Mechanisms of Wheat Allergenicity in Mice: Comparison of Adjuvant-Free vs. Alum-Adjuvant Models

Abstract

Wheat protein is considered a major type of food allergen in many countries including the USA. The mechanisms of allergenicity of wheat proteins are not well understood at present. Both adjuvant-based and adjuvant-free mouse models are reported for this food allergy. However, it is unclear whether the mechanisms underlying wheat allergenicity in these two types of models are similar or different. Therefore, we compared the molecular mechanisms in a novel adjuvant-free (AF) model vs. a conventional alum-adjuvant (AA) model of wheat allergy using salt-soluble wheat protein (SSWP). In the AF model, Balb/cJ mice were sensitized with SSWP via skin exposure. In the AA model, mice were sensitized by an intraperitoneal injection of SSWP with alum. In both models, allergic reactions were elicited using an identical protocol. Robust IgE as well as mucosal mast cell protein-1 responses were elicited similarly in both models. However, an analysis of the spleen immune markers identified strikingly different molecular activation patterns in these two models. Furthermore, a number of immune markers associated with intrinsic allergenicity were also identified in both models. Since the AF model uses skin exposure without an adjuvant, the mechanisms in the AF model may more closely simulate the human wheat allergenicity mechanisms from skin exposure in occupational settings such as in the baking industry.

Keywords: adjuvant; food allergy; food safety; immune markers; intrinsic allergenicity; mouse model; risk assessment; skin sensitization; wheat allergy; wheat protein allergenicity.

Figure 1

(A,B). Comparison of wheat protein-specific IgE antibody responses in the adjuvant-free (AF) vs. the alum-adjuvant (AA) mouse models of wheat allergenicity. (A) In the AF model, Balb/c mice were exposed to salt-soluble wheat protein (SSWP) once a week for 6 weeks via the transdermal route, as described in the methods. A group of control mice did not receive this exposure. Plasma collected after 6 weeks of exposure sensitization was used in the wheat-specific IgE (WSIgE) antibody analysis using an ELISA method described previously [32]. Figure shows the WSIgE levels in allergic mice vs. the control mice in the AF model. (B) In the AA model, Balb/c mice were injected with SSWP along with alum by the intraperitoneal route, as described in the methods. A group of control mice received alum only for the injection. Plasma collected after 6 weeks of sensitization was used in the WSIgE antibody analysis using an ELISA method described previously [32]. Figure shows WSIgE levels in allergic mice vs. the control mice in the AA model. * Student’s t test, p < 0.05.

Figure 2

(A,B). Comparison of wheat protein-elicited plasma total IgE antibody responses in the adjuvant-free vs. the alum-adjuvant mouse models of wheat allergenicity. (A) In the AF model, Balb/c mice were exposed to SSWP once a week for 6 weeks via the transdermal route, as described in the methods. A group of control mice did not receive this exposure. Plasma collected after 6 weeks of exposure sensitization was used in the TIgE antibody analysis using an ELISA method described previously [42]. Figure shows the TIgE levels in allergic mice vs. the control mice in the AF model. (B) In the AA model, Balb/c mice were injected with SSWP along with alum by the intraperitoneal route, as described in the methods. A group of control mice received alum only for the injection. Plasma collected after 6 weeks of sensitization was used in the TIgE antibody analysis using an ELISA method described previously [32]. Figure shows the TIgE levels in allergic vs. the control mice in the AA model. * Student’s t test, p < 0.05.

Figure 3

(A,B). Comparison of the wheat protein-specific IgG1 antibody responses in the adjuvant-free vs. the alum-adjuvant mouse models of wheat allergenicity. (A) In the AF model, Balb/c mice were exposed to SSWP once a week for 6 weeks via the transdermal route, as described in the methods. A group of control mice did not receive this exposure. Plasma collected after 6 weeks of exposure sensitization was used in the WSIgG1 antibody analysis using an ELISA method described previously [32]. Figure shows the WSIgG1 levels in allergic mice vs. the control mice in the AF model. (B) In the AA model, Balb/c mice were injected with SSWP along with alum by the intraperitoneal route, as described in the methods. A group of control mice received alum only for the injection. Plasma collected after 6 weeks of sensitization was used in the WSIgG1 antibody analysis using an ELISA method described previously [32]. Figure shows the WSIgG1 levels in allergic mice vs. the control mice in the AA model. * Student’s t test, p < 0.05.

