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.
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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
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