Source: CORNELL UNIVERSITY submitted to
ROLES OF UNIQUE SUBSETS OF THE INTESTINAL INTRA EPITHELIAL LYMPHOCYTES (IEL) IN THE DEVELOPMENT OF FOOD-BORNE ALLERGY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0226213
Grant No.
2011-67012-30698
Project No.
NYCV-433505
Proposal No.
2010-05177
Multistate No.
(N/A)
Program Code
A7201
Project Start Date
Sep 1, 2011
Project End Date
Aug 31, 2013
Grant Year
2011
Project Director
Huang, F.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Microbiology And Immunology
Non Technical Summary
Food allergy has been rising as a serious health problem in US. The prevalence of egg allergy is estimated to be 1.6 - 3.2% of the pediatric population. The interactions of the food borne allergen and gut mucosal immune system may play a critical role in food allergy. We recently found two specialized immune cells residing within the intestine that may regulate the sensitivity to food allergens. These are {alpha beta}-T cells bearing CD8{alpha alpha} and a specific population of {gamma delta}-T cells. In order to understand how the mucosal immunity in gut determines whether food allergy develops or not, we propose to build novel animal models of food allergy, and examine the role of specific immune cell populations in the development of food allergic responses. We will first develop a novel model of food allergy based on exposure to egg albumin and gluten, a wheat protein, which may enhance access of food allergens to the immune system. Through this novel model, we will further test the hypothesis that specific immune cell populations mentioned above regulate the development of food allergy. The rationale for this proposal is that once we know the mechanism by which food allergens develop, we can use this information to eventually develop strategies to interfere with the process. If successful, our work will provide an innovative and realistic model of food allergy development, which will allow us to develop effective strategies to treat and prevent the food allergy responses.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123270109050%
7023270109050%
Goals / Objectives
Our long-range goal is to provide a detailed understanding of the mechanism by which the mucosal immune system, particularly in the gut, determines the immune response between tolerance and allergy. In pursuit of that goal, the objective of this application is to determine the role of wheat gluten in allowing specific IEL populations access to food borne allergens, leading to the development of an allergic response to a food borne allergen. We hypothesize that specific T cell subsets within the IEL population along with exposure to wheat gluten in foods, regulate the development of food allergy. Using murine exposure to gluten and chicken egg albumin (OVA) as a model, we will test this hypothesis by performing experiments in the following 2 specific aims: Specific Aim #1: Determine the function of CD8{alpha alpha} (CD8aa) bearing {alpha beta}-T cells (ab-T cells) in suppressing the development of IgE mediated allergy to OVA. The objectives of aim #1 are to develop a novel murine model of food allergies, and determine whether the CD8aa bearing ab-T cells act to prevent the development of inappropriate immune response to food borne allergens in young mice. Our approach will be to utilize young mice and the egg allergen ovalbumin, along with exposure to gluten, to develop a model of egg allergy. We expect to find that young mice are highly susceptible to developing an IgE response to food borne allergens, and that this is controlled by immunosuppressive cells in the IEL population, the CD8aa ab-T cells. Specific Aim #2: Determine the role of gut {gamma delta} (gd)-T cells in controlling B cell class switch leading to the development of IgE mediated allergy to chicken egg albumin. In the first aim, we will address the role of IEL ab-T cell populations carrying the CD8aa as protective populations. This will help to understand why children who have food allergies most likely are sensitized at an early age to allergens, delivered via food products that contact the immune system through the gut mucosal immune system or in their mother's milk. In aim #2 of the proposal, we will further address another potential gd-T cell population that may be allergy promoting T cells in gut. Published work, along with our preliminary data, provide strong support for the idea that gd-T cells bearing specific T cell receptors are involved in the development of IgE responses. Therefore, the objective of aim 2 is to determine the role of these Vgamma1.1 (Vg1.1) gd-T cells in the development of inappropriate immune responses to food borne allergens in our mouse models. Our approach will be to utilize the models developed in aim 1, as well as mice lacking gd-T cells, or the Cgamma1-/- (Cg1-/-) mice carrying elevated Vg1.1+ T cell populations to address the objective. Based on the published data and our preliminary experiments, we expect to find that Vg1.1+ T cells promote the development of allergic response to food borne allergens.
