5...; i. F» (I) CHARACTERIZATION OF TYPE 2 CYTOKINE RESPONSE TO ALLERGENS IN A MOUSE MODEL OF FOOD ALLERGY AND (II) THE EFFECT OF SELECTED HERBAL SUPPLEMENTS ON ALLERGEN DRIVEN TYPE 2 CYTOKINE RESPONSE BY SPLEEN CELLS FROM FOOD ALLERGIC MICE BY SITARAM PARVATANENI A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science & Human Nutrition 2007 Copyright by Sitaram Parvataneni 2007 ABSTRACT CHARACTERIZATION OF TYPE-2 CYTOKINE RESPONSE TO ALLERGENS IN A MOUSE MODEL OF FOOD ALLERGY AND THE EFFECT OF SELECTED HERBAL SUPPLEMENTS ON ALLERGEN DRIVEN TYPE-2 CYTOKINE RESPONSE BY SPLEEN CELLS FROM FOOD ALLERGIC MICE By Sitaram Parvataneni Food allergy is a growing problem with potential for fatality. However, mechanisms underlying food allergy are not completely understood. The ‘ present study was conducted using a mouse model of food allergy. In the first part, studies were conducted to characterize hazelnut and sesame allergen driven Type-2 cytokine response by spleen cells. We found a CD4-dependent, recall IL-4, IL-5 and IL-13 responses in food allergic mice. These Type-2 cytokine responses were observed in mice strains with different H-2 genetics. Further studies using Stat6 and 114 knockout mice suggested that allergic and anaphylactic response was dependent on IL-4 but not IL-5 or IL-13. In the second part of the study, the impact of selected herbal supplements on type-2 cytokine responses was studied. Among the herbal supplements tested, only licorice (from three different sources) consistently inhibited Type-2 cytokine responses. In summary: (1) these results have advanced the knowledge on the mechanism of food allergy in this mouse model; and (2) licorice was identified as an inhibitor of Type-2 cytokine response in this model. ACKNOWLEDGEMENTS The work was accomplished with the help and support of many individuals. First I would like to thank my major advisor, Dr. Venu Gangur for his guidance and continuous moral and financial support throughout M.S program. His continuous support through out the project allowed me to finish this project succesfully. I would like to thank the members of my graduate committee, Dr. Gale Strasburg and Dr. James J Pestka for their support and guidance. Besides my advisors, I would like to thank my lab members Dr. Hanem Mahamood (Post-Doc), Neil Birmingham, Sridhar Sarnineni, Babu Gonipeta and John for their help in a number of ways. My special thanks to my friends, Lalitha Navuluri, Caleb Kelly, Venkat, and Anindya Chanda, for giving me moral support throughout the study. I would like to thank Dr. Gale Strasburg, Department of Food science and Human Nutrition Michigan State University, Graduate School for funding part of my studies. Finally I would like to thank my parents, my family and fiiends for their support. iv TABLE OF CONTENTS LIST OF TABLES vii LIST OF FIGURES vii INTRODUCTION 1 REVIEW OF THE LITERATURE 05 A. Food induced adverse reactions and food allergy 05 B. Allergenic foods vs. food allergens 08 C. Food allergy: Relevance to food industry 09 D. Prevalence of food allergy 11 E. Mechanism of food allergy 13 1. Coombs and gel classification of immune-mediated adverse reactions 13 i) Type-1 hypersensitivity reaction -------------- 13 ii) Type-II hypersensitivity reaction -------------- 14 iii) Type-III hypersensitivity reaction ------------- 14 iv) Type-1V hypersensitivity reaction ------------ 14 2. Mechanism of Food Allergy: An Immune Response Model ----- 15 3. The major histocompatibility complex class II and the allergic response 18 4. Role of CD4+ T helper (Th) - 2 cells in allergic responses-- 21 a. Interleukin-4 (IL-4) 21 b. Interleukin-5 (IL-5) 24 c. Interleukin-13 (IL-13) 25 5. STAT6: Potential role in food allergy 26 6. IgE antibody: A central role in most food allergies ---------- 28 7. Prevention and treatment of food allergy: potential role for herbal Supplements 3O 1. Herbal supplements: Potential utility in the prevention and/or therapy of food allergy 31 8. Mouse models of food allergy: valuable tools to elucidate disease mechanisms and to develop new preventive and therapeutic approaches 33 1. Adjuvant-based food allergy mouse model 34 2. An adjuvant-fi'ee mouse model of food allergy using hazelnut and sesame as model allergenic foods --- 35 H. Statement of the problem 42 CHAPTER 1 44 A. Abstract 44 B. Introduction 45 C. Materials and methods 47 D. Results 52 E. Discussion 64 CHAPTER 2 72 A. Abstract 72 B. Introduction 73 C. Materials and methods 75 D. Results 79 E. Discussion 102 SUMMARY AND FUTURE DIRECTIONS 106 ‘REFERENCES 108 vi LIST OF TABLES LIST OF TABLES PAGE NO Table 1 Impact of herbal supplements on Th1/Th2 cytokine production in various experimental model systems ------ 32 Table 2 Different strains of mice used in the study 48 Table 3 Systemic anaphylaxis symptom score 51 Table 4 Herbal supplements used in the study 76 Table 5 Irnmuno modulatory effects of licorice 104 Table 6 Dose calculations of herbal supplements 105 vii Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 LIST OF FIGURES Classification of food induced adverse reactions -------- 07 Mechanism of peanut allergy: Immune response model --- 17 Allergen presentation to the T lymphocyte: Role of MHC class H and CD4 molecules 19 Dose-response and time-course analyses of IgE antibody response to transdermal hazelnut protein exposure in BALB/c mice ----- 38 Immune response to sesame seed protein following transdennal exposure in BALB/c mice 39 Transdermal exposure to hazelnut protein is sufficient to sensitize BALB/c mice for systemic anaphylaxis following oral challenge with hazelnut 40 Systemic anaphylaxis to sesame seed protein in transdermally sensitized mice 41 Transdermal sensitization protocol 49 Hazelnut-driven IL-4, IL-5 and IL-13 responses of hazelnut sensitized mice vs saline exposed mice 53 Sesame-driven IL-4, IL-5 and IL-13 responses of sesame sensitized mice vs saline exposed mice 55 Hazelnut and sesame-driven IL-4 levels are CD4 depensdent --- 56 Hazelnut and sesame-driven IL-5 levels are CD4 depensdent - 57 Hazelnut and sesame-driven IL-l3 levels are CD4 depensdent - 58 Hazelnut-driven IL—4 response from three different mice strains; BALB/c, ASW and SJ L 60 Hazelnut-driven IL-5 response from three different mice strains; BALB/c, ASW and SJL 61 Hazelnut-driven IL-13 response from three different mice strains; BALB/c, ASW and SJL 62 viii Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Figure 24 Figure 25 Figure 26 Figure 27 Figure 28 Figure 29 Figure 30 Hazelnut-driven IL-4, IL-5 and IL-13 response in Stat6 knockout and wild type animals 63 Hazelnut-specific IgE response in BALB/c wild type animals and 114 knock out animals 65 Systemic anaphylaxis and rectal temperature before and after oral challenge with hazelnut protein in wild type and [14 knock out mice 66 Working model of type-2 cytokine response in our adjuvant-free mouse model of transdermal sensitization 70 Cell viability in spleen cell cultures in saline-exposed mice after culture with four different herbal supplements 81 Cell viability in spleen cell cultures in saline—exposed mice afier culture with five different herbal supplements 82 Cell viability in spleen cell cultures in hazelnut-sensitized mice after culture with four different herbal supplements ------------- 83 Cell viability in spleen cell cultures in hazelnut-sensitized mice after culture with five different herbal supplements -------------- 84 Impact of herbal supplements on hazelnut-driven H.-4 response by spleen cells from hazelnut allergic mice 85 Impact of herbal supplements on hazelnut-driven IL-4 response by spleen cells from hazelnut allergic mice 84 Impact of herbal supplements on hazelnut-driven IL-5 response by spleen cells from hazelnut allergic mice 85 Impact of herbal supplements on hazelnut-driven IL—5 response by spleen cells from hazelnut allergic mice 88 Impact of herbal supplements on hazelnut-driven IL-13 response by spleen cells from hazelnut allergic mice 90 Impact of herbal supplements on hazelnut-driven IL-13 response by spleen cells from hazelnut allergic mice 91 ix Figure 31 Figure 32 Figure 33 Figure 34 Figure 35 Figure 36 Figure 37 Figure 38 Cell viability in spleen cell cultures of hazelnut sensitized mice after culturing with licorice from three different sources -------- 92 Impact of licorice from three sources on hazelnut-driven IL-4 response by spleen cells from hazelnut allergic mice ---------- 94 Impact of licorice from three sources on hazelnut-driven IL-5 response by spleen cells from hazelnut allergic mice ----------- 95 Impact of licorice from three sources on hazelnut-driven IL-13 response by spleen cells from hazelnut allergic mice ----------- 96 Cell viability in spleen cell cultures of sesame sensitized mice after culturing with licorice from three different sources -------------- 97 Impact of licorice from three sources on sesame-driven IL-4 response by spleen cells from sesame allergic mice ------------ 99 Impact of licorice fiom three sources on sesame-driven IL-S response by spleen cells from sesame allergic mice ------------ 100 Impact of licorice from three sources on sesame-driven IL-1 3 response by spleen cells from sesame allergic mice ----------- 101 INTRODUCTION Allergic reactions to food (commonly called food allergy) are due to abnormal responses of the immune system to food proteins (1). The food allergies are classified as immediate hypersensitivity reactions because they occur within minutes after exposure to the food to which an individual has been sensitized (2). Significance of Food Allergy Food allergy is a growing public health problem of serious proportions in many Westernized countries including the United States of America (3). Recent epidemiological data suggests that nearly 4% of American adults are afflicted with food allergies; the disease is more prevalent in children with nearly 6-8°/o of them below the age of 3 years affected (4, 5). Food allergy has both psychological (6) and economic (7) impact on the society by causing nearly 200 deaths per year and 30,000 emergency room visits (8, 9). Similar to other chronic diseases such as asthma, diabetes, and psychiatric diseases, food allergy can impact the quality of life. For example, people with food allergies, constantly worry about the possibility of exposure to food allergens in a variety of places such as restaurants, schools and airplanes (10, 11). Food allergens pose a significant challenge to the food industry. Food allergens continue to be the major cause of Class-1 food recalls and negatively impact the food industry (12). According to Dick Dahl (2006), the restaurant industry might face an increasing number of food allergen related lawsuits in coming days, even though McDonald’s is the only corporation sued for the wheat and milk contents in their French fries (13). The agricultural biotech industry faces the challenge of potential allergenicity of novel foods produced by genetic engineering. For example, a recent episode of contamination of the human food chain by a genetically engineered corn (Star link) led to scores of food recalls; the total cost of cleanup has been estimated to be ~100 million dollars (14-18). Mechanism of Food Allergy The precise mechanism of food allergy is not completely understood at present (19). It is generally believed to be similar to other allergic disorders such as allergic rhinitis and allergic asthma (20). According to one proposed mechanism, peanut allergy develops as follows: first exposure of susceptible individuals to peanut results in the production of peanut-specific IgE antibodies. These antibodies bind to high affinity IgE receptor present on the mast cells and basophils. Once this occurs, this individual is considered ‘sensitized’ to peanuts. When this individual is re-exposed to peanuts, the I gE antibodies bind to the peanut protein and initiate a series of biochemical reactions leading to the clinical symptoms of food allergy such as hives, vomiting, diarrhea, respiratory distress or in rare cases, systemic anaphylaxis and death (21). As alluded earlier, specific mechanisms of food allergy are not completely understood at present. Mouse models of food allergy have been developed to facilitate investigation into the mechanisms. For example, an adjuvant-free mouse model has been recently developed in our laboratory of Food Allergy and Immunology at the Michigan State University (19, 22). During the first part of my thesis work, I used this model to study the role of Type—2 cytokines in tree nut and sesame allergy. Prevention and Therapy of Food Allergy Despite the potential for a fatal outcome, no effective preventive methods (e. g., a vaccine) are available for food allergy. Currently, avoiding the offending food is the only proven way to prevent food allergic reaction. Therefore, researchers have been examining a number of novel methods for prevention and therapy of food allergy (23-32). Many of these studies use mouse models of food allergy for testing. There is growing interest in the potential of herbal supplements in the prevention and therapy of allergies and asthma including food allergies. A recent study demonstrated that a mixture of Chinese herbal supplements was effective in preventing systemic anaphylaxis in a mouse model of peanut allergy (23, 29). Thus, the potential of herbal supplements in food allergy remains to be examined in detail. Mouse models of food allergy provide a valuable tool for such studies. In the second part of my thesis work, I tested a number of herbal supplements for their potential utility using the mouse model of food allergy developed in our laboratory. An Adjuvant-free Mouse Model of Food Allergy Our laboratory has previously developed an adjuvant-free mouse model of food allergy using hazelnut and sesame as model allergenic foods (19, 22). The major features of this model are: (1) induction of food-specific IgE antibody response following transdermal exposure to food allergens without any adjuvant; and (2) induction of clinical signs of systemic anaphylaxis following oral challenge with a food allergen. Using this mouse model, the present study was conducted in two parts. In Part- I, allergen-driven Type-2 cytokine responses were characterized in hazelnut and sesame allergy mouse models. In Part-II, the impact of selected herbal dietary supplements on allergen-driven Type-2 cytokine responses by spleen cells from food allergic mice was studied. In the next section, I have presented an extensive review of the literature relevant to my studies. In Chapters 1 and 2 I have presented findings from part- I and part-II of my studies in a manuscript style. REVIEW OF THE LITERATURE In this chapter I have reviewed the relevant published work available on the following topics in food allergy: food induced adverse reactions and food allergy; allergenic foods vs. food allergens; food allergy - relevance to food industry; prevalence of food allergy; mechanism of food allergy; prevention and treatment of food allergy - potential role for herbal supplements; mouse models of food allergy - valuable tools to elucidate disease mechanisms and to develop new preventive and therapeutic approaches. Food Induced Adverse Reactions and Food Allergy Food induced adverse reactions are broadly classified into two types: 1) toxic and 2) non-toxic reactions (Fig. 1). Whereas, toxic adverse reactions are mediated by biologically active microbial toxins, non-toxic reactions are due to specific food constituents such as allergens or lactose. Non-toxic reactions are further classified as immune mediated and non-immune mediated. In this system of classification, food allergies are classified as non-toxic, immune- mediated adverse reactions (33). The mechanism underlying food intolerance such as lactose intolerance is different fi'om that of food allergy. Thus, as opposed to food allergy, food intolerance is not caused by the immune system. For example, lactose intolerance is due to a precise metabolic defect such as absence and/or deficiency