspirin for Enhanced Detection of Hypersensitivity Manifestations and Immunotherapy Monitoring

Abstract

Food allergy is one of the major existing health problems, but no effective treatment is available. In the current work, a murine model that closely mimics pathogenesis of human food allergy and its quantifiable diagnostic parameter design, even for mild hypersensitivity reactions, were established. BALB/c mice were epicutaneously sensitized with 1 mg chicken egg ovomucoid (OVM) or cow's milk casein, free of adjuvants, five times a week for two consecutive weeks. Eleven days later, allergen-specific IgG1 and IgE in serum were measured by ELISA. On day 25, 20 mg OVM or 12 mg α-casein was administered orally, and allergic reactions such as the fall in rectal temperature, symptom scores during 90-120 min, serum mast cell protease-1 and cytokine levels were monitored. The detection of mild allergic reactions due to adjuvant-free allergen sensitization and oral allergen challenge routes was amplified by the combination of oral allergen and aspirin administration simultaneously or aspirin administration within 15-30 min before an allergen challenge. Quantification of the maximum symptom score and the frequency of symptoms during the monitoring period improved evaluation accuracy of food allergy signals. Based on these results, efficacy of casein oral immunotherapy for cow's milk allergies, which are generally difficult to detect, was monitored adequately.

Keywords: aspirin; chicken’s egg OVM allergy; cow’s milk casein allergy; enhanced detection; food allergy; murine model; oral allergen challenge; skin sensitization; symptom score.

Figure 1

Experimental design. For epicutaneous sensitization, the shaved back skin of BALB/c female mice (n = 5–10/group) was treated with 4% SDS and 1 mg OVM, without adjuvant, five times a week in the first 2 weeks. On day 25, mice were orally challenged with OVM and allergic reactions were monitored. (A) Experimental protocol; (B) Serum levels of OVM sIgE and sIgG1 on day 11; (C) ΔT after intraperitoneal challenge with 1 mg OVM; (D) ΔT after oral challenge with 20 mg OVM. Data are mean ± SEMs. ** p < 0.01.

Figure 2

Effects of oral pre-medication with ASA and its derivative, L-ASA on oral-OVM-challenge-induced fall in rectal temperature. (A) Oral challenge protocol (n = 10/group); (B) ΔT after 20 mg OVM challenge in sensitized and naïve mice with and without 30 min pre-medication of ASA at 50 mg/kg, an equivalent dose of L-ASA at 91 mg/kg and 50% EtOH; (C) ΔT without oral OVM challenge in sensitized mice pre-medicated with ASA at 50 mg/kg or 50% EtOH and ΔT in naïve mice treated with 50 mg/kg ASA and OVM challenge as a comparison group. Data are mean ± SEMs.

Figure 3

Dose-dependent effect of ASA on fall in rectal temperature after oral allergen challenge. (A) ΔT after 20 mg OVM challenge in allergen-sensitized and naïve mice with and without 30 min pre-medication with each ASA dose (n = 5–10/group); (B) ΔT correlations between sensitized and naïve mice with and without pre-medication of ASA; (C) Dose-dependent effect of pre-medication of ASA on the lowest point ΔT in sensitized mice compared with naïve mice after OVM challenge. Data are mean ± SEMs. * p < 0.05, ** p < 0.01.

Figure 4

Optimal timing of ASA administration relative to oral allergen challenge. (A) Effects of sub-maximal dose at 50 mg/kg ASA before and after 20 mg OVM challenge on ΔT in sensitized mice; (B) Effects of timing of 50 mg/kg ASA medication on the lowest point ΔT in sensitized mice; (C) Effects of timing of 50 mg/kg ASA medication on the maximum symptom score in sensitized mice. Data are mean ± SEMs (n = 10/group). * p < 0.05, ** p < 0.01.

Figure 5

Effects of ASA pre-medication on the modified symptom score evaluation system after oral allergen challenge in sensitized mice. Allergic reactions after OVM challenge in sensitized mice were compared with and without ASA-pre-medication at 50 mg/kg. (A) Comparison of the effects on the lowest point ΔT; (B) Comparison of the effects on the maximum symptom score; (C) Total number of recorded symptom score 1 after allergen challenge for 90 min. Data are mean ± SEMs. (n = 6–8/group) ** p < 0.01.

Figure 6

Serial changes in serum mMCPT-1 levels before and after oral administration of 20 mg OVM and ASA pre-medication at 50 mg/kg. (A) Changes in mMCPT-1 levels in sensitized mice (n = 5/group); (B) Changes in mMCPT-1 levels in naïve mice (n = 6/group). Data are mean ± SEMs. * p < 0.05, ** p < 0.01.

Figure 7

Design of adjuvant-free cow’s milk allergy experiments. (A) Experimental protocol; (B) Oral challenge protocol of 12 mg α-casein combined with 30 min ASA-pre-medication; (C) Serial changes in ΔT in casein-sensitized and naïve mice pre-treated with different doses of ASA (n = 6–8/group); (D) Dose-dependent effect of ASA pre-medication on the lowest point ΔT in casein-sensitized mice. The following results (E–G) were comparisons between casein-sensitized and naïve mice treated with oral α-casein challenge with ASA pre-medication at 100 mg/kg; (E) Comparison of the lowest point ΔT; (F) Comparison of the maximum symptom score; (G) Comparison of total number of recorded symptom score 1 after allergen challenge for 90 min. Data are mean ± SEMs. * p < 0.05, ** p < 0.01.

Figure 8

Changes in α-casein sIgE and sIgG1 levels by oral casein immunotherapy in adjuvant-free casein-sensitized allergy model mice. (A) Experimental protocol. As an oral immunotherapy, 0.1 µg/head of casein was administered orally two times on day 25 and 28 in sensitized mice. As a control, saline was administered orally. On day 72 or 73, mice were challenged with ASA and α-casein, then rectal temperature and symptom score were monitored (n = 6/group); (B) Fold changes in serum α-casein sIgE and sIgG1 levels after immunotherapy of casein or saline in the sensitized and naïve mice. Serum samples were collected before immunotherapy on day 24 and, after, on day 64 (n = 6/group).

Figure 9

Effects of allergic symptoms after casein immunotherapy in adjuvant-free casein-sensitized allergy model mice. (A) Comparison of the lowest point ΔT and (B) the maximum symptom score after oral α-casein challenge with ASA pre-medication in casein- and saline-treated sensitized and naïve mice; (C) Total number of recorded symptom score 1 after oral α-casein challenge in casein- and saline-treated sensitized and naïve mice; (D) Fold changes in serum mMCTP-1 levels after oral α-casein challenge. Serum samples were collected before on day 64 and after α-casein challenge on day 72 or 73 (n = 6/group). Data are mean ± SEMs. * p < 0.05.