Grantee Research Project Results

Final Report: Assessment of Allergic Responses to Food Proteins Using a Novel and Sensitive Adjuvant-Free Ingestion-Based Mouse Model

EPA Grant Number: R834825
Title: Assessment of Allergic Responses to Food Proteins Using a Novel and Sensitive Adjuvant-Free Ingestion-Based Mouse Model
Investigators: Oettgen, Hans C
Institution: Children’s Hospital, Boston
EPA Project Officer: Aja, Hayley
Project Period: September 15, 2010 through September 14, 2012 (Extended to September 14, 2013)
Project Amount: $424,803
RFA: Approaches to Assessing Potential Food Allergy from Genetically Engineered Plants (2009) RFA Text | Recipients Lists
Research Category: Human Health

Objective:

The overall goal of this project was to test the utility of a novel adjuvant-free mouse model of food allergy as a biological probe for food protein allergenicity. We had previously shown that mice harboring and activating mutation of the IL-4 receptor chain (F709) could be sensitized to develop food allergy by ingestion of the model allergen, ovalbumin (OVA). For this EPA STAR project we tested the effects of this mutation on mast cell responses and whether these animals could similarly be sensitized to clinically relevant food allergens (like peanut) and further whether the animals might serve to distinguish allergenic from non-allergenic food proteins. Such an ability to discriminate allergenic from non-allergenic proteins might prove valuable in the analysis of genetically engineered plants.

Summary/Accomplishments (Outputs/Outcomes):

We have previously shown that F709 mice, which harbor an activating mutation of the IL-4R chain, have an intrinsically allergic phenotype with very high IgE levels and strong specific IgE responses to both inhaled and ingested antigens. In addition to generating robust IgE responses and Th2 cellular reactions to ingested proteins, the animals exhibit intense anaphylaxis upon ingestion challenge, a unique phenotype in mouse models of food allergy and one that recapitulates the type of reaction that is most concerning with food allergens. We hypothesized that such animals could be used to discriminate between allergenic and non-allergenic foods in a natural setting of purely enteral immune sensitization and challenge.

In order to test this hypothesis, egg white and peanut were identified as model allergenic foods while chicken and rice were chosen as non-allergens. Various protocols of sensitization and challenge were tested in pilot experiments. The most consistent protocol was determined to be the following: F709 mice were sensitized once per week for 5 weeks by oral gavage with 2.5 milligrams food protein and 10 micrograms cholera toxin, as adjuvant. Seven days after the last sensitization dose, all mice were challenged with 150 milligrams of the relevant food protein. Temperatures were monitored for 60 minutes using subdermal microchips, and levels of mMCP-1 and protein-specific IgE were measured by ELISA in serum collected 60 minutes after challenge. A decrease in core body temperature and a release of mMCP-1 into the serum are evidence of mast cell degranulation. Mast cell degranulation results in the release of mediators of allergic reactions including anaphylaxis and individuals undergoing food anaphylaxis exhibit increased levels of the mast cell mediator, tryptase, consistent with degranulation.

As expected, the relatively non-allergenic proteins, rice and chicken, resulted in no significant drop in temperature in experimental mice, and very little rice-specific IgE. Unexpectedly, mice sensitized and challenged with chicken protein released significantly more mMCP-1 into the sera compared to their mock-sensitized counterparts, although these levels are lower than those usually observed in mice that have just experienced an anaphylactic reaction.

In subsequent experiments we further delineated the biology of mast cell expansion in this model, showing that IL-4 has direct growth promoting effects on cultured mast cells as well as on intestinal mast cells in the setting of food allergy in vivo. These results represent an important advance in our understanding of mechanisms of food allergy, establishing that enhanced IL-4 signaling (which is observed in allergic diseases) directly drives expansion of mast cells, the effectors of food anaphylaxis.

Conclusions:

In a final significant phase of work we have further developed the peanut sensitization protocol in order to elicit more robust immune sensitization and anaphylactic responses. We have shown that intragastric instillation of peanut butter (containing both peanut oils and protein, unlike the protein-only approach initially tried) gives rise to a strong peanut allergy.

The intense expansion of mast cells observed in the peanut treated animals suggested that they play a key role both in immune sensitization and in the anaphylactic response to food allergen. In order to assess this possibility, we undertook the breeding of mast cell-deficient F709 mice by crossing F709 animals with the c-Kit-mutant strain, Kit^Wsh, which completely lacks intestinal mast cells. The mast cell deficient F709 mice showed no anaphylaxis to peanut. In order to prove that mast cells were indeed responsible for this loss of responsiveness, we injected mast cell-deficient F709/Kit^Wsh mice and then subjected them to peanut sensitization and challenge. Mast cell reconstituted mice exhibited robust anaphylactic responses consistent with a central role for mast cells in peanut allergy.

In our most recent experiments on the peanut food allergy model we evaluated the effect of the F709 mutation on the generation of peanut specific regulatory T cells (Treg), cells which are thought to be critical in preventing food allergy under physiologic conditions. By staining for the Treg marker FoxP3 in peanut responsive T cells we found that F709 mice, as expected, have markedly fewer peanut specific Treg than do their wild-type counterparts.

In additional studies under this EPA project, we have worked to determine the most effective and consistent protocol for evaluating the allergenicity of food proteins in F709 mice. We have gathered data for peanut, egg white, rice, and chicken meat proteins using the originally proposed sensitization and challenge protocol.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Publications Views
Other project views:	All 2 publications	2 publications in selected types	All 2 journal articles

Publications
Type	Citation	Project	Document Sources
Journal Article	Bartnikas LM, Gurish MF, Burton OT, Leisten S, Janssen E, Oettgen HC, Beaupré J, Lewis CN, Austen KF, Schulte S, Hornick JL, Geha RS, Oyoshi MK. Epicutaneous sensitization results in IgE-dependent intestinal mast cell expansion and food-induced anaphylaxis. Journal of Allergy and Clinical Immunology 2013;131(2):451-460.e1-6.	R834825 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Full-text: JACI-Full Text HTML Exit Other: JACI-Full Text PDF Exit
Journal Article	Burton OT, Darling AR, Zhou JS, Noval-Rivas M, Jones TG, Gurish MF, Chatila TA, Oettgen HC. Direct effects of IL-4 on mast cells drive their intestinal expansion and increase susceptibility to anaphylaxis in a murine model of food allergy. Mucosal Immunology 2013;6(4):740-750.	R834825 (2012) R834825 (2013) R834825 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Full-text: Nature-Full Text HTML Exit Other: Nature-Full Text PDF Exit

Supplemental Keywords:

Food allergy, allergen, IgE, anaphylaxis, IL-4

Relevant Websites:

http://www.childrenshospital.org/research-and-innovation/research-labs/oettgen-laboratory

Progress and Final Reports:

Original Abstract

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.