of lactase (34). Fig 1: Classification of food induced adverse reactions Food induced adverse reactions are classified primarily into two types: toxic and non-toxic reactions. Non-toxic reactions are further classified as immune mediated and non-immune mediated reactions. Immune mediated reactions are subdivided as IgE mediated and non- IgE mediated reactions. Most food allergies are examples of IgE mediated immune reactions, where as celiac disease and some milk allergies are examples of non-IgE mediated reactions. Lactose intolerance is an example of non-immune mediated adverse reaction due to enzyme deficiency. Scambroid fish poisoning is an example of adverse reaction due to preformed chemicals (such as histamine) in TOXIC ADVERSE REACTION TO FOOD IgE Mediated (e.g., Peanut allergy) NON-TOXIC IMMUNE MEDIATED Non-IgE Mediated (e.g., Celiac disease) Enzyme deficiency (e.g., Lactose intolerance) NON-IMMUNE _ Preformed histamine MEDIATED (e.g., Scambroid fish poisoning) Undefined reactions (e.g., Irritable bowl syndrome) Allergenic Foods vs. Food Allergens Any food that triggers an allergic reaction in a sensitized individual is called an allergenic food. More than 160 different types of foods have been documented to trigger allergic reaction in sensitized individuals (35). However, according to the Food and Agriculture Organization (F AO), the Food Allergy Task Force of the International Life Sciences Institute (ILSI), and the United States Food and Drug Administration (US FDA), nearly 90% of all food allergies are caused by eight main food types called “red flag” allergenic foods. They are: Chicken egg, cow’s milk, peanut, soybean, wheat, tree-nut, fish, and shell-fish (36-39). However, in addition to these eight foods, the Canadian Food Inspection Agency (CFIA) has added sesame seeds and sulphites to their list of allergenic foods (40). The European Food safety Authority (EFSA) considers cow's milk, eggs, fish, peanuts, tree nuts, soy, celery, cereals containing gluten, crustacean (crab, crayfish, lobster and shrimps), sesame, mustard, and sulphites as major allergenic foods (41). Tree nuts have been identified as a major group of red flag allergenic foods by the US FDA, the CFIA as well as the EFSA (39-41). Tree nuts, along with peanuts and sesame, are the leading cause of life-threatening allergic reactions such as systemic anaphylaxis (42, 43). Accordingly, US FDA has identified the following tree nuts as “red-flag” tree nuts for regulatory purpose: Almond; beech nut; Brazil nut; butternut; cashew; chestnut (Chinese, American, European, Seguin); chinquapin; coconut; filbert/hazelnut; ginko nut; hickory nut; lichee nut; macadamia nut/bush nut; pecan; pine nut/pinon nut; pili nut; pistachio; sheanut; and walnut ( English, Persian, black, Japanese, California) (44)- The active ingredient present in the allergenic food that is responsible for triggering an allergic reaction is called a food allergen. Typical food allergens are either proteins or glyco-proteins (45, 46). These allergens elicit IgE antibodies and bind to them specifically. Food allergens are usually: 1) proteins of 10-70 kDa; 2) heat resistant (90°C, 5 minutes); 3) acid stable (pH 5 2.0); 4) resistant to pepsin digestion; and 5) water soluble (21). However, these properties have little value in predicting allergenic potential of a given protein (47). Following are some examples of major food allergens: Ara h], Ara h2, and Ara h3 are major peanut allergens; casein, a lactalbumin, and B lactoglobulin are major milk allergens (48, 49). Food Allergy: Relevance to Food Industry Food industry is facing several challenges because of food allergy. Some of these challenges are: 1) food sensitized consumers are at risk of potentially fatal allergic reactions; 2) compensation to food allergy victims, lawsuits; 3) product recalls due to food allergen contamination; 4) allergen labeling issue; 5) potential allergenicity of exotic foods and genetically engineered foods; 6) lack of consensus on the threshold for allergenic foods to elicit a clinical reaction; and 7) the effect of processing on food allergenicity (21). According to the United States Food and Drug Administration (US FDA), presence of “red flag” allergenic foods in a product should be identified on the label (50). Failure to include such information on the label is the main reason for class I food recalls in the United States of America (12). To protect food allergic consumers, a new law (Food Allergen Labeling and Consumer Protection Act) came into effect in January 2006. It is intended to make the food allergen labeling much more consumer friendly than it was before. Thus, all food products containing the major allergenic foods must identify them with common names on the label (e. g., ‘milk’ instead of ‘casein’; ‘peanut protein extract’ instead of ‘protein extract’ etc). Furthermore, in the case of tree nuts, fish and crustacean shellfish, more specific information on the type of nut and species of fish/shellfish must be identified on the label (50). Availability of commercial rapid immunoassays (such as enzyme linked immunosorbent assay, often called ELISA) to detect allergenic foods in field samples has helped the identification of allergen contamination. However, a major issue is about acceptable levels of food allergen contamination. According to the US FDA there are no recommended threshold levels for allergenic food contamination of food products. Consequently, zero tolerance is the current policy of US FDA for food allergen contamination (21, 51, 52). 10 Potential impact of food processing on food allergenicity is another area of concern for the food industry (53, 54). However the specific impact of different processing methods on specific allergens in different food types is not well studied (21). Potential allergenicity of novel foods such as genetically engineered foods is another significant problem for the food biotechnology industry. Since genetically engineered foods express new non-indigenous proteins, potential allergenicity of the newly introduced protein is an issue. During 2000-2001 , contamination of food with a genetically engineered corn (StarLink) occurred. This corn was originally approved for use as animal feed. Subsequent consumer complains of potential allergies to this corn led to food recalls. Total cost of the cleanup from this episode of contamination has been estimated be ~100 million dollars (14-18). Prevalence of Food Allergy Overall prevalence of food allergy in the United States of America is ~ 4% in adults and ~6-8 % among children (55). Among children, cow’s milk with a prevalence of 2.5%, is the leading cause of food allergy. Prevalence rate of other food allergies among children are as follows: egg at 1.3%; peanut at 0.8%; wheat and soy at 0.4%; tree nuts at 0.2%; and fish as well as shell fish at 0.1% (55). Children usually outgrow milk, egg, wheat and soy allergies by the age of 3 years. Other food allergies such as peanut, tree nut, fish and shell fish 11 allergies are rarely outgrown. Among adults, the prevalence rate of the later four food allergies is as follows: shell fish at 2%; peanut at 0.6%; tree nuts at 0.5%; and fish at 0.4% (55). The overall prevalence of tree nut allergy in the United States of America is ~0.5% (55, 56). According to the peanut and tree nut registry, which is the largest source of self-reported allergic reactions in the USA, walnut is the leading cause of tree nut allergies (at 34%). It is followed by cashew (at 20%), almond (at 15%), pecan (at 9%), pistachio (at 7%), and other nuts (at 5%) (5 7). In another report, where 54 tree nut allergic pediatric patients were followed at Johns Hopkins, walnut was again the leading allergenic nut at 26% followed by almond (13%), pecan (13%), cashew (11%), hazelnut (7%), pine nut (7%), pistachio (7%), and Brazil nut (4%)(58). Notably, hazelnut allergy has been reported to be more common in Europe than in the USA. It is thought to be due to increased exposure to hazel pollen in Europe (59, 60). Currently the US FDA, as opposed to CFIA and EFSA, does not consider sesame as a major allergenic food (39-41). However, a previous study fi'om our laboratory found that the reporting of sesame allergy is growing world-wide (61). An Australian study found that sesame was the fourth (0.42%) leading cause of food allergy among children after egg (3.2%), milk (2%), and peanut (1.9%). This study also found that sensitivity to sesame was greater than any single tree nut studied (62). Prevalence of severe allergic reactions to sesame 12 was estimated at 0.05% in the general population of the United Kingdom (63). A recent study on food allergy among Israeli children concluded that sesame was the third most common food causing sensitization at (0.18%) after egg (0.5%) and cow’s milk (0.3%) (64). Thus, there is considerable evidence to suggest that the sesame allergy may be a growing food allergy with serious life- threatening consequences such as systemic anaphylaxis (43). Mechanism of Food Allergy Coombs and Gel Classification of Immune Mediated Adverse Reactions Immune mediated adverse reactions in general were originally classified by Coombs and Gel as hypersensitivity reactions (21, 65). Based on the type of immune component involved, the time of onset of the reaction afier exposure to the antigen, and the type of antigen involved, they classified hypersensitivity reactions into four groups: Type-I; Type-II; Type-III; and Type-IV reactions. Major features of these reactions are briefly reviewed below. Dipe-I HypersensitivityReaction Type I hypersensitivity is also called as the immediate hypersensitivity reaction because it occurs within minutes afier exposure to a type of antigens called allergens. It is mediated by IgE antibodies produced in response to allergens. Allergen binding to the IgE antibody fixed onto mast cell leads to cellular activation and the release of chemical mediators such as histamine—a 13 major mediator of clinical symptoms of allergy such as anaphylaxis. Most food allergies, and allergic asthma are examples of Type-I hypersensitivity reactions. T vpe-II Hypersensitivity Reaction Type H hypersensitive reaction, also known as cytotoxic reaction, is mediated by the IgG antibody. Here antibodies bind to the insoluble antigen (e. g. antigen present on cells and/or bound to cells) and activate the complement system leading to tissue damage. Role for this mechanism in food allergy is unclear at present. Examples include autoimmune hemolytic anemia and some drug allergies. Type-III Hzpersensitivitv Reaction In type HI hypersensitivity, IgG and IgM antibodies bind to the circulating antigen and form immune-complexes. Deposition of these immune- complexes leads to tissue damage. It is not known at present whether this mechanism may play a role in food allergy. Lupus—an autoimmune disease, is a common example of type-III hypersensitivity reaction. T vpe-I V H wersensitivitv Reaction Type-IV hypersensitivity reaction is also called as delayed hypersensitivity reaction. This is because, after exposure to the antigen, it takes at least 24 hrs for the reaction to manifest. It is mediated by antigen specific T lymphocytes. Although food allergies in general are considered as immediate 14 hypersensitivity reactions, it is proposed that milk allergy may involve both Type-I and Type-IV mechanisms (66). Furthermore, Type-IV mechanism is considered to be a major player in gluten enteropathy (or the Celiac disease). Mechanism of Food Allergy: An Immune Response Model The mechanism underlying food allergy is not completely understood at present. It is generally thought to be due to, Type-I hypersensitivity reactions similar to other allergic diseases such as airways allergies (20). Accordingly, an immune response model has been proposed to explain how foods such as peanuts trigger allergic reactions. The major feature of this model is illustrated (Fig. 2) (21). According to this proposed mechanism, there are two general stages in the development of food allergy: (1) sensitization phase; and (2) disease expression phase. When a genetically susceptible individual is exposed to peanut for the first time, peanut proteins are phagocytosed by antigen presenting cells (such as dendritic cells/macrophages) and peanut allergens are presented along with MHC class II molecules to the CD4 positive T cells. The latter undergo differentiation into T helper (Th)-2 subset and help the allergen specific B cells to produce IgE antibodies. Most of these antibodies are taken- up and displayed by mast cells and basophils that express the high affinity receptor for IgE (called Fc epsilon receptor-I). Once an individual has produced IgE antibodies and displayed them on their mast cells, he/she is considered to be ‘sensitized’ to that food. At this point there are no clinical symptoms. However, following re—exposure to the same food (in this case, the peanut), 15 MMeChanism of peanut allergy: Immune response model. Exposure of a genetically susceptible individual to peanuts results in presentation of peanut allergens by antigen presenting cells (APC) to T helper (Th) lymphocytes. A consequence of this is the proliferation and activation of peanut specific Th-2 lymphocytes that help peanut specific B cells to produce peanut specific IgE antibodies. IgE antibodies bind to the high affinity IgE receptor present on cells such as mast cells that are rich in histamine and other proinflammatory mediator containing granules. Subsequent exposure to the same allergen (peanut in this case) results in cross linking of surface IgE receptors on mast cells results in cellular activation, degranulation. Host response to histamine and other mediators results in the expression of clinical symptoms of food allergy within minutes of exposure to allergens (Source: Ref. 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This results in clinical symptoms such as hives, anaphylaxis etc. Thus, allergen presentation via MHC class II molecules, activation of CD4 positive Th2 cells, and production of IgE antibodies are thought to be critical mechanisms underlying food allergy development. Therefore, I have reviewed these events in detail in the next section. The Major Histocompatibility Complex Class H and The Allergic Response The major histocompatibility complex (MHC) in humans is called the human leukocyte antigen (HLA) and in mice it is called the H-2 complex (67- 69). It is located on the human chromosome 21q and the mouse chromosome 17 (69). The H-2 complex consists of several major genes including MHC class II encoding genes (70). As discussed earlier, MHC class II molecules are involved in presenting antigen/allergen peptides to the CD4 positive T cells. The CD4 positive T cells are able to recognize only those antigens that are presented with MHC class II molecules (Fig. 3). 18 , - Alleraen phage .( Class II histocompatibility molecule Pemide (Allergen) T cell receptor Fi . 