Project Methods
Specific Aim #1: We will first establish a mouse food allergy model by using OVA/gluten for induction of food allergy in young mice. Once it is established, we will determine the function of gut CD8aa ab-T cells in this food allergy model. The response of food allergy will be determined by measuring the increase in IgE production, increase in serum histamine concentration, body temperature loss, diarrhea, induction of Th2 cytokines, and the profiles of immune cells, such as Th2 cells, eosinophils and basophils that are associated with allergic responses, and the histological scores of intestinal inflammation. The number of mice used for the experiments will be determined by power analysis based on preliminary data to ensure sufficient mice are used to obtain statistically confident data, and significance determined by ANOVA and student T test. At the completion of the first aim, we expect to have developed 2 novel models of murine food borne allergy based on OVA/gluten exposure. This will provide a significant advancement for the study of food allergy in murine models. Through the novel food allergy models, as well as the model of Rag KO mice receiving naive T and B cells described in aim#1, we will be able to determine whether CD8aa ab-T cells in IEL play a role in protecting against the inappropriate development of allergic response to food borne allergen. These findings will add support to our hypothesis that specific T cell subsets within the IEL population regulate the development of food allergy. Specific Aim #2: We will use gd-T cell KO and Cg1 KO mice as well as adoptive transfer techniques of gd-T cells and Vg1.1 bearing gd-T cells to determine the function of these specific cells in food allergy models developed in aim 1. At the completion of this aim, we expect to have determined the role of gd-T cells, and more specifically, Vg1.1 bearing gd-T cells, in the development of murine food borne allergy. The allergy responses will be measured and evaluated by the methods similarly to those in aim 1. These findings will add support to our hypothesis that specific T cell subsets within the IEL population regulate the development of food allergy. The work proposed in this application will allow us to develop experiments aimed at determining if humans have similar mechanisms of protection and promotion of development of allergic responses to food borne allergens. Upon completion of the experiments in this proposal, we expect to add a significant amount of information to the body of knowledge regarding not only the mechanism by which food allergies develop, but also the regulation of this response by specific T cell populations in the IEL. This work will have implications for the development of approaches to prevent food allergies from occurring in young children, but also to utilize specific cellular populations to manipulate the response. Therefore, we can use this information to eventually develop strategies to interfere with the process, providing novel options of therapies for food allergy.

Progress 09/01/11 to 08/31/13

Outputs
Target Audience: The project has been presented as a poster presentation in 1st Annual “Nutrition, Metabolism, and Disease” symposium, attended by expert nutrition scientists and immunologists at Cornell University. The symposium had strong emphasis on nutrient metabolism and disease model systems. The project also has been discussed with immunologists, biologists, and pediatricians in two other meetings: American Association of Immunologists Conference and Upstate New York Immunology Conference in 2012. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? I was able to have funding support to attend some immunology meetings: American Association of Immunologists conference 2012; Upstate New York Immunology conference 2012, and discussed about the studies with other immunologists in the food allergy field. How have the results been disseminated to communities of interest? I presented one poster of this novel animal model for childhood food allergy in a symposium titled 1st Annual “Nutrition, Metabolism, and Disease” symposium, attended by expert nutrition scientists and immunologists at Cornell University. The symposium had strong emphasis on nutrient metabolism and disease model systems. The project was also discussed with some immunologists, biologists, and pediatricians in two other international meetings: American Association of Immunologists Conference and Upstate New York Immunology Conference in 2012. