Digestion and Transport across the Intestinal Epithelium Affects the Allergenicity of Ara h 1 and 3 but Not of Ara h 2 and 6

Abstract

Scope: No accepted and validated methods are currently available which can accurately predict protein allergenicity. In this study, the role of digestion and transport on protein allergenicity is investigated.

Methods and results: Peanut allergens (Ara h 1, 2, 3, and 6) and a milk allergen (β-lactoglobulin) are transported across pig intestinal epithelium using the InTESTine model and afterward basophil activation is measured to assess the (remaining) functional properties. Additionally, allergens are digested by pepsin prior to epithelial transport and their allergenicity is assessed in a human mast cell activation assay. Remarkably, transported Ara h 1 and 3 are not able to activate basophils, in contrast to Ara h 2 and 6. Digestion prior to transport results in a significant increase in mast cell activation of Ara h 1 and 3 dependent on the length of digestion time. Activation of mast cells by Ara h 2 and 6 is unaffected by digestion prior to transport.

Conclusions: Digestion and transport influences the allergenicity of Ara h 1 and 3, but not of Ara h 2 and 6. The influence of digestion and transport on protein allergenicity may explain why current in vitro assays are not predictive for allergenicity.

Keywords: allergenicity prediction; basophil and mast cell activation; food allergy; intestinal protein transport; protein digestion.

Figure 1

Percentage of Ara h 1, Ara h 2, and β‐lactoglobulin transport across the intestinal epithelium of three pigs. Per allergen the data is presented as mean percentage (n = 3) of transport ± SD.

Figure 2

Basophil activation after stimulation with a serial dilution of peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6, and β‐lactoglobulin retrieved from the apical (1:10³, 1:10⁴, 1:10⁵, 1:10⁶) compartment and from the basolateral (1:3, 1:10, 1:50, 1:10²: 1:10³) compartment after transport across the intestinal epithelium of two pigs (pig 4 and pig 5) in the InTESTine model. Serum of three peanut‐allergic patients was used to reload the basophils. The data is presented as the percentage of CD63⁺ ± SD of the means of the serial dilutions (2 pigs and 3 patients sera, n = 6). The specific reactivity for each dilution is given in Figures S1 and S2, Supporting Information. Reactivity of the basolateral samples compared to the apical samples was evaluated by a Mann–Whitney U test. The number of symbols indicates the level of significance between the apical and basolateral CD63 expression: *p < 0.05.

Figure 3

Digestion kinetics of Ara h 1, 2, 3, and 6 visualized using Coomassie stained 15% SDS‐PAGE gels and IgE immunoblots incubated with the serum from peanut‐allergic patient 4. The samples were collected after 0, 15 and 30 s and 1, 5, 10, 30 and 60 min of digestion, respectively.

Figure 4

Percentage CD63⁺ basophils after stimulation with a serial dilution of digests from peanut allergens Ara h 1, 2, 3, and 6. Serum of three peanut‐allergic patients (1, 2, and 4) was used to reload the basophils. The data is presented as the mean percentage of CD63⁺ ± SD and all peanut allergen digests showed reactivity.

Figure 5

Mast cell activation after stimulation with digested (t = 0, 30 s, or 5 min, and 60 min) and transported Ara h 1, 2, 3, and 6 across the intestinal epithelium of three different pigs (pig 6, 7, and 8). Data is presented as the percentage β‐hexosaminidase release ± SD with respect to the release induced by incubation with anti‐IgE. The means of the serial dilutions (1:1, 1:2.5, 1:5, 1:10, 1:10², 1:10³) are displayed and the β‐hexosaminidase release of the specific dilution points can be found in Figures S3 and S4, Supporting Information. The reactivity between the different digestion times was evaluated by a Kruskal–Wallis test followed by Dunn's multiple comparison test. The number of symbols indicates the level of significance between the digestion times: * p < 0.05, ** p < 0.005.