3: Aller en resentation to the T 1 hoc e: Role of MHC class II and CD4 molecules Antigen presenting cells (such as macrophages) present the processed allergen-derived peptides along with MHC class II molecules to T lymphocytes. The T cell receptor can recognize the allergen only when it is presented with MHC class II molecules. The CD4 molecule expressed on the surface of CD4+ T cells act as co-stimulatory molecules in the allergen presentation (Modified fiom http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AntigenPresenta tion.html). Polymorphism is a major feature of the MHC class II molecules. Thus, different allelic forms are inherited by different subjects (and by different strains of mice). Different allelic forms of MHC class II molecules present different sets of peptide antigens/allergens to T cells (2). Consequently, it is hypothesized that some individuals (and some mice strains) with particular types of MHC class II molecules may be able to present allergens much better while other individuals (and mice strains) may be unable to do so due to their having other polymorphic variants of MHC class II molecules (71). Consequently, polymorphisms in MHC class II is considered as an important genetic factor that may render susceptibility for allergic disorders (72). There is extensive evidence from studies conducted during 1980 and 1990’s implicating the role of MHC class II molecules in the production of specific IgE antibodies to pollen and house dust mite allergens (73). Similarly, it is well established that whereas, some strains of mice with certain MHC class II haplotypes (e. g., Balb/c, H-2 d) are ‘high IgE responders’ to allergens, other strains with different MHC class II variants (e. g., C57BL/6, H-2 b ) are intermediate responders, and some other strains (e. g., SJ L, H-ZS) are low/non- IgE responders (74-76). As opposed to airways allergies and asthma, role of MHC class 11 polymorphism in food allergy has not been well studied. A recent study suggested the possible role of HLA class II genetic polymorphism in 20 determining susceptibility to peanut allergy (77). Similar to human disease, role of MHC class 11 genes in mouse models of food allergy has not been examined in-depth. Role of CD4 Positive T helper (Th) - 2 Cells in Allergic Respon_sas_ The CD4 positive T cells play an important role in allergic immune responses by helping B cells to produce the IgE antibody (2). The CD4 molecules expressed on the surface of CD4 positive T cells enable them to bind to antigen/allergen presenting cells. Once activated, CD4 positive T cells proliferate and differentiate into two main subsets of cells--T helper Type 1 (Th1) and T helper Type 2 (Th2) cells. Whereas Th1 cells play a major role in cell mediated immunity (e.g., delayed type hypersensitive reaction), Th2 cells are key players in allergic responses. Mossman et a1 (1986) originally named the mouse T helper cells into Th1 and Th2 based upon cytokines they secrete. Whereas, secretion of IFN Y defines a Th1 cell, Th2 cells secrete mainly IL-4, IL-5 and IL-13 (78). A similar profile was also recognized later in humans (79). Other studies found that in addition to CD4 positive Th2 cells, other immune cells can also produce Th2 cytokines. For example, IL-4 can be produced by mast cells (80), Natural Killer (NK) cells (81) , basophils (82) and eosinophils (83); IL-5 can be produced by eosinophils (83) and NK cells (84); IL-13 can be produced by B cells (85), mast cells (86), NK cells (87), dendritic cells (88) and basophils 21 (89). Recognizing that diverse types of immune cells can produce these cytokines, a Th2 cytokine is also sometimes referred to as a Type-2 cytokine when the cellular source is not clear. It is well established that there is excessive production Type-2 cytokines such as IL-4, IL-5 and IL—13 in allergic disorders such as allergic rhinitis and asthma (90). Several studies have demonstrated that the transcription factor, STAT6, plays a major role in the regulation of Th2 cytokine responses (91, 92). Some studies indicate that Type-2 cytokines are important in food allergies such as peanut allergy (20, 93), although other studies do not support this (94). Furthermore, relative contribution of these cytokines to disease expression and the mechanism of their regulation are not completely understood in food allergy. It is also unclear whether different types of food allergies may exhibit different profile of Type-2 cytokine responses. Thus, because of their importance in allergic disease in general and their potential role in food allergy, I have briefly reviewed each individual Type-2 cytokine followed by a discussion on the role of STAT6 in food allergy in the next section. Interleukin-4 (IL-4) Interleukin (IL)-4 is a highly pleiotropic cytokine produced primarily by activated CD4 positive Th2 cells (95, 96). It is also produced by mast cells (78), NK cells (79), basophils (80) and eosinophils (81). Consistent with its pleiotropic nature IL-4 receptor is present on T cells, other hematopoietic cells 22 (e. g., basophil, eosinophil) as well as non-hematopoietic cells (e.g., fibroblasts and epithelial cells) (95). Molecular and Structural Characteristics Human IL-4 is a 129 amino acid long, 18 kD protein. The IL-4 gene spans about 10 kb and is located on chromosome 5 (5q31.1—31.2) (95). Mouse IL-4 is a 120 amino acid long, 18 kD protein and its gene is located on chromosome 11 (95, 97). The crystallographic structure of recombinant human IL-4 revealed that, it is a highly compact globular protein with a predominantly hydrophobic core (98). Major Functions of IL-4 Interleukin-4 was first described as a B cell growth factor in the mouse system. It is vital for B cell growth, immunoglobulin class switch to IgE as well as IgGl classes (96). IL-4 induces the expression of MHC class-II molecules and the high affinity IgE receptor (FceRI) on B cells (95). It acts as a T cell growth factor; it induces proliferation of both human and murine T lymphocytes (99). It also differentiates T cells into Th2 cells (100, 101). It increases the growth of mast cells (102). It also increases the vascular responsiveness to mediators of allergic inflammation (103). The phenotype of mice deficient in IL-4 gene demonstrates the major role of this cytokine in IgGl and IgE production (104). However specific role of IL-4 in food allergy is unknown. 23 Interleukin-5JIL-5) Interleukin (IL)-5 is produced mainly by activated Th2 cells, as well as mast cells, NK cells, and eosinophils (83, 84). The major target cells for IL-5 are eosinophils which express IL-5 receptor (105, 106). Consequently, IL-5 is a major player in chronic inflammation associated with eosinophil infiltration such as asthma (107). Studies using IL—5 gene knockout mouse model have demonstrated a key role for IL-5 in allergic eosinophlic esophagitis --an inflammatory condition often associated with food allergy in some individuals (108). Molecular and Structural Characteristics Human IL-5 is a 117 amino acid long protein. The IL-5 gene is located on chromosome 5 (5q3l.l) (109). Mouse IL-5 is a 111 amino acid long protein and its gene is located on chromosome 11 (109). Compared to IL-4, IL-5 is a bigger protein with a molecular weight of 45 kDa. IL-5 exists as a dimer with two identical polypeptide chains wound together to achieve functional confirmation (1 10). Major Functions of IL-5 The major target cell for IL-5 is an eosinophil. Stimulation of eosinophils with IL-5 leads to their activation and degranulation (111). It also inhibits the apoptosis of eosinophils (112, 113). IL-5 is the key cytokine in the production 24 and survival of eosinophils. Importance of IL-5 in the production of eosinophils is evidenced by the dramatically reduced number of eosinOphils in IL-5 knockout mice (114). IL-5 also facilitates entry of eosinophils from the circulation into the tissue (115). Since eosinophilia is a characteristic feature of chronic allergic inflammation, IL-5 is considered as a major player in allergic disorders (107). Notably, the specific role of IL-5 in food induced systemic anaphylaxis is unknown. Interleukin-13 (IL-1 3) Interleukin (IL)-13 is a pleiotropic cytokine produced by activated Th2 cells, B cells (83), mast cells (84), NK cells (85), dendritic cells (86) and basophils (87). It plays a critical role in allergic inflammation of the airways associated with asthma (116, 117). IL-13 shares many functions similar to that of IL-4 because these two cytokines share a common component in their receptor called IL-4Ra (118). The shared actions of IL-4 and IL-13 include: 1) induction of MHC class-H expression; 2) helping B cells to produce IgE in humans (but not in mice) (119). Molecular and Structural Characteristics ofIL-13 Human IL-l3 gene is located on chromosome 5 along with genes of IL-3, IL-4, IL-5, IL—9, and GM—CSF (120). The mouse IL-13 gene is located on chromosome 11 (121). The high resolution multidimensional NMR structure of 25 human IL-13 found that it has a short chain lefi handed four-helix bundled structure (120). Major Functions of IL-1 3 Along with the common fimctions it shares with IL-4, researchers found that IL-13 has an important role in pathophysiology of asthma (116, 117). For example blocking of IL-13 can inhibit pathophysiological changes in asthma such as excess mucus secretion (122). The phenotype of mice deficient in IL-1 3 gene revealed its importance in expulsion of helminth parasites from the gastrointestinal tract (123). In addition to IL-4, IL-13 is important in IgE production in humans; but its role in IgE production in mice is controversial. Notably, specific role of IL-13 in food allergy and anaphylaxis is unknown. STAT6: Potential Role in Food Alleagy Signal transducers and activators of transcription (STATS) are the members of a family of transcription factors that activate gene transcription in response to a signal from cytokines (124). STATS are cytoplasmic proteins that are activated by tyrosine phosphorylation by the cytokine receptor associated Janus kinases after the binding of cytokine to its receptor (125). Phosphorylation leads to dimerization of individual STAT proteins. The resulting dimer then migrates to the nucleus and activates the transcription by binding to the target DNA sequence (125). 26 STAT6 is a member of this group of transcription factors. It is especially important in the regulation of IL-4 gene transcription (126). Studies using Stat6 knockout mice found that STAT6 was necessary for the gene activation signal in response to IL—4 stimulation. Lymphocytes from Stat6 deficient mice are unable to express MHC Class H expression in response to IL-4 (91). Stat6 deficient B lymphocytes were unable to switch to the immunoglobulin isotypes IgG1 and IgE in response to IL-4 (127). Stat6 deficient T helper cells were also unable to differentiate into Th2 cells in vitro or in vivo (91). Studies using mouse models have shown that STAT6 also plays an important role in IL-13 responses to helminth infection in mice (128, 129). Thus, it is well established that the signaling molecule, STAT6 plays a key role in the Type-2 immune responses in general. However, specific role of STAT6 in food allergy is not well studied. There is one study suggesting a potential role for STAT6 in nut allergy in particular in humans. Thus, Amoli et al (2000) initially identified a single nucleotide polymorphism (SNP) in 3’ UTR of the STAT6 gene in a cohort in the United Kingdom. Subsequently they studied the frequency of this SNP in 71 nut allergic patients and compared it with 45 atopic patients without nut allergy and 184 healthy controls. They found that a polymorphic variant of STAT6 (called as G allele) frequency was significantly increased in nut allergic patients when compared to atopic individuals without nut allergy and healthy controls. Based on these data, they concluded that STAT6 3' UTR polymorphism might play an important role in 27 determining susceptibility and severity among nut allergic patients (130). Notably, the role of STAT6 in mouse models of food allergy has not been examined previously. IgE Antibody: A Central Role in Most Food Allergies Prausnitz and Kustner (1921) first demonstrated the presence of a serum component responsible for an allergic reaction (131). Later, Ishizaka and Ishizaka (1966) discovered that IgE antibody was the serum component responsible for an allergic reaction (132). This antibody was later named as ‘IgE (‘E’ for erythema or a rash)’. Immunoglobulin (Ig) E (also known as the reaginic antibody), is one of the five isotypes of immunoglobulins (2). IgE plays a central role in allergic reactions (2). However, it is thought to have evolved to protect hosts against helminth parasite infestations (133). Among immunoglobulins, IgE is the least abundant isotype in the blood, normally present in nanogram per mL of blood. These levels are significantly lower than other isotype such as IgG that is present in milligram per mL of blood. Among immunoglobulins, IgE has shortest serum half—life (2 days) (2). After its production, IgE binds to the high affinity IgE receptor (F ceR—I) present on the surface of mast cells and basophils (134). Even though it has very short serum half-life, IgE remains for weeks or months when bound to chR-I on the 28 surface of mast cells and basophils. Binding of allergen to the IgE-IgE receptor complex leads to the cellular activation and release of inflammatory mediators such as histamine. These reactions are important in the clearance of parasites as well in triggering allergic reactions as described earlier (Fig. 2) (133). Although central role of IgE in airways allergies is well known, the key role of IgE antibodies in food allergy was demonstrated only recently in peanut allergic subjects (28). Thus, neutralization of circulating IgE using an anti-IgE vaccine in peanut allergic children was found to confer clinical protection against peanut allergy (28, 135). Furthermore, other studies demonstrated that circulating levels of food specific IgE correlated with food induced clinical reactions and higher IgE levels were predictive (~95% chances) of food allergy (136). The predictive IgE level for egg allergies at S 2 years age is 2 kIU/L and at age 5 is 7 kIU/L. In case of milk allergies, the predictive levels are 5 and 15 kIU/L at ages 2 and 5 respectively (136). Predictive IgE level for peanut allergy is 14 kIU/L at mean age of 5 years (136). Notably, predictive values for other food allergies such as tree nut allergies have not been established as yet. It is noteworthy that there are reports of a subset of food allergies independent of IgE antibodies. For example some milk allergic patients do not have demonstrable milk specific IgE antibodies in the blood, but continue to react to oral exposure to milk (66). However, mechanisms underlying such IgE independent food allergies are not completely clear at present (21). 29 Prevention and Treatment of Food Allergy Currently effective preventive methods (such as a vaccine) are not available for food allergy, except an experimental peanut allergy vaccine that was reported to confer partial protection (28). Although exclusive breast feeding has been suggested to provide some protection against food allergy, it continues to be an intense research area to clarify doubts raised by others (137, 138). Thus, the only recommended and effective preventive method for food allergy is to avoid exposure to the causal food. Although this is a very useful method, many food allergy cases occur due to accidental exposure to the offending food (e. g., in a restaurant or in a processed/packaged food without adequate labeling). Thus, novel methods for prevention of food allergy are highly desirable. Currently available treatment methods for food allergies include: 1) use of epinephrine injections to save life in case of systemic anaphylaxis; 2) use of anti-histamines to control symptoms such as hives; and 3) corticosteroids to control inflammation (55). Several novel approaches to treat and prevent food allergies are under investigation. These include: 1) the reduction of IgE by the infusion of anti-IgE antibodies; 2) the use of probiotics; (3) the use of gene therapy; 4) use of cytokines such as IL-12; and 5) immunotherapy with mutated allergen derived proteins and peptides; and 6) herbal supplements (24-30, 32, 139-142). 30 Since the second part of my thesis work involved the testing of herbal supplements, I have briefly reviewed the potential of herbal supplements for application in food allergy in the next section. Herbal Supplements: Potential Utility in The Prevention and/or Therapy of Food Allergy A large body of evidence in the literature suggests that herbal supplements can modulate immune responses (Table. 