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? I). Major activities: Establish a novel food allergy model in mice and characterization of immune cells in this model. In this study, we established a novel food allergy model for mice, by feeding young mice (starting at 2 week of age), a diet containing OVA and gluten. Our results show that 2-week-old mice fed a diet containing OVA and gluten developed much stronger immune response to the OVA allergen than mice fed the same diet starting at 6-weeks of age. This new model of food allergy is described in detail as follows: 1. Diet regimen: 2-week-old and 6-week-old mice are fed a diet containing 10 mg gluten and 100 mg OVA every other day for 10 days. Following a 3 week rest period, all mice are fed a diet containing 20 mg gluten and 200 mg OVA every other day for 7 days. 24 hours after the last feeding, mice were euthanized and immune tissues were collected. 2. In vitro cell stimulation and analysis Lymphoid cells from each tissue were isolated and analyzed for their response to stimulation with OVA and PMA/ionomycin. To determine the responses of the CD4+ T cells and other inflammatory cells, isolated cells were cultured with 1000 ng/ml OVA in complete RPMI for 3 days, and restimulated with PMA/ionomycin for 6 hours in the presence of Brefeldin A. Cells were characterized by flow cytometry for their surface markers and cytokines of interest. II, III). Objectives and Significant Results: Characterization of the immune response in food allergy model. We examined the response of several immune cell types in different tissues that potentially responsible for food-borne allergy reaction in our model: 1. Overall cell number in related immune tissues: The major immune tissues involved in the gut immune responses are: IEL, payer’s patch, mesenteric lymphoid tissues (mLM), and spleen. Therefore, we first determined whether cells from all these tissues exhibited differential proliferation in response to OVA/gluten in these two groups of mice. We did not find any significant difference in total lymphatic cells in the tissues tested, suggesting that overall immune cell numbers were not affected by the timing of OVA and gluten exposure in the diet of these mice. 2. CD4+ T cells response to OVA in different immune tissues: We found that mLN CD4+ T cells exhibited significantly higher inflammatory cytokine production (TNF-alpha and IFN-gamma) in 2 weeks old mice than those in 6 weeks old mice exposed to OVA/Gluten. CD4+ T cells in other tissues tested show no higher TNF-alpha or IFNG-gamma production in other tissues from mice exposed to OVA/gluten at the younger age. There was no significant difference in Th2 cytokine response in the CD4+ T cells from any of the tissue tested. 3. gd T cells in different immune tissues. We found that splenic gd T cells were able to produce more IL-4 and IL-13 cytokines when mice were exposed to OVA/gluten at a younger age. However, there was no significant difference in production of Th2 cytokine, IFN-gamma or TNF-alpha secretion in gd T cells residing in IEL, Peyer’s Patch or mLNs in mice treated with OVA and gluten at the different ages. 4. CD8aa T cell development and cytokine response. The population of CD8aa T cells in IEL has been found to play a suppressive role in the immune response. In this study, we found that CD8aa T cells significantly expands after 3 weeks and reaches levels close to adult mice after 4 weeks. Therefore, mice younger than 3 weeks have few suppressive immune regulatory CD8aa T cells. This finding drove the development of our hypothesis that young mice lacking suppressive CD8aa immune cells are more susceptible to food allergen due to reduced regulatory mechanisms. We tested the cytokine production of the CD8aa T cells in IEL of the challenged mice. We found that these cells did not produce much cytokine after OVA and PMA/ionomycin stimulation. In addition, we did not find any difference of cytokine production of these cells from mice exposed to OVA/gluten at age 2- or 6-weeks-old. 5. gd T cell response. As gd T cells also play important roles in the local immune response, we tested the gdT cell response in four tissues. In spleen gd T cells, we found a significantly higher production of IL-4 and IL-13 in the mice exposed to OVA/gluten at younger age. Production of cytokine from gdT cells from other tissues was similar between two mice groups. 