Figure 4

(A,B). Comparison of the wheat protein-specific IgG2a antibody responses in the adjuvant-free vs. the alum-adjuvant mouse models of wheat allergenicity. (A) In the AF model, Balb/c mice were exposed to SSWP once a week for 6 weeks via the transdermal route, as described in the methods. A group of control mice did not receive this exposure. Plasma collected after 6 weeks of exposure sensitization was used in the WSIgG2a antibody analysis using an ELISA method described previously [32]. Figure shows the WSIgG2a levels in allergic mice vs. the control mice in the AF model. (B) In the AA model, Balb/c mice were injected with SSWP along with alum by the intraperitoneal route, as described in the methods. A group of control mice received alum only for the injection. Plasma collected after 6 weeks of sensitization was used in the WSIgG2a antibody analysis using an ELISA method described previously [32]. Figure shows the WSIgG2a levels in allergic mice vs. the control mice in the AA model. * Student’s t test, p < 0.05.

Figure 5

(A,B). Comparison of the mucosal mast cell protease-1 responses in the adjuvant-free vs. the alum-adjuvant mouse models. (A) In the AF model, Balb/c mice were exposed to SSWP once a week for 6 weeks via the transdermal route, as described in the methods. A group of control mice did not receive this exposure. Plasma collected at 1 h after the challenge with SSWP was used in the MMCP-1 protein analysis using an ELISA method described previously [42]. Figure shows the MMCP-1 levels in allergic mice vs. the control mice in the AF model. (B) In the AA model, Balb/c mice were injected with SSWP along with alum by the intraperitoneal route, as described in the methods. A group of control mice received alum only for the injection. Plasma collected at 1 h after the challenge with SSWP was used in the MMCP-1 protein analysis using an ELISA method described previously [42]. Figure shows the MMCP-1 levels in allergic vs. control mice in the AA model. * Student’s t test, p < 0.05.

Figure 6

Analysis of in vivo activation of cytokines in the spleen in adjuvant-free vs. alum adjuvant mouse models of wheat allergy. Spleen tissues were collected from the experimental groups of mice in the AF vs. AA models, and used in the cytokine protein analysis, as described in the methods. Using the pg/mg spleen protein content, the fold changes in the cytokines in the AF vs. AA models were determined as shown. Data shows that different sets of cytokines are overexpressed in vivo in the AF vs. AA models, indicating the different mechanisms of wheat allergenicity in these two types of models.

Figure 7

Analysis of in vivo activation of the chemokines in spleen tissue in the adjuvant-free vs. alum adjuvant mouse models of wheat allergy. Spleen tissues were collected from the experimental groups of mice in the AF vs. AA models, and used in the chemokine protein analysis, as described in the methods. Using the pg/mg spleen protein content, fold changes in chemokines in the AF vs. AA models were determined as shown. Data shows that different sets of chemokines are overexpressed in vivo in the AF vs. AA models, indicating the different mechanisms of wheat allergenicity.

Figure 8

(A,B). Analysis of the in vivo activation of the adhesion molecules and other allergy-relevant immune markers in the spleen tissue of adjuvant-free vs. alum adjuvant mouse models of wheat allergy. Spleen tissues were collected from the experimental groups of mice in the AF vs. AA model, and used in the analysis of the adhesion molecules and other immune markers, as described in the methods. Using the pg/mg spleen protein content, fold changes in these markers in the AF vs. AA models were determined as shown. Data shows that the adhesion molecules (Figure A) and other immune markers (Figure B) are differently overexpressed in vivo in the AF vs. AA models, indicating the different mechanisms of wheat allergenicity.