1). Consequently, it is possible that some herbal supplements may be useful in the prevention and/or therapy of immune mediated disorders such as allergies and autoimmune disorders (143). Many studies demonsu'ate that several herbal supplements can modulate immune responses especially Th1/Th2 cytokine production in mouse models of diseases (Table 1). Consequently, there is potential for application of herbal supplements in immune mediate disorders characterized by Th1 or Th2 polarized cytokine responses. There is a growing interest in the potential utility of herbal supplements in the prevention and treatment of allergies and asthma (143). However, the potential utility of herbal supplements for food allergy has not been explored in-depth. There are few recent studies testing the potential of Chinese herbal supplements in a mouse model of peanut allergy (23, 29). Thus, researchers at the Mount Sinai school of medicine (New York), using a cholera-toxin based peanut 31 Table 1: Impact of herbal supplements on Thl/Th2 cytokine production in various experimental model systems Herbal Experimental Results Reference supplement model system Garlic/Alliin Leishmania Increase in IFN gamma (145) infected and decrease in IL—4 and BALB/c mice IL-10 in spleen cell cultures Echinacea Rat Increase in IFN gamma (146) macrophages Licorice/ Peritoneal Increase in IL-12 (147) Glycyrrhizin macrophages production Milk Murine mixed Increase in IFN gamma (148) thistle/Silymarin lymphocyte production culture Neem leaf Rat/ Oral Increase in IFN gamma (149) extract feeding production in lymph node cultures Rumex NC/Nga Decrease in IL-4 and no (142) japonicus mice/oral change in the IFN administration gamma levels in serum Ailanthus BALB/c Decrease in IL-4 and IL- (150) altissima mice/ Oral 13 mRNA expression in feeding lung tissue 32 allergy mouse model, have been testing the utility of Chinese herbal supplements in their model. They found that a Chinese herbal formula—that they named Food Allergy Herbal Formula (FAHF), was effective in completely blocking the peanut induced systemic anaphylaxis in their mouse model (23, 29). Thus, these studies using a peanut allergy mouse model, in principle, demonstrates the potential utility of herbal supplements in food allergy prevention and therapy. However, in addition to these few herbs, there are a large number of other herbal supplements available and their potential remains to be tested. Furthermore, effectiveness of herbal supplements against different subsets of food allergy (e. g., tree nut allergy etc) also remains to be tested. This was the focus of the second part of my thesis work. Mouse Models of Food Allergy: Valuable Tools to Elucidate Disease Mechanisms and to Develop New Preventive and Therapeutic Approaches A number of animal species have been used to develop food allergy models including rats, mice, dog and swine (150-152). However, mouse model of food allergy has been identified as a model of choice by regulatory agencies such as United States Environmental Protection Agency (US EPA) (153). This is because, mouse models offer several advantages including: 1) availability of inbred strains, large litter size and short generation time facilitate studies on the role of genetics in disease susceptibility; 2) availability of gene knockout mice 33 assist in elucidating the role of a specific gene in disease pathogenesis; 3) relatively lower cost; and 4) the availability of immunological reagents such as antibodies to study disease markers and mechanisms. A significant obstacle in the development of a mouse model of food allergy is the development of oral tolerance to ingested food antigens. Researchers have used two major strategies to overcome induction of oral tolerance: 1) use of adjuvant (e.g., oral adjuvant, cholera toxin; or systemic adjuvant, alum); and 2) the use of non-oral route (e. g., transdermal route) for sensitization without the use of any adjuvant. I have reviewed these two approaches in the next section below. Adiuvant-based Food Allergy Mouse Models A group at the Mount Sinai school of medicine developed a mouse model of cow’s milk and peanut allergy using cholera toxin as an adjuvant (154, 155). They found that C3H/Hej mice developed peanut and milk allergy only when orally exposed to peanut or milk protein along with cholera toxin but not peanut or milk protein alone (154, 155). They demonstrated that cholera toxin facilitated allergic response to peanut and milk by overcoming the innate oral tolerance. Subsequently, a number of other researchers have followed the same principle of using cholera toxin to develop mouse models for other food allergies such as 34 soybean allergy, buckwheat allergy, and shrimp allergy (156-158). Roy et al (1999) used a complex approach of using a combination of oral cholera toxin and intra-peritoneal injection with alum to develop a mouse model of peanut allergy (140). Thus, adjuvant based model of food allergy has been very popular among researchers. They are useful to understand food allergy development in the presence of adjuvant such as cholera toxin. However, it is very difficult to separate the effects of adjuvant from that of the food allergen in such models. Furthermore, there is a concern that the use of cholera toxin may enhance sensitivity of the model at the cost of specificity (l 59). That is, any protein— food allergen or not, administered with cholera toxin may lead to an allergic response. Consequently, it is reported that adjuvant based mouse models may not be useful to assess the allergenic potential of novel food such as genetically modified foods (159). An adjuvant-free Mouse Model of Food Allergy Using Hazelnut and Sesame as Model Allergenic Foods Although the adjuvant based models may be usefirl in studying mucosal immune mechanisms induced by dietary proteins in the context of adjuvant as a co-factor, their relevance to human disease is difficult to assess. Consequently, our laboratory has focused on developing an adjuvant-free method for food protein induced allergic and anaphylactic reactions. 35 The mechanisms of sensitization to dietary proteins in humans are not yet completely understood. Although the original route of exposure to food proteins leading to initial sensitization in humans is largely suspected to be oral, it is not clear whether all human food allergies results fiom oral route of sensitization. However, as noted earlier, in most animal models, oral exposure results in tolerance rather than sensitization (160-162). Since it is very common to see infants and children getting exposed to food proteins via skin (hands, face, neck, chest, etc) while eating, it is plausible that Langerhan’s cells present in the skin function as potent antigen presenting cells and initiate food allergen sensitization (163, 164). Investigating factors associated with the development of peanut allergy in childhood, Lack et al (2003) concluded that that “sensitization to peanut protein may occur in children through the application of peanut oil (containing small amounts of peanut protein) to inflamed skin” (165). Furthermore, there are reports of sensitization to food proteins via occupational exposure to food in food industries (166, 167). Consequently, we and others have proposed that some food allergies may actually develop following initial sensitization to food proteins via skin exposure (19, 22, 168). Based on the rationale presented above, previous workers in our laboratory tested whether transdermal exposure to dietary proteins (in the absence of 36 adjuvant) in mice leads to allergenic sensitization. They found that found that transdermal exposure to Lipo Poly Saccharide (LPS)-free allergenic food proteins (sesame and hazelnut) but not a non-allergenic food protein (vanilla), in the absence of adjuvant, elicits specific IgE antibody response in BALB/c mice (Fig. 4 and 5) (19, 22). To test whether transdermal exposure to food protein sensitizes mice for a clinical reaction, they examined clinical responses of sesame and hazelnut sensitized mice to oral food protein challenge. As evident, oral challenge with sesame and hazehiut elicited clinical symptoms of systemic anaphylaxis (Figs. 6 and 7). In addition, mice undergoing systemic anaphylaxis also exhibited a significant hypothermia (i.e., drop in rectal temperature) at 30 minutes following oral food challenge (Figs. 6 and 7) (19, 22). Using IgE-Westem blot and protein sequencing techniques, they studied sesame and hazelnut allergens in this model and found them to be identical to human allergens (19, 22). These data further add validity to the approach and the model that has been established to study food allergy in our laboratory. Taken together, our laboratory has established an adjuvant-flee transdermal allergic sensitization method for food proteins that yield three readouts highly consistent with human allergic sensitization: (1) food protein specific IgE antibody response (an immune marker); (2) systemic anaphylaxis to oral food allergen challenge (a clinical marker); and (3) hypothermia to oral food 37 , xxxxx Hazelnut (O ug/mouse) —9— 3%: 23% g flélmgflggi 77h")! Hazelnut (5 ug/mouse) + aze nut 8 g/mous Hazelnut (50 ug/mouse) " ‘7 aze "Ut 5 0 ug/mouse) Hazelnut (500 ug/mouse) E10000 A E 25 B '55 - a: .3 Z 3 ,3 a; 20 *" - 2 g 1000: 5‘ '15" ’ :8 15 3 100;- L3) 10 :2 = a 3 E C a 8 5 g '5. I 10E 76 5 l' 1 1 4 1 1 1 "5 0 pre 1R 2R 3R 4R 5R "' Pre 3R 6R Timepoint of analysis Timepoint of analysis Fig. 4: (A & B): Dose-responaqaad time-course analyses of IgE antibody resngse to hagglemazemut exposure in BAL_B/c mice. Groups of mice (n=4/ group) were transdennally exposed to indicated dose of hazelnut protein extract (0, 5, 50 or 500 ug/mouse). A. Specific IgE antibody levels were measured before (pre) and after 18’, 2nd, 3‘d’ 4‘“’ 5th and 6‘“.exposure (IR-6R). Data are shown as geometric means :1: SE. B. Total IgE levels were measured before (3 days before 1St exposure, Pre) and 3 days after 3rd (3R) and 6th (6R) exposure. Data shown as means i SE). ANOVA test: bars with different letters are significantly different (P<0.05) (Ref. No. (19)). 38 Food specific plasma antibody levels (OD 405-690nm) —1— pre —9— 2R + SR IgE, sesame E. sums lgE, Vanilla V nil (mm-om) (gauzmalss) (Guelmouui (W) _ t _ .. W ::H=1_.l L$=I=I=4 AAAAAA O O O O O O O O O O O s§§§e$ s§§§ei ssééei s§§§e§ ‘- s- 1- s- 1- ‘- s- s- s- 1— v- ‘- IeG1. 8am '951- 30““ 1961. Vanilla I961. Vanilla (omnnouu WW) (owmolm) (mammal 00 i w l (A) I 0) l 2— 2- 2. 2~ 1. 1— 1~ 1- MALIAI M- xxxxxxxxxx °sss a °§s 8 088 8 8 088 8 :esgég ze§§8g :e§e§g 2Q§§§g Plasma dilutions Fig. 5: Immune response to sesame seed protein following transdermal exposure in BALB/c mice Groups of mice (n=4 per group) were exposed to saline or sesame seed extract or control food (vanilla) extract by transdermal applications. Food specific IgE (upper panel) and IgG] (lower panel) antibodies were measured using pre-immune plasma and following five weeks of exposure. Data is shown from pre treatment, secondary response (2R) and fifth response (5R) (Ref. No. (22)). 39 Systemic anaphalyxis 5 4 A p<0.001 - —— W 3 _ 2 .- 1 .. 0 L Saline Hazelnut Saline Hazelnut Saline exposed mice Hazelnut sensitizede challenged with mice challenged with .v '1 0.‘ O O 0.. lVf ffi? so. so \‘C WV? 0... .0. 201.20! Rectal temperature ( °F) .0 see. v5‘3'gaaagag b flflfifififl% s ”3%gg5. édfififi OVVVVVV @fififi§fi’ o 0.900 fi§fififififl avvv’ap ‘\ '0'. 3%%%% §g§¢§ 900 so see. JW%%V%%F . D 0. l :d% ‘1” O O O O O O ‘ dot-2932.2- A . ‘0' .( 0. ..€. . .;. 0 O O O O. O . .9...‘ O O .0 O O O O O O s o. 3’ ..... .0 Q Q .0 0 o. ”a 2&9 so g; o . .?£ 0 a so 0000 // . ° iv“?% 4‘ 0 O O O o§q$ 0 egg &\ vvvv .6030 O O 0 so 0 O C so ’8’ see .0..... Sali_ng___HLeIrNt Salirle—Hazelnut Saline exposed 'mice Hazelnut sensitizede challenged With mice challenged with Fi . 6. Transdermal ex osure to hazelnut is sufficient to sensitize BALB/c mice for s stemic ana h laxis followin oral challen e with hazelnut. Groups of BALB/c mice (16 mice/group) were sensitized with hazelnut (500 pg/ mouse) or saline by transdermal exposure as described in Materials and Methods. After 6 cycles of exposure, IgE induction was confirmed and then orally challenged with hazelnut (13 mg/mouse) or saline and examined for indicators of systemic anaphylaxis. Data are shown as means :1: SE. A. Clinical symptoms are shown as scatter plot with each symbol representing 1 mouse. Kruskal-Wallis test results: hazelnut sensitized mice with hazelnut challenge, p<0.05 vs. all other groups. B. Changes in rectal temperature ANOVA test results: bars with different letters are significantly different (P<0.0l) (Ref. No. (19)). 40 .9 5 A X 1%: 4 - p<0.001 '8 0 g 3 - 1 Median cu O .2 2 r O E 3 1~ (I) Saline Sesame 9L: n.s. p<0.001 9 p<0.01 2. i! an a E 9 E ’8' 85 . 0‘ Sallne Sesame Pre 7////A 30 minutes after challenge fig. 7: Systemic anaphylaxis to sesame seed protein in transderrnally sensitized mice. Groups of BALB/c mice (n=4 per group) were sensitized with sesame seed protein (500 ug per mouse) or saline via transdermal exposure for 6 weeks and confirmed for IgE response. A. Clinical scores of systemic anaphylaxis. B. The profile of rectal temperature before and at 30 min following oral challenge with saline or sesame protein (13mg/ mouse). Differences were compared using Student’s t test (Ref. No. (22)). 41 challenge (a physiological marker). This model was used in the present study as discussed in the next section. Statement of the Problem As alluded earlier, specific mechanisms of food allergy are incompletely understood at present. Also, the potential of herbal supplements for treating or preventing food allergy has not been completely explored. The work reported in this thesis addressed these two specific problems using an adjuvant-free mouse model developed in our laboratory (19, 22). This work was conducted in two parts. In the first part, allergen-driven Type-2 cytokine responses were studied in hazelnut and sesame allergy mouse models. In the second part, impact of selected herbal supplements on allergen- driven Type-2 cytokine responses was studied. Specific hypotheses and objectives of this study were as follows: Park! Hypothesis and Objectives “Transdermal exposure to food allergens leads to activation of allergen- driven CD4 dependent, Type-2 cytokine responses in hazelnut and sesame allergy mouse models” 42 This hypothesis was tested with the following objectives: 1) to study allergen-driven recall Type-2 cytokine responses in hazelnut and sesame allergy mouse models; 2) to test the role of CD4 in Type-2 cytokine responses; 3) to test the role of H-2 complex in Type—2 responses using mouse strains with different H-2 genetics. Additional follow-up studies were conducted using Stat6 and 114 knockout mice to further characterize the Type-2 cytokine responses in this model. Part-II Hypothesis and Objectives “Selected herbal supplements will inhibit allergen-driven Type-2 cytokine responses by spleen cells from food allergic mice” This hypothesis was tested with the following objectives: 1) to study the impact of selected herbal dietary supplements on spleen cell viability; 2) to study the impact of above supplements on allergen-driven recall Type-2 cytokine responses by spleen cells from food allergic mice. 43 CHAPTER 1 CHARACTERIZATION OF TYPE-2 CYTOKINE RESPONSE TO ALLERGENS IN A MOUSE MODEL OF FOOD ALLERGY Abstract Backgxound Food allergy is a growing problem of serious proportions with potential for fatality. The specific role of Type-2 cytokines in food allergy is not completely understood. It is critical to understand such mechanisms as this may lead to better methods for prevention and treatment of food allergies. Our laboratory has developed an adjuvat-free mouse model to study food allergy. Hypothesis We hypothesized that, transdermal exposure to food allergens leads to activation of allergen-driven CD4-dependent, Type-2 cytokine responses in this mouse model. Approach and Results Initially, the allergen-driven recall Type-2 cytokine responses using spleen cells from hazelnut and sesame-sensitized BALB/c mice were studied. In both models robust allergen-driven, dose-dependent, recall IL-4, IL-5 and IL-13 responses were observed. Using anti-CD4 antibodies in the culture, it was found that, Type-2 cytokine response to allergens were largely CD4 dependent. To test the role of H-2 genetics in Type-2 response, mice strains 44 with different H-2 locus (BALB/c, H-2d; ASW, H-2’, and SJ L, H-2’) were used. Whereas BALB/c and ASW exhibited significant allergen-driven IL-4 and IL- 13 responses, SJ L did not. However, all three strains produced significant IL-S responses. Furthermore, follow-up studies using Stat6 and [14 gene knockout mice provided evidence that allergic and anaphylactic responses in this model are dependent on IL—4 but not IL-5 or IL-13. Conclusion These data argue that transdermal exposure to food allergen tested results in activation of IL-4, IL-5 and IL-13 responses by positive spleen cells. However, only IL-4 response, that is STAT6 dependent, is required for allergic response and systemic anaphylaxis. Introduction Specific role of Type-2 cytokines in food allergy is not completely understood. It is critical to understand such mechanisms as this may lead to better methods for prevention and treatment of food allergies. Mossman et a1 (1986) originally divided and named the mouse T helper cells into Th1 and Th2 based upon cytokines they secrete (79). Thus, Th1 cells in general secrete IL—2, Interferon gamma (IFN v) and Tumor Necrosis Factor alpha (TNF 0), whereas Th2 cells secrete mainly IL-4, IL-5 and IL-13. Later studies identified that some of these cytokines can also be produced by non-T 45 cells that lead to classification of cytokines as Type-l and Type-2 instead of Th1 and Th2 respectively. Type-1 cytokines have main role in delayed type of hypersensitivity reactions (DTH) and class switch factor for IgG2a antibodies in mice, where as Type-2 cytokines IL-4, IL-5 and H.