6. Specific subsets of gdT cells: Vg1.1 and Vdelta(d)6.2/3 gdT cells There are two specific subsets of gdT cells that have been shown to play a role in the gut immune response: Vg1.1 and Vd6.2/3 gd T cells populations. In the model, we also determined the cytokine secretion pattern of these cells in mLN and spleen, but found no significant number difference between these two subtypes of gd T cells. In conclusion, we established a new model to of food-borne allergy in young mice. The timing of the allergen treatment for induction of allergy supports our hypothesis that the deficiency of the suppressive CD8aa cells in IEL of 2 week old mice results in higher susceptibility to developing food allergy. Our model showed that different subsets of T cells, CD4+ and gd T cells, differentially secrete various inflammatory cytokines, which may be responsible for allergic reactions after secondary exposure to food-borne allergens. We also suggest while CD8aa T cells may play an important role in the prevention of the initiation of allergy response, we did not find any cytokine production in these cells in both groups of mice. In addition to the proposed objectives, we coincidently found that mice reared in different rooms in our animal facility responded differently in this model. We found that mice which came from our “clean” room (only specific pathogens approved) failed to respond differently to differential timing of the exposure to OVA and gluten. Mice that came from our “dirty” room, were able to respond differently to differential timing of exposure to OVA and gluten. These results suggest that development of food allergy model may be dependent on where the mice reared in our facility, and this is very likely to be associated with different microflora in intestine of mice from different rooms. As a number of papers have previously shown, the characteristics of intestinal microflora may change the in vivo immune response of T cells to foreign antigens. Since both the initial and subsequent food allergen are taken places in the gut, we suspect that the microflora in the intestine play a big role in the discrepancies between mice from different rooms in our facility. Further studies need to be performed to characterize and compare the differences of the microflora from the mice of different rooms to determine if these could be the reasons for the different immune reactions from the mice from the two rooms. IV). Key outcomes or other achievements. It has been shown that childhood allergy poses a big problem in the food safety for children in the U.S. In this project, we successfully established a new food allergy model of childhood allergy in mice. This model may provide a novel tool for researchers who are interested childhood food allergy. This model was created and developed based on the hypothesis that deficiency in immune suppressive CD8aa T cells in IEL populations in mice prior to 3-weeks of age may regulate subsequent development of food allergy in young mice. In addition, we used gluten, a common ingredient in wheat and also a common allergen, to enhance the induction of food allergy to OVA, which is rich in chicken eggs. Our findings suggest that this early exposure to gluten and OVA leads to enhanced production of inflammatory cytokines by CD4+ T cells and Th2 cytokine production by gd T cells. We hope that this novel animal model can be used for studies of common allergens that induce childhood allergy in US.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: A poster presentation in 1st Annual Nutrition, Metabolism, and Disease symposium. Poster Abstract: Food allergies are more prevalent in children, and may be due to sensitization via food borne allergens at a young age. Wheat gluten, commonly found in food, can increase access to food borne allergens to the immune system. Our objective is to determine the role of wheat gluten in allowing specific intestinal intraepithelial lymphocyte (IEL) populations access to chicken egg albumin (OVA), a food allergen, leading to the development of food allergy in young subjects. First, we developed a murine model using gluten and OVA diet for mice at a young age. With this model, we determined whether age is a critical determinant for the development of food allergy, and further examine the role of specific IEL populations that vary by age, in regulating tolerance and allergic response. Our results show that 2-week old mice fed gluten and OVA for 2 weeks developed much stronger immune response to the allergen than mice fed the same diet starting at 6 weeks old. Based on these data, we conclude that the age of first exposure to food allergen and the presence of gluten may be critical for the development of food allergy. Finally, we found that susceptibility to food allergy in young mice with wheat gluten diet correlates with the lack of intestinal CD8aa suppressive T cells in mice younger than 3 weeks old. Further experiments are needed to determine the role of intestinal CD8aa suppressive T cells in development of food allergy during childhood using this murine food allergy model.


Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: The prevalence and severity of food borne allergies has increased 18% in the last 10 years, and in the United States, generates high morbidity among children and young adults (Branum, A and Lukacs, S, NCHS Data Brief, CDC, Oct 2008). In particular, the prevalence of egg allergy is estimated to be between 1.6 and 3.2% of the pediatric population, making it the second most common cause of food allergy in children. Food borne allergy is the result of an inappropriate immune response characterized by skin itching, or swelling or gastrointestinal system symptoms, including vomiting, diarrhea and colitis. In some cases, exposure to the allergen can cause severe anaphylactic shock resulting in death. Therefore, it is a very important issue in food safety of United States. Our long-range goal is to provide a detailed understanding of the mechanism by which the mucosal immune system, particularly in the gut, determines the immune response between tolerance and allergy in young subjects. In pursuit of that goal, the objective of this application is to determine the role of wheat gluten in allowing specific IEL populations access to food borne allergens, leading to the development of an allergic response to a food borne allergen. During the first year of the project, we successfully establish a new food borne allergy model in mice. With this model, we determined whether age is a critical determinant for the development of food allergy, and further examined the role of specific IEL populations that vary by age, in regulating tolerance and allergic response. Based on our result, we determined that oral exposure of 2 week-old mice to both gluten and OVA induce a more robust inflammatory response after rechallenge, compared to adult mice. The novelty of this model is that gluten is an important component for induction of food borne allergy in mice as young as 2 weeks old. Using this new model, we not only seek to find mechanisms of regulating childhood food allergy but also provide an experimental model for other researchers to study childhood food allergy. In order to increase the profile of this finding and to bridge the field of immunology and food safety, I became the member of American Association of Immunologists and attend their annual meeting this year to meet experts in gut immunity and childhood allergy. Details of the project design and the direction of the mechanisms of childhood food allergy are discussed with experts in the field. In addition, I also plan to attend a meeting organized by the Division of Nutrition Science at Cornell University to present the current findings to the experts in nutrition so that we could develop collaborations that could lead to rational treatment or preventive methods for childhood food allergy. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: The project aims to determine the role of specific IEL population in intestine in childhood food allergy by using a newly developed food allergy model in mice. Therefore, the results will bring insights to medical doctors, and childhood food allergy researchers who have patients with food allergy or who are doing research on food allergy field. The finding in this project also will impact on the field of infant food production and general public knowledge of food safety. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Food allergies are more prevalent in children, and may be due to sensitization via food borne allergens at a young age. In this study, we used wheat gluten, which can increase access of food borne allergens to the immune system in order to establish a new model of food allergy in mice. As described in the output section, we used this new model to determine whether age is a critical determinant for the development of food allergy, and further examine the role of specific IEL populations that vary by age, in regulating tolerance and allergic response. Our results show that 2-week old mice orally exposed to gluten and OVA for 2 weeks developed much stronger immune response to the allergen than mice fed the same diet starting at 6 weeks old. By flow cytometry analysis, we found that in CD4 T cells in younger mice produce more inflammatory cytokines such as IFN-gamma and TNF-alpha, compared to older mice. In addition, we found that gamma/delta T cells from the spleens of 2 week-old mice fed with gluten and OVA produced more Th2 cytokines, mainly IL-4 and IL-13, which are responsible for many allergic responses. Finally, we found that susceptibility to increased immune response to oral allergen in young mice that are fed with wheat gluten diet correlates with the lack of intestinal CD8-alpha alpha suppressive T cells in mice younger than 3 weeks old. Based on these data, we conclude that the age of first exposure to food allergen and the presence of gluten may be critical for the development of food allergy. The impact of the result and conclusion provides a useful animal model for childhood allergy, and potential mechanisms of the cause of the imbalance of immune system when exposed to food antigens in young age. Using this model, we and others can now examine the roles of different immune cells in IEL and surrounding lymphoid tissues that mediate the allergic reaction in young subjects. This USDA fellowship has provided tremendous support for allowing the development of this new model to study childhood food allergy. With this fellowship, I was able to go to the top immunology conference to meet and interact with experts in immunology and get their insights about childhood food allergy, that further help us to improve and strengthen our experimental designs for current and future studies.

Publications

  • No publications reported this period