Is Wheat Glutenin Extract Intrinsically Allergenic? Evaluation Using a Novel Adjuvant-Free Mouse Model of Systemic Anaphylaxis

Abstract

Wheat is a prominent allergenic food that can trigger life-threatening anaphylaxis. Presently, it remains unclear whether wheat glutenin (WG) extract possesses inherent sensitization potential independently, without the use of adjuvants, and whether it can sensitize mice to the extent of inducing life-threatening systemic anaphylaxis. In this study, we tested the hypothesis that repeated skin exposures to WG extract without adjuvant will sensitize mice with the resultant anaphylactic reaction upon systemic WG challenge. Balb/c mice were bred and maintained on a strict plant protein-free diet and were repeatedly exposed to a WG extract or vehicle once a week for 9 weeks. WG-specific (s)IgE and total (t)IgE levels were quantified. Mice were challenged with WG extract to induce anaphylactic reactions as measured by hypothermic shock response (HSR) and mucosal mast cell degranulation response (MMCR). We also conducted proteomic analysis of 120 spleen immune markers. These skin-sensitized mice exhibited exposure-dependent IgE responses and near-fatal anaphylaxis upon challenge. Proteomic analysis identified seven dramatically elevated immune biomarkers in anaphylactic mice. These data reveal that WG is intrinsically allergenic, and that chronic skin exposure to WG extract can prime the mice for potentially fatal anaphylaxis.

Keywords: IgE; glutenin allergy; mouse model; skin sensitization; systemic anaphylaxis.

Figure 1

Chronic skin exposure to wheat glutenin (WG) extract elicited specific IgE antibody responses and elevation of total IgE in Balb/c mice. Mice were exposed to WG or to vehicle, as described in the Materials and Methods section. Blood was collected before first exposure (Pre) and after sixth exposure. Plasma was used in measurement of WG-specific IgE levels (OD 405–690 nm) using an ELISA method described previously. Each dot represents one mouse data. (A) WG-specific IgE antibody levels in control mice. (B) WG-specific IgE antibody levels in sensitized mice. (C) Total IgE levels in vehicle sensitized control mice. (D) Total IgE levels in WG-sensitized mice. Student’s two-tailed t-test: *** p < 0.001.

Figure 2

Pearson correlation analysis between wheat glutenin (WG)-specific IgE antibody levels and total IgE levels. Mice were treated as described in the Materials and Methods section. Pearson correlation analysis was used to test the relationship between WG-specific IgE antibody and total IgE levels in the plasma after 6th transdermal exposure to WG. Each dot represents one mouse data.

Figure 3

Chronic skin exposure to wheat glutenin (WG) is sufficient to clinically sensitize Balb/c mice for systemic anaphylaxis. Groups of Balb/c mice were sensitized with either vehicle or WG as described in the Methods section. After nine transdermal exposures mice were challenged with either vehicle or WG (0.5 mg/mouse) by intraperitoneal injection. Each symbol represents an individual mouse.

Figure 4

Induction of hypothermic shock responses upon systemic challenge with wheat glutenin (WG). Mice exposed to WG or to vehicle were systemically challenged by intraperitoneal injection as described in the Materials and Methods section. (A) Rectal temperatures (°C) at indicated time points in vehicle-sensitized mice challenged with WG or vehicle. (B) Change in rectal temperature (∆°C) at indicated time points in vehicle-sensitized mice challenged with WG or vehicle. (C) Rectal temperatures (°C) at indicated time points in WG-sensitized mice challenged with WG or vehicle. (D) Change in rectal temperature (∆°C) at indicated time points in WG-sensitized mice challenged with WG or vehicle. * p < 0.05.

Figure 4

Induction of hypothermic shock responses upon systemic challenge with wheat glutenin (WG). Mice exposed to WG or to vehicle were systemically challenged by intraperitoneal injection as described in the Materials and Methods section. (A) Rectal temperatures (°C) at indicated time points in vehicle-sensitized mice challenged with WG or vehicle. (B) Change in rectal temperature (∆°C) at indicated time points in vehicle-sensitized mice challenged with WG or vehicle. (C) Rectal temperatures (°C) at indicated time points in WG-sensitized mice challenged with WG or vehicle. (D) Change in rectal temperature (∆°C) at indicated time points in WG-sensitized mice challenged with WG or vehicle. * p < 0.05.

Figure 5

Systemic anaphylaxis induced by wheat glutenin (WG) is associated with degranulation of mucosal mast cells in this model. Mice were treated as described in the Materials and Methods section. Their plasma mucosal mast cell protease-1 (MMCP) levels (ng/mL) were measured using an ELISA-based method described in the texts. (A) MMCP-1 levels in control mice challenged with vehicle. (B) MMCP-1 levels in vehicle-sensitized control mice challenged with WG. (C) MMCP-1 levels in WG-sensitized mice challenged with vehicle. (D) MMCP-1 levels in WG-sensitized mice challenged with WG. Each dot represents one mouse data. Student’s two-tailed t-test: *** p < 0.001.

Figure 6

Heat map analysis of 120 spleen immune biomarkers in glutenin-induced systemic anaphylaxis. Using spleen extracts from control mice and anaphylactic mice, a proteomic microarray analysis was conducted using RayBiotech system cytokine panels, (A) CYT-4, (B) CYT-5 and (C) CYT-6 as described in the methods. Background levels of immune biomarkers are shown in green. Upregulated biomarkers are shown in red, and down-regulated biomarkers are shown in blue.

Figure 7

Identification of differentially expressed immune biomarkers in the spleen of control vs. anaphylactic mice. (A) Immune biomarkers that are significantly elevated (2-fold or higher, p < 0.05) in anaphylactic mice are shown. (B) Immune biomarkers that are significantly decreased (2-fold or lower, p < 0.05) in anaphylactic mice are shown. The dotted lines indicate a 4-fold and 6-fold changes in protein expression. Immune biomarkers that show 4-fold or higher changes are identified with names.