-13 have role in immediate hypersensitive reactions. Importance of these Type-2 cytokines in allergy relevant phenotypes is evident from various gene knock out studies. Several researchers showed the importance of Type-2 cytokines such as IL-4, IL-5 and IL-13 in allergic diseases. These cytokines play many different roles and participate in allergic reactions as summarized in review of the literature section. Because of their crucial role in IgE class switching IL-4 and IL-13 have gained special significance. To understand the patho- physiology of any disease good animal models are desirable. Several efforts were made in the past few years for developing animal models which allow the researchers to study the disease mechanism and also predict the allergencity of new compounds. These include using dogs, pigs, and mice. While each model offers various advantages it also suffers from several limitations. The hypothesis that transdermal exposure to food allergens leads to activation of systemic allergen-driven CD4-dependent, Type-2 cytokine responses was tested in the mouse model. There were three specific aims: (1) To study allergen-driven recall Type-2 cytokine response in hazelnut and 46 sesame allergy mouse models; (2) To test the role of CD4 in Type-2 cytokine response; and (3) To test the role of H-2 complex in Type—2 response using mice strains with different H-2 genetics. Additional follow-up studies were conducted using Stat6 and 114 knockout mice (KO) to further characterize the Type-2 cytokine response in this model Material and. Methods Hazelnut protein extract, Sesame seed extract (Greer Labs, Lenoir, NC, USA); Protein content of these three protein extracts was measured by Lowry’s method (170). Normal saline was prepared in our lab (0.85 % W/V NaCl solution); Bovine serum albumin, L-glutamine, Penicillin, Streptomycin and neomycin liquid, Inomycin, Phorbol myristate acetate (PMA), p—nitro- phenyl phosphate and MTT assay kit (Sigma, St Louis, MO, USA); 2 Mercapto-ethanol (Gibco BRL Grand Island NY USA); Anti-CD4 antibody, Isotype control antibody and Paired antibodies, recombinant standards for mouse IL-4 and IL-5 (BD PharMingen, San Diego, CA, USA); Paired antibodies and recombinant standards for mouse IL-13 (R&D Minneapolis MN); Streptavidin alkaline phosphatase (Jackson ImmunoResearch, West Grove, PA); 96-well cell culture plates (FALCON, Franklin Lake NJ USA); 96-well ELISA plates (Costar, Corning NY USA). Reagents prepared for ELISA werelOX PBS (NaHzPO4.H20, NazHPO4, NaCl), Wash buffer (10XPBS, Tween 20, 2%NaN3), Dilution buffer (10XPBS, Tween 20, 2%NaN3, BSA), Blocking buffer (10XPBS, 2%NaN3, BSA), 5% gelatin 47 blocking buffer (Gelatin), Coating buffer (NazCO3, NaHCO3, 2%NaN3, BSA), Substrate buffer (2M MgClz.6HzO, Di ethnolamine, pH- 9.8) All mice (Table 2) were purchased from Jackson Laboratories (Bar Harbor, Maine, USA). All animals were females and used at 7 weeks of age. All procedures involving mice were in accordance with Michigan State University animal use policies. Mice were acclimated for one week to their new environment before starting the experiment. Table 2: Different strains of mice used in the study Mice strain MHC Age at arrival Age at the start Background (In weeks) (In weeks) BALB/c H-2‘I 5 7 BALB/c Stat6 KO H-26 5 7 BALB/c 114 KO H-zd 5 7 ASW H-2‘ 5 7 SJL H-25 5 7 Transdermal Sensitization Transdermal exposure experiments were performed using the method described previously (22). Groups of mice were exposed to saline, hazelnut protein or sesame protein extract (500 ug/l 00 ul in saline per mouse per application); each mouse had the reagent applied to the skin of the back that had the hair clipped-off and covered with non-latex bandage for 1 day. Mice were kept idle for 5 days. Then the cycle of exposure to saline or Food allergen was continued for a total of six times (Fig. 8). 48 Figure 8: Transdermal sensitization protocol Treatment cycle Day 49 F A W e—_ Mice Age Pre- Day 51 Day 52 Day 58 Repeat the bleed/ Transdermal Transdermal Transdermal treatment cycle clip hair Patch on (18‘) Patch off (13‘) Patch on (2“) 6 times 1 1 l [I l 2 days 5 day!’ I rest rest Animal sacrifice and spleen cell cultures Spleen Cell Culture Spleen cells were harvested and standard cell cultures were set up essentially as described. Briefly, pooled spleen cells from either saline-exposed mice or food allergen sensitized mice were cultured (7.5X106 cells/ml) in duplicates in the absence and presence of food allergen (100 and 500 ug/ml). Cells cultured in presence of culture medium alone served as a negative control whereas cells cultured with PMA (1 ug/ml) plus ionomycin (1 pg/ml) served as .a positive control. Cells that were cultured for 3 days, the duplicate cultures were removed from the 37°C incubator at the end of day 3 and pooled in appendorf tube afier through mixing, whereas cells that were cultured for 5 days the duplicate cultures were pooled at the end of 5 days. Later the supematants were separated and stored separately from the mixtures by spinning the tubes at 2000 rpm for 5 minutes. 49 In the experiments where role of CD4 molecule was tested the pooled splenocytes from both hazelnut and sesame-sensitized mice were cultured in duplicates in the presence of 500 ug of food allergen, with or without Anti-CD4 antibody (20 ug/ml) and Isotype control antibody (20 ug/ml). Kinetics of cytokine production was studied by culturing the cells for 3, 4 and 5 days. Cell culture supematants were collected in similar fashion described above. Composition of culture medium: Fetal bovine sermn -10%, Antibiotic - 01%, L-Glutanine- 1%, Beta mercapto ethanol- 0.1% make the volume to 100% with RPMI 1640. Induction of Systemic Anaphylaxis, Clinical Symptoms Scoringfla Measurement of Rectal Tempefliture Ten days after the last transdermal application mice were orally gavaged with either hazelnut for hazelnut-sensitized and saline-exposed mice (15 mg/mouse; 0.5 ml volume) sesame for sesame-sensitized mice (13 mg/mouse; 0.5 ml volume) and saline for saline exposed and hazelnut-sensitized mice (0.5 ml/mouse) and observed for clinical signs of systemic anaphylaxis during the next 60 minutes by 2 individuals in a blinded manner according to the method described previously (156) (Table 3). Rectal temperature was measured by using temperature probe (Yellow Springs Instrument, Yellow Springs, Ohio, USA) before and 30 minutes after oral challenge. 50 Table 3: Systemic anaphylaxis symptom score Score Symptoms 0 No symptoms 1 Scratching and rubbing around nose and head 2 Puffiness around the eyes and mouth, pilar erecti, decreased activity with increased respiration 3 Wheezing, labored respiration, cyanosis around mouth and tail 4 Slight or no activity after prodding, tremor and convulsion 5 Death ELISA Method of Cytolgne Estimation All cytokine measurements were done using sandwich ELISA method. The sandwich ELISA protocol is a 3 day protocol starting with coating the 96- well plate with 50 ul/well of antibody against the protein of interest in coating buffer on day one. After 12-16 hours of keeping the coated plate in the refrigerator, blocked with blocking buffer (75ul/well) and kept them in the 37°C incubator. After 3 hours of incubation wash the plates thrice with wash buffer and add samples and standard in duplicates with a final volume of 50 ul/well. After adding the samples keep the plate in the refrigerator (+4°C) over night. On day 3 remove the plate from refiigerator and add 50 ul/well biotin labeled secondary antibody against protein of interest after wash the plate three times. Incubate the plate for 90 minutes in 37°C incubator. After 90 minutes remove the plate from the incubator and add 50 ul/well enzyme (Streptavidin conjugated alkaline phosphatase) after washing the plate for three times. Incubate the plate for 30 minutes in 37°C incubator before washing and adding 51 50 ul/well pnp substrate. After adding the substrate read the absorbance at 405- 690 nm. Statistical Analysis AN OVA and Student’s t test were used to evaluate significance using GraphPad Instat TM software program (GraphPad software, San Diego, USA). The statistical significance level was set at 0.05. Results T_ransdermal Exposure to Hazelnut and Sesame Proteins Results in Activation of Type-2 kaine Respon_ses in BALB/c Mice: Groups of BALB/c mice were exposed to either saline or hazelnut protein extract transderrnally for 6 times as mentioned in materials and methods section. Splenocytes from saline-exposed mice and hazelnut-sensitized mice were cultured in the presence or absence of hazelnut protein for 5 days. Supematants from the cultures were collected on day 3 and day 5. There was significant production of IL-4, IL-5 as well as IL-1 3 in hazelnut-sensitized mice when compared with saline-exposed mice (Fig. 9). Within the hazelnut- exposed group there was significant difference (p<0.05) between culture medium alone and those that were cultured with hazelnut protein extract (Fig. 9). 52 lL-4(pg/ml) 8 8 8 8 O O O O O 9000 IL-5 (pg/ml) 8 8 o 8 8 N O O O 1000 IL-13 (pg/ml) Fig. 9: Hazelnut-driven IL-4. IL—5 and IL-13 responses of hazelnut- sensitized mice vs saline exposed mice Groups of BALB/c (n=5 to 6 per group) were sensitized with hazelnut protein (500 ug/mouse) via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured with the indicated dose of hazelnut protein. Cell culture supematants were collected on days 3 and 5; samples were analyzed for IL-4 (top panel), IL-5 (middle panel) and IL-13 (bottom panel) using ELISA. Data shown are average +/- SE of duplicate analyses from hazelnut-sensitized mice (n=6) (A) and saline-exposed mice (n=5) (B). All the differences were compared using ANOVA and the statistical - OHN W 100 HN 7////2 500 HN 400 **, 300 - 200 — 100 — 0 d5 9000 6000 7 3000 ‘ d5 2000 rue: 1000 " d3 d5 significances are expressed as * P<0.05 ; ** P<0.01 ; *** P<0.001 53 Groups of mice were evaluated for antigen driven memory Type-2 cytokine responses to sesame proteins in sesame-sensitized vs saline-exposed mice. Protocol for this experiment was similar to the one used for hazelnut proteins. The only difference was sesame protein extract was used in place of hazelnut protein extract. Sesame allergic mice but not control mice exhibited sesame-driven recall Type-2 cytokine production (Fig. 10). Food Allergen-driven Type-2 Cytokine Responses in this Mouse Model is CD4-Dependent Splenocytes were cultured as described above along with anti-CD4 antibody or an isotype-matched control antibody. Hazelnut and sesame proteins were used at 500 11ng and with and without anti-CD4 antibody and a control antibody. Supematants were collected on days 3, 4 and 5 and then stored at - 80°C till they were analyzed for cytokines IL—4, IL—5 and IL-13 levels by ELISA. The production of IL-4 (Fig. 11), IL-5 (Fig. 12) and IL-13 (Fig. 13) were significantly inhibited by the anti- CD4 antibody in both hazelnut and sesame allergy mouse models. Type-2 Cytokkine Respon_ses to Hazelnut Allergens Mouse with Different Maior histocompatibility (H-2) Genetic Locus The protocol used for transdermal sensitization followed by spleen cell culture was identical to those described in previous experiments. There was a significant difference between all three different strains in the production of 54 IL-4 (pg/ml) lL-5 (ng/ml) - Oses W 100 ses m 500 ses 400 400 300 L B 200 - 100 - 0 d3 d5 20 B 10 7 0 d3 d5 4000 3000 7 B 2000 ' 1000 r O -‘- —m____ d3 d5 Fig. 10: Sesame-driven IL-4. IL-5 and IL-13 respon_ses of sesame- sensitized mice vs saline exposed mice Groups of BALB/c (n=7 to 8 per group) were sensitized with sesame protein extract (500 ug/mouse) via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured with the indicated dose of sesame protein extract. Cell culture supematants were collected on days 3 and 5; samples were analyzed for H—4 (top panel), IL-S (middle panel) and IL-13 (bottom panel) using ELISA. Data shown are average +/- SE of duplicate analyses from sesame -sensitized mice (n=8) (A) and saline-exposed mice (n=7) (B). All the differences were compared using ANOVA and the statistical significances are expressed as * P<0.05 ; ** P<0.01 ; *** P<0.001 55 Food 0dallegehuanti CD4 Ab Food allergen+lsotype control Ab 1 0 v/uun Days of culture t 0.) 01 lL—4 concentration (pg/ml) Days of culture Fi . 11: Hazelnut and sesame-driven IL-4 levels are CD4 d endent Groups of BALB/c mice were sensitized with sesame seed extract (A) and hazelnut protein extract (B) (500 ug/mouse) via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured with indicated dose of sesame, hazelnut proteins or isotype control antibody and anti-CD4 antibodies. Cell culture supematants were collected on days 3, 4 and 5 and analyzed for IL-4 using ELISA. Data shown are average +/- SE of duplicate analysis from sesame—sensitized mice (n=8) and hazelnut-sensitized mice (n=6). The differences between different groups on same day of collection were compared using ANOVA. Bars labeled with different letters are significantly different (p<0.05) 56 y Food allergen ///////% Food allergen+anti CD4 Ab Food allergen+lsotype control All lL-5 concentration (pg/mi) 1 00 Days of culture 1 00 A B 1 0 A A A A ‘ 1 _w: B B 0.1 3 4 Days of culture flg._l_2_: HazelnuL and me-driven IL-5 levels are CD4 dependent Groups of BALB/c mice were sensitized with sesame seed extract (A) and hazelnut protein extract (B) (500 ug/mouse) via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured with indicated dose of sesame, hazelnut proteins or isotype control antibody and anti-CD4 antibodies. Cell culture supematants were collected on days 3, 4 and 5 and analyzed for IL-5 using ELISA. Data shown are average +/- SE of duplicate analysis fi'om sesame-sensitized mice (n=8) and hazelnut-sensitized mice (n=6). The differences between different groups on same day of collection were compared using ANOVA. Bars labeled with different letters are significantly different (p<0.05) 57 7 Food allergen //////////, Food allergen+anti CD4 Ab - Food allergen+lsotype control All 10000 A A ,K 1000 E 100 g 10 c 1 a 0.1 4 '5' Days of culture 8 510000 0 (O 1000 3 100 - 10 1 0.1 4 Days of culture fig. 13: Hazelnut apd sesame-driven IL-13 levelsJare CD4 dependent Groups of BALB/c mice were sensitized with sesame seed extract (A) and hazelnut protein extract (B) (500 ug/mouse) via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured with indicated dose of sesame, hazelnut proteins or isotype control antibody and anti-CD4 antibodies. Cell culture supematants were collected on days 3, 4 and 5 and analyzed for IL-13 using ELISA. Data shown are average +/- SE of duplicate analysis from sesame-sensitized mice (n=8) and hazelnut-sensitized mice (n=6). The differences between different groups on same day of collection were compared using ANOVA. Bars labeled with different letters are significantly different (p<0.05) 58 IL-4, IL-5 and IL-13. In the case of IL-4 production, there was a significant difference between saline group and hazelnut group in BALB/c and ASW strains but that difference was not present in SJ L mice implying that IL-4 production has genetic factor in its etiology (Fig. 14). In the case of IL-5 production there was no significant difference between BALB/c and SJ L strains where as ASW strain produced significantly higher amount of IL-5 when compared with the other 2 strains (Fig. 15). In the case of IL-13 production in SJ L mice there was no significant allergen-driven production and it was not significantly different from ASW mice, where as BALB/c produced significantly higher amount of IL-13 when compared to ASW and SJ L strains (Fig. 16). IL-4 but not IL-5/ IL-13 Responses to Hazelnut Allergen is STAT 6 Dependent in our Mouse Model The protocol used for transdermal sensitization, followed by spleen cell culture was identical to what has been described earlier. There was a 90% reduction in the production of IL-4 in Stat6 Knock out mice was observed, where as that reduction was only 40% in the case of IL-5 and 15% in the case of IL-l3. Interestingly the difference in IL-13 production was not significantly different between wild type and knock out mice. Thus, only hazelnut-driven IL- 4 but not IL-5/ IL-13 responses was found to be largely STAT6 dependent in this model (Fig. 17). 59 300 9200 E. C) e a too A A A A CM CM+HN CM CM-i-HN CM CM+HN CM CM+HN CM CM+HN CM CM-i-HN Saline Hazelnut Saline Hazelnut Saline Hazelnut (n=5) (n=10) (n=6) (n=6) (n=6) (n=6) BALB/c ASW SJL H-2“ H-25 H-28 Fig. 14: Hazelnut-driven IL-4 respon_se from three different mouse strain_s; BALE/cg ASW and SJL Groups of BALB/c, ASW and SJL mice (n=5-10 per group) were sensitized with hazelnut protein extracts (500 ug/mouse) via transdermal exposure for 6 times. One week following the last exposure, mice were intraperitoneally challenged with hazelnut protein and 1 hour later spleen cells were harvested and cultured with 0, 100 or 500 ug/ml dose of hazelnut proteins. Cell culture supematants were collected on days 1, 3 and 5. Samples were analyzed for IL-4 using optimized ELISA. Data shown is average +/- SE of duplicate analysis from peak response. All differences were compared using ANOVA. Bars labeled with different letters are significantly different (p<0.05) 60 20 iL-5 (ng/ml) 8 61‘ 0'1 A A CM CM+HN CM CM+HN CM CM+HN CM CM+HN CM CM+HN CM CM+HN Saline Hazelnut Saline Hazelnut Saline Hazelnut (n=5) (n=10) (n=6) (n=6) (n=6) (n=6) BALB/c ASW SJL H-2d H-2‘ H-28 Fig. 15: Hazelnut-driven IL-5 respopse from three different mouse straig; BAL_B/c. ASW and SJL Groups of BALB/c, ASW and SJL mice (n=5-10 per group) were sensitized with hazelnut protein extracts (500 ug/mouse) via transdermal exposure for 6 times. One week following the last exposure, mice were intraperitoneally challenged with hazelnut protein and 1 hour later spleen cells were harvested and cultured with 0, 100 or 500 ug/ml dose of hazelnut proteins. Cell culture supematants were collected on days 1, 3 and 5. Samples were analyzed for IL-5 using optimized ELISA. Data shown is average +/- SE of duplicate analysis from peak response. All differences were compared using ANOVA. Bars labeled with different letters are significantly different (p<0.05) 61 4000 A CM CM+HN CM CM+HN CM CM+HN CM CM+HN CM CM+HN CM CM+HN Saline Hazelnut Saline Hazelnut Saline Hazelnut (n=5) (mm (n=6) (n=6) (n=6) (n=6) BALB/C ASW SJL H-2" H-28 H-25 Fig. 16: Hazelnut-driven IL-13 respopsye from three different moug strain_s_; BAL_B/c. ASW and SJL Groups of BALB/c, ASW and SJL mice (n=5-10 per group) were sensitized with hazelnut protein extracts (500 ug/mouse) via transdermal exposure for 6 times. One week following the last exposure, mice were intraperitoneally challenged with hazelnut protein and 1 hour later spleen cells were harvested and cultured with 0, 100 or 500 ug/ml dose of hazelnut proteins. Cell culture supematants were collected on days 1, 3 and 5. Samples were analyzed for IL-13 using optimized ELISA. Data shown is average +/- SE of duplicate analysis from peak response. All differences were compared using ANOVA. Bars labeled with different letters are significantly different (p<0.05) 62 IL-4 (pg/ml) - BALB/c m BALB/c stat6 KO 400 A 10 B 2000 C 9 _ . 8 i— 300 7 .- 7. . c 6 _ 511:7;1‘7. c E E B: ...... 5 200 5 5 - *-;;;:1;‘ 31000 "a :2 _l I "' =‘ 100 CM CM+HN Fig. 17: Hazelnut-driven IL-4, IL-5 and IL-13 respon_se in Stat6 knockputand wild type animals Groups of BALB/c (n=6 per group), BALB/c stat6 KO (n=4 to 6 per group) mice were sensitized with hazelnut protein extracts (500 ug/mouse) via transdermal exposure for 6 times. One week following the last exposure mice were intraperitoneally challenged with hazelnut and one hour later spleen cells were harvested and cultured with two doses of hazelnut proteins for three different days. Cell culture supematants which collected on days 1, 3 and 5 were analyzed for levels of IL-4 (A), IL-5 (B) and IL-13 (C) using ELISA. Data shown are average +/- SE of duplicate analysis of peak response. All the differences between wild type and knockout mice were compared by ANOVA. Bars labeled with different letters are significantly different (p<0.05) 63 Allergic Respm and Systemic Anaphvlagis to Hazelnut is IL-4 Dependent in this Mouse Model The protocol used for transdermal sensitization followed by spleen cell culture was identical to what has been described earlier. 114 KO mice failed to exhibit significant allergic (IgE) response to transdermal hazelnut exposure (Fig. 18) or systemic anaphylaxis following oral challenge with hazelnut (Fig. 19). These data suggest that allergic and anaphylactic response to hazelnut in this model is dependent on IL-4. Discussion This study was undertaken with the aim of characterizing the Type-2 cytokine responses in an adjuvant-free mouse model that our laboratory has developed. There are five novel and important findings from this study: (1) transdermal exposure to hazelnut and sesame allergens was sufficient to activate recall Type-2 cytokine responses by spleen cells; (2) recall allergen- driven Type-2 cytokine responses was largely CD4-dependent in both hazelnut and sesame allergy models; (3) Type—2 cytokine responses to hazelnut was H-2s independent; (4) hazelnut allergen-driven IL-4 but not IL-5/IL-l3 is STAT6 dependent; and (5) IL-4 is required for allergic and systemic anaphylaxis to hazelnut in this mouse model. Initially this study looked at the Type-2 cytokine responses using two different food allergens to determine if there will be any major differences 64 + Pre —0- 3R —V— 6R 3 A 3 A B E c... L— 02" 2 O) “P “o’ “-1- 1'- 8 v _0 0% n) 0000000 0000000 WVQCON 0 NVQ‘ON 0 Essessgg sessgéxg a h K <3 3 LLI 2» C D (I) 52F 2- E a) «'B' I1“ 1' 0%! 0W N V m to N no N v- 00 <0 N CD \ \ \ v~ to (EN \ \ \ v~ to (EN Plasma dilution Fig. 18: Hazelnut-specific IgE response in BALB/c wild type anima_l_s am 114 knockout animals Groups of mice (n=6 per group) were sensitized with saline (B&D) (100 ul) or hazelnut protein extract (A&C) (500 ug/mouse in 100 ul) via transdermal exposure for 6 times. Plasma samples were collected before transdermal application (Pre), after 3 applications (BR) and after 6 applications (6R). Hazelnut- specific IgE levels were evaluated using ELISA. Data shown are hazelnut-specific IgE values (OD 450-690) average +/- SE of duplicate analysis from wild type BALB/c mice (A&B) in comparison to 114 Knockout mice (C&D) 65 5 p<0.0001 4 ' 0‘. .3 3 r O 2 — A .13 u. >><. 1- 1, a A a, .c a) AAA].- L L l I— 8' g- 0 Saline Hazelnut *3 Ha(::lg)ut g _ (n=5) (n-6) 5 o 8 O. -- s E E <2 5 2 ‘63 E > C .5 (I) 4 - SE 3 - n.s O 2 .— 1 .— 0 3 3l 3 3 3 3 1 ‘3‘: Saline l-i'azefilut H (n=6) (n=5) (n=4) (n=5) Fi . 19: S stemic ana h laxis and rectal tern erature before and afier oral challen c with hazelnut rotein in wild t e and [[4 knock out mice BALB/c-Wild type (A&B)(n=5-6 per group) and BALB/c-Il4 Knockout (C&D) (n=4-5 per group) were sensitized with saline and hazelnut (500 ug/mouse) via transdermal exposure for 6 times. One week following the last exposure hazelnut-sensitized mice were orally challenged with hazelnut (15 mg/mouse in 500 ul) and saline-exposed mice were challenged with saline (500 ul). Rectal temperatures were recorded before and 30 minutes after oral challenge by using rectal probe (B&D). Data shown are average +/- SE. Differences were compared using ANOVA in B&D and Student’s t test in A&C. Bars labeled with different letters are significantly different (p<0.05) 66 between food allergens in the way they activate the immune system. However, the data suggest that, although hazelnut and sesame belong to two different food groups (tree nuts and seeds respectively), they activate the Type-2 cytokine responses in a similar fashion. We are not aware of previous studies examining hazelnut or sesame allergen-driven cytokine responses in mice or other animals or in humans. There are previous studies examining Type-2 cytokines in mouse models of other allergens such as ovalbumin and peanut (23, 151, 152, 157, 158, 168, 170-172). These models either used tape-stripping skin exposure method or cholera toxin to elicit of allergen sensitization. Nevertheless, they reported that ovalbumin and peanut activated IL-4 and IL-4, IL-5 and IL-13 cytokine responses by spleen cells respectively. However, [14 or Stat6 gene knockout studies were not conducted. There are several cell types that were present in spleen cell suspension such as basophils, eosinophils, NK cells and mast cells that are able to produce Type-2 cytokines. Therefore, the role of CD4 positive cells was tested in this model. Type—2 cytokine responses were found to be largely CD4-dependent. We are not aware of previous food allergy model studies characterizing Type-2 cytokines in this manner (15, 150-152, 157, 158, 168, 170-175). 67 In order to examine the role of host genetics in Type-2 cytokine responses to hazelnut allergens, the immune response in mouse strains with different MHC genetics was studied. The classical major histocompatibility complex (MHC) molecules have a unique function in the complex immunological dialogue that must occur between T cells and other cells of the body. T cells can able to recognize only those antigens that were presented along with MHC molecules. Researchers showed that MHC molecules has critical role in antigen presentation and cytokine production in different systems. H—2 is a complex of genetic loci on chromosome 17 that define the major histocompatibility complex (MHC) of the mouse (67). H—2 is homologous to HLA in man (68). The H—2 complex consists of several major genes whose products are important in immune system, auto immunity and grafi rejection (70). As evident, in contrast to SJ L, only BALB/c and ASW exhibited significant allergen-driven IL-4 and IL-13 responses. However, all three strains produced significant IL—5 responses. These data argue that Type-2 cytokine responses to hazelnut allergens is dependent on mice strain and are H- 25 independent. The reason why SJ L did not produce robust IL-4 response is not known at this point although previous reports demonstrate that SJ L lack NK 1.1 cells that are required for robust IL-4 (176). Furthermore, SJ L mice are also unable to produce IgE response to allergens(177). The difference between ASW and BALB/c might be due to generally more Th2 response by the BALB/c mice 68 compared to the ASW. However, both produce robust IL-4 and allergic response. Since previous studies in allergy mouse models and in humans implicated a critical role for STAT6 in Type-2 cytokine responses, this study examined Stat6 gene knockout mice (91, 125, 127, 130, 178). Hazelnut-driven IL-4 but not IL-5 and IL-13 responses was almost completely abrogated in Stat6 KO mice. Furthermore, these mice were unable to develop allergic (IgE) response to transdermal hazelnut exposure or systemic anaphylaxis to i.p. challenge with hazelnut (179). These data together implicated STAT6 is a critical player in this mouse model. It also suggested that IL—4 may be critical in this model that led to the follow-up study using [14 KO mice. To directly test the role of IL-4 in our model, allergic response to transdermal hazelnut exposure and systemic anaphylaxis following oral hazelnut allergen challenge was studied. As evident, [14 KO mice were resistant to both allergic response as well as systemic anaphylaxis arguing the critical role of this prototypic Type-2 cytokine in our adjuvant-free mouse model of food allergy. Taking all these data into consideration, a working model was developed to explain how transdermal exposure to food allergens might shape the immune response leading to food allergy disease in this model (Fig. 20). 69 I Food allergen ] i Skin 1 CD4’ T Cells a Stat6 independent I Stat6 dependent ———> lL-4 lL-5, lL-13 H-2‘ independent IgE —) Systemic anaphylaxis M0: Working model of Type-2 cytokine responses in our adiuvan_t_- free mouse model of transdermal sensitization Exposure to food allergens via skin leads to activation of Type-2 cytokine (IL-4, IL-5 and IL-13) responses by CD4 positive spleen cells. However, in contrast to IL—5 and IL-13, only IL-4 response is STAT6 dependent. Furthermore, both IL—4 and STAT6 are required for allergic response and systemic anaphylaxis in this model. 70 According to this model after expose the animal through skin, allergen is activating the CD4 positive T cells. Afler activation CD4 positive T cells producing allergen-driven IL-4, IL-5 and IL-13 cytokines. Out of these three cytokines IL-4 production is dependent on STAT6 and IL-4 is crucial cytokine for IgE production and systemic anaphylaxis. 71 CHAPTER 2 THE EFFECT OF SELECTED HERBAL SUPPLEMENTS ON ALLERGEN- DRIVEN TYPE-2 CYTOKINE RESPONSE BY SPLEEN CELLS FROM FOOD ALLERGIC MICE Abstract Background Food allergies afflict nearly 4% adults and 6-8% children in most developed countries including USA. Despite the potential fatal outcome, no specific therapies except symptomatic treatment are available for food allergy. Currently avoiding the food is the only sure way to prevent food allergy. Therefore, improved methods are needed for prevention and/or treatment of food allergies. Using a food allergy mouse model, here attempts were made to identify potential anti-allergy herbal supplements using a novel approach. Hypothesis and approach Here the hypothesis that selected herbal supplements will inhibit allergen- driven Type-2 cytokine response by spleen cells from food allergic mice was tested. There were two specific objectives for this study: (1) To study impact of selected herbal dietary supplements on spleen cell viability; (2) To study the impact of above supplements on allergen-driven recall Type-2 cytokine response using spleen cells fi'om food allergic mice. 72 m Except neem leaf extract, none of the herbal supplements studies exhibited cytotoxic effect at dilutions of 1/1000 or 1/5000 on spleen cells from hazelnut allergic mice. Among the herbal supplements tested only licorice consistently inhibited allergen-driven IL-4, IL-5 and IL-13 responses by spleen cells fi'om hazelnut and sesame allergic mice. Furthermore, licorice from two additional sources also exhibited inhibition of all three Type-2 cytokine responses in both models. Conclusion These studies demonstrate a novel approach for identifying herbal supplements and phyto chemicals that inhibit allergen-driven recall Type-2 cytokine responses in mice. Furthermore, these data suggest the need for future studies to test the potential of licorice to inhibit Type-2 cytokine responses in vivo and its impact on allergic (IgE) and anaphylactic responses in this mouse model. Introduction Food allergies afflict nearly 4% adults and 6-8% children in most developed countries including USA. Despite the potential fatal outcome, no therapies except symptomatic treatment are available for food allergy. Currently avoiding the food is the only sure way to prevent food allergy. But 73 the major problem in food avoidance is the hidden nature of food allergens in several processed foods. Researchers are trying to find newer methods for treatment or prevention of food allergies. Recent clinical trail showed increase in the threshold amount of peanut required to induce systemic anaphylaxis after monthly injections of humanized recombinant anti-IgE antibodies, however taking monthly injections is laborious, time consuming, expensive and not fool- proof .Therefore there is need to develop alternative therapeutic strategies (e.g. use of herbal supplements). There is growing interest in the potential utility of herbal dietary supplements in the prevention and treatment of several human disorders including allergies and asthma. Many studies demonstrate that several herbal supplements can modulate immune response especially Thl/Th2 cytokine production. However, the potential of individual herbal supplements in modulating food allergen-driven Type-2 cytokine response has not been adequately studied. This part of the study examined the possibility of identifying herbal supplements that might be potentially useful for food allergy. The hypothesis that selected herbal supplements will inhibit allergen-driven Type-2 cytokine response by spleen cells from food allergic mice was tested. There were two 74 specific objectives for this study: (1) To study impact of selected herbal dietary supplements on spleen cell viability; (2) To study the impact of above supplements on allergen-driven recall Type—2 cytokine response using spleen cells from food allergic mice. Material and methods Herbal supplements and their sources were as listed in table 4. This study used both whole extracts of herbal supplements and active ingredients. The whole extracts used in this study were from Echinacea, Garlic, Licorice (three different sources), and Milk Thistle. All these supplements were supplied in capsule form and they were added to the culture by making extract from these supplements. The extracts were made by mixing each capsule in 2.5 m1 of culture media and after through mixing the whole mixture was centrifuged at 2500 rpm for 10 minutes. Supernatant was collected and filter sterilized before use it in the culture as stock solution. This supernatant was used in the culture at 1 in 1000 and 1 in 5000 dilutions. The active ingredients were dissolved as per the instructions given by the manufacturers (Table 4). All the stock solutions were made at 10 mg/ml concentration and used in the culture at l in 1000 (10 ug/ml) and 1 in 5000 (2 ug/ml) dilutions. Neem leaf extract and Licorice from Herbs Pharrna which were provided by the manufacturer as liquid preparation and they were considered as stock solutions and used in the culture after filter sterilization at 1 in 1000 and 1 in 5000 dilutions. 75 Spleen cell culture Spleen cells were harvested and standard cell cultures were set up essentially as described. Briefly the pooled spleen cells from food allergen sensitized mice were cultured in quadruplicates in the presence of 100 ug of food allergen, with and without herbal supplements at two different dilutions that is at l in 1000 and 1 in 5000. Cell culture supematants were harvested as described above at the end of 4 days. Cell culture supematants from all the cell culture experiments were stored at -70°C till they were analyzed for levels of different cytokines. Table 4: Herbal supplements used in this study Herbal Extract Extract Stock Source supplement prepared made concentartion with At Echinacea Culture 160 mg/ml Supernatant solaray®, Medium Foods for Living store, East Lansing, MI, USA Garlic Culture 128 mg/ml Supernatant solaray®, Medium Foods for Living store, East Lansing, MI, USA Genistein Ethyl 10 mg/ml 10 mg/ml Sigma, St alcohol Louis, MO, USA 76 Herbal supplement Milk Thistle Alliin Glycyrrhizin Licorice Licorice Neem Leaf Extract Silymarin Extract prepared with Culture Medium Water Aluminum Hydroxide Culture Medium Culture Medium DMSO Extract made At 240 mg/ml 10 mg/ml 10 mg/ml 180 mg/ml Liquid preparation Liquid preparation 10 mg/ml 77 Stock concentartion Supernatant 10 mg/ml 10 mg/ml Supernatant Neat preparation Neat preparation 10 mg/ml Source solaray®, Foods for Living store, East Lansing, MI, USA Sigma, St Louis, MO, USA Sigma, St Louis, MO, USA solaray®, Foods for Living store, East Lansing, MI, USA Herbs Pharma®, Foods for Living store, East Lansing, MI, USA NATURAL S®, Foods for Living store, East Lansing, MI, USA Sigma, St Louis, MO, USA MTT assay for cell viabiliil Cell viability was measured by MTT based assay following manufacturers’ instructions: Initially cultures which were in quadruplicates were removed from incubator and placed in laminar flow hood. At the same time MTT to be used was reconstituted with 3 ml of balanced salt solution without phenol red and serum, and then add reconstituted MTT in an amount equal to 10% of the culture volume. Cultures along with MTT solution were incubated for 3 hours in the 37°C incubator. After the incubation period, cultures were removed from incubator and dissolve the resulting fonnazan crystals by adding an amount of MTT solubilization solution equal to the original culture volume. While mixing make sure formazen crystals were completely dissolved. After mixing measure the absorbance Spectrophotometrically at a wavelength of 570 nm. Measure the background absorbance of multi well plates at 690 nm and subtract from the 570 nm measurement. ELISA method of cytoer estimtion All cytokine measurements were done using sandwich ELISA method. The sandwich ELISA protocol is a 3 day protocol starting with coating the 96- well plate with 50 ul/well of antibody against the protein of interest in coating buffer on day one. Afier 12-16 hours of keeping the coated plate in the refiigerator, blocked with blocking buffer (75ul/well) and kept them in the 37°C incubator. After 3 hours of incubation wash the plates thrice with wash 78 buffer and add samples and standard in duplicates with a final volume of 50 ul/well. Afier adding the samples keep the plate in the refrigerator (+40C) over night. On day 3 remove the plate fiom refrigerator and add 50 ul/well biotin labeled secondary antibody against protein of interest after wash the plate three times. Incubate the plate for 90 minutes in 37°C incubator. After 90 minutes remove the plate from the incubator and add 50 ul/well enzyme (Streptavidin conjugated alkaline phosphatase) after washing the plate for three times. Incubate the plate for 30 minutes in 37°C incubator before washing and adding 50 ul/well PNP substrate. Afier adding the substrate read the absorbance at 405-690 nm. Statistical Analysis AN OVA was used to evaluate significance using GraphPad Instat TM software program (GraphPad software, San Diego, USA). The statistical significance level was set at 0.05. Results Impact of herbal supplements on spleen cell viability In the case of saline-exposed control mice, there was a significant decrease in cell viability when co-cultured with neem leaf extract at lin 1000 dilution. In contrast, there was a significant increase in cell viability when co- cultured with licorice at both dilutions (Figs 21 and 22). In the case of hazelnut-sensitized mice there was no significant decrease in cell viability for all the herbal supplements at both dilutions (Fig. 24). But there was a 79 significant increase in the cell viability in case of echinacea and milk thistle at both dilutions tested (Figs 23 and 24). Impact of herbal supplements on hazelnut-driven Type-2 cfiokine (IL-4, IL-5 and IL—l3) responses by spleen cells from hazelnut allergic mice There was no significant decrease or increase in IL-4 production at both dilutions in the case of Echinacea, Genistein, Glycyrrhizin, Neem Leaf Extract and Silymarin (Figs. 25 and 26). There was a significant increase in the production of IL-4 when co-cultured with Alliin at both dilutions (Fig. 26). There was a significant decrease in the production of IL-4 at 1 in 5000 dilution for garlic and milk thistle, but these herbs didn’t show any decrease at l in 1000 dilution (Fig. 25 and 26). Only licorice exhibited dose-dependent decrease in the production of allergen-driven IL-4 (Fig. 26) Results showed that there was no significant effect on the production of IL-5 by Milk thistle, Alliin, Glycyrrhizin and Silymarin at both dilutions (Figs. 27 and 28). There was a significant increase in the production of IL-5 at both dilutions by herbal supplement Echinacea. There was a decrease in the production of IL-5 by garlic at 1 in 1000 dilution but not at 1 in 5000 dilution. There was a significant decrease in the production of Interleukin-5 by Genistein and Neem Leaf Extract at 1 in 1000 dilution and Licorice at both dilutions in a dose dependent manner (Figs. 27 and 28). 80 Cell viability (MTT assay; OD 570—690 nm) 1 .00 E to .9 g m a: Q S ‘1’ .9 8 t a b 8 t a E .5 if; <9 S it; <9 S 3 CM+Herb(1/5000) CM+Herb(1/1000) Fig. 21: Cell viability in spleen cell cultures in saline exposed mice afier culture with four different herbal supplements Mice (n=6) were exposed to saline via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone and with different herbal supplements Echinacea, Garlic, Genistein and Milk Thistle at 1 in 1000 and 1 in 5000 dilutions. Cells were cultured for 4 days and cell viability at the end of 4th day was measured by MTT assay. Data shown are OD mean +/- SE of quadruplicate analysis. Differences of Culture Medium vs others were analyzed by ANOVA and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 81 .1. o O 0.50 Cell viability (Ml l assay; OD 570-690 nm) .0 O O s .g .s g a g ,5 .s g a .g .9 x a: e .u - a 3 ? LE § § 5 5 E .5? 3: S s c} :i .E’ 5‘ g. :1 .S’ .é .9 a ”’ a .3 ‘° 2 __ 3__ CM+Herb(1/5000) CM+Herb(1/1000) Fig. 22: Cell viability in spleen cell cultures in saline exposed mice after culture with five different herbal supplements Mice (n=8) were exposed to saline via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone and with different herbal supplements Alliin, Glycyrrhizin, Licorice, Neem Leaf Extract and Silymarin at l in 1000 and 1 in 5000 dilutions. Cells were cultured for 4 days and cell viability at the end of 4th day was measured by MTT assay. Data shown are OD mean +/- SE of quadruplicate analysis. Differences of Culture Medium vs others were analyzed by ANOVA and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 82 Cell viability (MTT assay; 0D 570-690 nm) 0.50 0.00 E -~ 5 0b m N .5 o s5 w: ‘3' E u? a s é" CM+Herb(1/5000) CM+Herb(1/1000) Fig. 23: Cell viability in spleen cell cultures in hazelnut sensitized mice after culture with four different herbal supplements Mice (n=8) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone and with different herbal supplements Echinacea, Garlic, Genistein and Milk Thistle at 1 in 1000 and 1 in 5000 dilutions. Cells were cultured for 4 days and cell viability at the end of 4th day was measured by MTT assay. Data shown are OD mean +/- SE of quadruplicate analysis. Differences of Culture Medium vs others were analyzed by ANOVA. * P<0.05; ** P<0.01; *** P<0.001; others non significant. 83 Cell viability (MTT assay; OD 570-690 nm) 1.00 CM+Herb(1/5000) CM+Herb(1/1000) Fig. 24: Cell viability in spleen cell cultures in hazelnut sensitized mice after culture with five different herbal supplements Mice (n=8) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone and with different herbal supplements Alliin, Glycyrrhizin, Licorice, Neem Leaf Extract and Silymarin at l in 1000 and l in 5000 dilutions. Cells were cultured for 4 days and cell viability at the end of 4th day was measured by MTT assay. Data shown are OD mean +/- SE of quadruplicate analysis. Differences of Culture Medium vs others were analyzed by ANOVA and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 84 300 lL-4 (pg/ml) s‘ .5 2° 5’ 5’ 5 5’ 5 5 5 5’ s s a <5" 5 is s <5 a is s 5 g «> a '5 w a a g m 0 5 la 0 s 3? £7: CM+Hazelnut100+Herb(1/5000) CM+Hazelnut 100+Herb(1/1000) o 0 Fig. 25: Impact of herbal supplements on hazelnut-driven IL—4 response by spleen cells from hazelnut allergic mice Mice (n=6) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with hazelnut protein 100 ug and with different herbal supplements Echinacea, Garlic, Genistein and Milk Thistle at 1 in 1000 and 1 in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-4 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Hazelnut was compared with other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 85 lL—4 (pg/ml) 150 75 z CM+Hazelnut 100+Herb(1/5000) CM+HazeInut 100+Herb(1/1000) Fig. 26: Impact of herbal supplements on hazelnut-driven driven IL-4 response by spleen cells from hazelnut allergic mice Mice (n=6) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with hazelnut protein 100 ug and with different herbal supplements Alliin, Glycyrrhizin, Licorice, Neem Leaf Extract and Silymarin at l in 1000 and 1 in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-4 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Hazelnut was compared with other conditions the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 86 lL-5 (pg/ml) (Thousands) o 8’ ‘5 .5 <9 '5” ,5 .‘c" O '5” if E as 5‘ a: \ .5‘ o -: E’ {y u: 0 5 lu ‘9 5 (‘33 £7: CM+Hazelnut100+Herb(1l5000) CM+Haze|nut100+Herb(1/1000) c Fig. 27: Impact of herbal supplements on hazelnut-driven driven IL-5 response by spleen cells from hazelnut allergic mice Mice (n=6) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with hazelnut protein 100 ug and with different herbal supplements Echinacea, Garlic, Genistein and Milk Thistle at 1 in 1000 and l in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-5 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Hazelnut was compared with other conditions the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 87 lL-5 (pg/ml) 3000 1500 0 x: :1“ 0 C 3 C C 8 C E := E _-: .S g g 5 -LV 5? LU «’5 3. A! L8 a «'5 q, :: E 8 3? E E 8 3? E s 5 a :1 .1” a a :1 .S’ s .. {5’ a a a a 5 .5 .5 o z z CM+HazeInut100+Herb(1/5000) CM+HazeInut 100+Herb(1/1000) Fig. 28: Impact of herbal supplements on hazelnut-driven driven IL—5 response by spleen cells from hazelnut allergic mice Mice (n=6) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with hazelnut protein 100 ug and with different herbal supplements Alliin, Glycyrrhizin, Licorice, Neem Leaf Extract and Silymarin at 1 in 1000 and 1 in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-5 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Hazelnut was compared with other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 88 Results showed that Echinacea, Genistein, Alliin, and Silymarin had no major effect on the production of IL-13 when tested at 1 in 1000 and l in 5000 (Figs. 29 and 30). Glycyrrhizin significantly increased the production of IL-1 3 at l in 1000 dilution (Fig. 30). Garlic at 1 in 5000 dilution but not at 1 in 1000 and Neem Leaf Extract, Milk thistle at 1 in 1000 but not at 1 in 5000 dilutions significantly decreased the production of IL-13 (Figs. 29 and 30). Licorice significantly decreased the production of IL—1 3 at both dilutions tested in a dose dependent manner (Fig. 30). Impact of licorice from three different sources on hazelnut-driven Type-2 cytokine (IL-4. IL-5 and IL-13) respon_sg Licorice was the only herbal supplement tested that significantly decreased the production of all three Type-2 cytokines in a dose dependent manner. Since we had tested the licorice from only one company, we examined the effect of licorice fi'om two other sources. Our hypothesis was that the effect of licorice on memory Tpe-2 cytokine production was independent of the source of licorice. Cytotoxic effect of licorice supplements from two other sources on splenocytes from hazelnut-sensitized mice was tested by MTT assay. As evident, there was no significant effect at either dilutions on cell viability (Fig. 31). 89 lL-13 (pg/ml) 2000 1000 Gil/fie Genistein M E . G CM+HazeInut 100+Herb(1/5000) CM+HazeInut 100+Herb(1l1000) .5 8’ 5 .5 3 0 CM +HaZG/Inlt1oo Fig. 29: Impact of herbal supplements on hazelnut-driven driven IL-l3 response by spleen cells from hazelnut allergic mice Mice (n=6) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with hazelnut protein 100 ug and with different herbal supplements Echinacea, Garlic, Genistein and Milk Thistle at 1 in 1000 and 1 in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-13 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Hazelnut was compared with other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 90 lL—13 (pg/ml) 3000 1500 0 Q C t C Q '~ '\ ~ :\ -5 ~ t .5 g g .u 59 5’ «’5 $ A’ '53 $9 5 5 = E 5 5 E E 5 a S S B: 3‘ ~I .._ t 3' ~I ._ t e a a 5 ”i a a a 0 O 2 CM+HazeInut 100+Herb(1/5000) CM+HazeInut 100+Herb(1/1000) Fig. 30: Impact of herbal supplements on hazelnut-driven driven IL-l3 response by spleen cells from hazelnut allergic mice Mice (n=6) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with hazelnut protein 100 ug and with different herbal supplements Alliin, Glycyrrhizin, Licorice, Neem Leaf Extract and Silymarin at 1 in 1000 and l in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL—13 levels by ELISA. Data shown are mean +/— SE of duplicate analysis. ANOVA: Culture medium+ Hazelnut was compared with other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 91 .3 o O Cell viability (MTT assay; OD 570-690 nm) 0 8 0.00 E S _\ 3T S c? A 3 it? E r‘“ 93 g: 5 233 a 5 4. 5’ a 5’ 5 .5” 5 8 5’ 6 a “ a .5 “ 5 ~I x! 5’ 5’ .5 .8 ~l ~l CM+Herb(1/1000) CM+Herb(1/5000) Fig. 31: Cell viability in spleen cell cultures of hazelnut sensitized mice after culturing with licorice from three different sources Mice (n=6 per group) were sensitized with hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, cells with 100 ug of hazelnut protein and with licorice from 3 different sources (solaray, now and herbas pharma) at 1 in 1000 and 1 in 5000 dilutions. Cell viability on day 4 was measured by optimized MTT assay. Data shown are mean +/— SE of quadruplicate analysis. ANOVA: Cell viability of culture medium was compared to other conditions and and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 92 Culture supematants were screened for IL- 4 by using ELISA. Results showed that licorice from the other two sources were also able to reduce the production of IL—4 significantly at both dilutions (Fig. 32). Results showed that licorice from one another source was able to significantly reduce the IL-5 production at both 1 in 1000 and 1 in 5000 dilutions (Fig. 33). Whereas licorice from third source Herbs Pharrna was able to reduce the IL-5 production only at higher concentration (1 in 1000) (Fig. 33). As evident, licorice from one source along with the licorice from the original source was able to significantly reduce the IL-13 production at both dilutions (Fig. 34). Licorice from the third source was able to significantly reduce the IL-13 production only at 1 in 1000 dilution, but not significantly at 1 in 5000 (Fig. 34). M of licorice from three different sources on sesa_m_e-driven T ype-2 cytokine (IL-4. IL-5 and IL-13) respon_aes by spleen cells from sesame- sensitized mice Cell viability was measured by using MTT assay when the splenocytes from sesame-sensitized mice were cultured with licorice from three different companies. Results showed that licorice had no significant effect on cell viability (Fig. 35). 93 150 75" lL-4 (pg/ml) *** $5 «2 0 .5 3 C) CM+HazeInut100ug+Herb(1/5000) CM+HazeInut100ug+Herb(1/1000) flg._32: Impact of licorice from three sources on hazelnut-driven driven IL-4 W by spleen cell_s from hazelnut allergic mice Mice (n=6) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with hazelnut protein 100 ug and with supplement licorice from three different sources (solaray, now and herbas pharrna) at 1 in 1000 and l in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-4 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Hazelnut was compared with other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 94 lL-5 (pg/ml) (Thousands) s o a n a a n e .5 § 8 3 g LU 5 E g D g 5 (u L!) \C— to f g“ {0. .5 5 a" a 5 0) U) 5 .I 5 5 5 5 J ~l ~l o 5 .5 $ L8 :75 0 s1 ‘1 m ’53 .g 5’ .5 ~l ;, CM+Hazelnut100ug+Herb(1/5000) CM+HazeInut100ug+Herb(1/1000) Fig. 33: Impact of licorice from three sources on hazelnut-driven driven IL- 5 response by spleen cells from hazelnut allergic mice Mice (n=6) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with hazelnut protein 100 ug and with supplement licorice from three different sources (solaray, now and herbas pharrna) at l in 1000 and l in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-5 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Hazelnut was compared with other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 95 3000 2 g 2000 or o. v on ‘T 1000 :' 0 E o -\ -\ -\ -\ '\ .9 § 5‘ g g 5‘ 5 g 5 5 5 5 m 5 5 m f f? 3; .§ if {a {3 if §$.§.§5.§5§ 6 s .5 " 5 .8 " a: Q ‘1 ~l {3 ,5 .8 .5? ~l xl CM+HazeInut100ug+Herb(1/5000) CM+HazeInut100ug+Herb(1/1000) Fig. 34: Impact of licorice fi'om three sources on hazelnut-driven driven IL- 13 response by spleen cells from hazelnut allergic mice Mice (n=6) were exposed to hazelnut protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with hazelnut protein 100 ug and with supplement licorice from three different sources (solaray, now and herbas pharrna) at l in 1000 and l in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-13 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Hazelnut was compared with other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 96 Cell viability (MTT assay; OD 570-690 nm) 1.00 0.50 0.00 E 0 - . E 3? ‘S a? 3T 3 7!? 5‘? if it" 5‘? 5 3%" § § 5 $9 § 53 If g; [:3 f e .3 3 .12” s 8 .é’ ~I ~I \I CM+Herb(1/5000) CM+Herb(1/1000) Fig. 35: Cell viability in spleen cell cultures of sesame sensitized mice after culturing with licorice from three different sources Mice (n=8 per group) were sensitized with sesame seed protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, cells with 100 ug of sesame seed protein and with licorice from 3 different sources (solaray, now and herbas pharma) at 1 in 1000 and l in 5000 dilutions. Cell viability on day 4 was measured by MTT assay. Data shown are mean +/- SE of quadruplicate analysis. ANOVA: Cell viability of culture medium was compared to other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 97 Culture supematants of the spleen cell cultures from sesame-sensitized animals with licorice from three different sources were screened for IL—4 with sandwich ELISA. As evident, licorice from all three different sources was able to reduce the IL-4 production in a dose dependent manner. The decreased production of IL-4 by Licorice fiom solaray and now was a significant at both dilutions whereas the decrease was not significant in case of Licorice from herbs pharma at 1 in 5000 dilution (Fig. 36). IL-5 levels were tested in the culture supematants of spleen cell cultures of sesame-sensitized animals with licorice from different sources. Licorice from solaray and now were able to significantly reduce this cytokine production at both dilutions tested, whereas Licorice from herbs pharma was able to reduce only at 1 in 1000 dilution (Fig. 37). IL-13 levels were screened in the supematants of the spleen cell cultures from sesame-sensitized mice with licorice from different sources. As evident, that only Licorice from now was able to significantly reduce the IL-13 production at both dilutions tested. Licorice from solaray reduced IL-13 production at 1 in 1000 but not at 1 in 5000. Licorice from herbs pharma had no effect at either dilution (Fig. 38). 98 lL-4 (pg/ml) 200 100 CM+SES100ug+Herb(1/5000) CM+SES100ug+Herb(1/5000) fig. 36: Impact of licorice from three sources on sesame-driven IL-4 respon_se bv spleen cells fi'om segue allergic mice Mice (n=6) were exposed to sesame seed protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with sesame seed protein 100 ug and with supplement licorice from three different sources (solaray, now and herbas pharma) at 1 in 1000 and 1 in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-4 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Sesame was compared with other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 99 IL-5 (pg/ml) 8000 4000 Lico - nee (So/aray) LIcOrIOe (new) Q ‘\ 5 2 s? a . . E B 5 a £0. 9 o s :0 .8 :l o 5 :38 - (U .8 f 0) .19 ~15. .8 a? ~I a .E 3? +3 é" 8 It .3 ~l CM+SES100ug+Herb(1/5000) CM+SE81 00ug+Herb(1/5000) Fig. 37: Impact of licorice fiom three sources on sesame-driven IL-5 response by spleen cells fiom sesame allergic mice Mice (n=6) were exposed to sesame seed protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with sesame seed protein 100 ug and with supplement licorice from three different sources (solaray, now and herbas pharma) at 1 in 1000 and 1 in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-5 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Sesame was compared with other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 100 IL-13 (pg/ml) 4000 2000 0 E Q '\ ‘x —\ -\ ‘\ -\ . 8 E" 8 .8 a é’ é" ~ .8 é" ~ g f .8 a .‘9 g e 715’ . ‘0 .s g: .g .5 g {.9 .8 g 5' S 8 " I 8 " I ° . r -. r 't ’1: .3 .5 ~l ~l CM+SES100ug+Herb(1/5000) CM+SES100ug+Herb(1/5000) Fig. 38: Impact of licorice from three sources on sesame-driven IL—13 response by spleen cells from sesame allergic mice Mice (n=6) were exposed to sesame seed protein via transdermal exposure for 6 times. One week following the last exposure spleen cells were harvested and cultured the cells alone, with sesame seed protein 100 ug and with supplement licorice from three different sources (solaray, now and herbas pharma) at 1 in 1000 and 1 in 5000 dilutions. Supematants were collected at the end of day 4 and screened for IL-13 levels by ELISA. Data shown are mean +/- SE of duplicate analysis. ANOVA: Culture medium+ Sesame was compared with other conditions and the statistical significances are expressed as * P<0.05; ** P<0.01; *** P<0.001; others non significant. 101 Discussion As aim two this study looked at the possibility of identifying potential anti-allergy herbal supplements using a novel approach was tested. Thus herbal supplements that inhibit the allergen-driven Type-2 cytokine responses in hazelnut and sesame allergy models were tested. Out of 9 herbal supplements tested only licorice consistently exhibited a dose dependent inhibition of the production of all three Type-2 cytokines (IL-4, IL-5 and IL-13) tested. Since licorice from one source tested, whether licorice from different sources might exhibit similar activity and found that to be the case. Later the question whether licorice might inhibit Type-2 cytokine responses to another food allergen using sesame allergy mouse model was addressed. Interestingly licorice from all three sources showed similar activity in sesame allergy model as well. This study used extracts of herbal supplements from commercial sources (Table 4). Therefore, identities of component responsible for the observed inhibition of Type-2 cytokine responses by licorice remain to be determined. However, glycyrrhizin, one active component present in licorice was not able to inhibit Type-2 cytokine responses (Fig. 25, 27 and 29). This 102 data suggest that active ingredient of licorice responsible for inhibition of Tytpe—Z cytokine responses is other than glycyrrhizin. This study examined the Type-2 cytokines as target cytokines. Because these cytokines exhibit several pro-allergic functions and participate in allergic disorders. As discussed, IL-4 was identified as critical Type-2 cytokine in this model. Since licorice inhibits IL-4 response to allergens in both hazelnut and sesame allergy model, one hypothesis to test would be that licorice prevents de novo allergic response and/or established allergic response in our model. There are two published papers (Hugh Samson’s lab Mount Sinai School of Medicine, NY, USA) that discussed the role of herbal supplements in an animal model of food allergy. They tested the efficacy of Chinese herbal formula, Food Allergy Herbal Formula (FAHF) in their adjuvant based mouse model of peanut allergy. They found the original Chinese formula (FAHF -1) and modified Chinese formula (FAHF-Z) were efficient in reducing the all three Type-2 cytokines; IgE levels and there by reduce the anaphylactic symptoms in this model. This study however, used cholera toxin based food allergy model and they looked at a cocktail herbal formula (23, 29). A number of previous studies demonstrate that licorice inhibits IL-4 response in mouse models of other disorders. Some studies also suggest that licorice might be useful in alleviating disease symptoms of allergies as summarized (Table 5). 103 Table 5: Immunomodulatory effects of licorice Form of Model system Result Refer Licorice used ence Glycyrrhizin Mouse model of Decrease in IL-4 and 5 in (181) asthma BAL fluid Glycyrrhizin Mouse model of Induces CD4 positive (182) pulmonary CD28 positive TCR carcinoma alpha/beta positive T cells (which counteracts the production of Type-2 cytokines) Glycyrrhizin Mouse model of Decreases all Type-2 (183) Candida albicans cytokine production. infection Glycyrrhizin Mouse model of Decreases all Type-2 (184) Candida albicans cytokine levels in serum. infection Glycyrrhizin In-vitro mouse Decrease in IL—4 and IL- (185) spleen cell cultures 10 and increase in IFN Glycyrrhizin Peritoneal Increase in IL-12 (147) macrophages Glycyrrhizin Thermally injured Increase in the IL-12 (186) mice levels in both serum and spleen cell cultures Glycyrrhizin Human lung fibro Decrease in EOTAXIN-l (187) derivative - blasts production hetero-30-OH- GL Licorice Passive Inhibition of ear swelling (188) roasted vs un sensitization model by both roasted and un roasted roasted licorice Licorice gel Human clinical Effective agent in treating (189) trial atopic dermatitis Glycyrrhizin, Contact Inhibited the passive (190) Glycyrrhetinic hypersensitivity cutaneous anaphylaxis and acid mouse model skin contact inflammation Glycyrrhizin Patient with Resolved with eosinophilic (191) eosinophilic peritonitis problem peritonitis 104 Even though this study identified an herbal supplement that inhibited all Type-2 cytokines it can not be extrapolated to humans. The dose recommended in human beings for the herbal supplements and the estimated doses for mouse based on body weight were summarized in table 6. Table 6: Dose calculations of herbal supplements S.No Name Recommended Estimated human dose Mouse dose 1 Echinacea 1600 mg/day 4.5 ug/day 2 Garlic 320 mg/day 1.0 ug/day 3 Milk Thistle 1800 mg/day 5 ug/day 4 Licorice- solaray 900 mg/day 2.5 ug/day 5 Licorice- now 900 mg/day 2.5 ug/day 6 Licorice- Herbs Pharma 15 to 30 drops/day 7 Neem Leaf Extract 15 to 30 drops/day Since this study was done in ex-vivo system and whole extracts were used the data can not be extrapolated to human studies. There are no previous studies that examined the potential of licorice in food allergy. Data from this work along with the literature discussed above strongly supporting the idea of conducting further in vivo studies using our mouse model to evaluate the potential of licorice as an anti-food allergy herbal supplement. 105 SUMMARY AND FUTURE DIRECTIONS This study was conducted in two parts using an adjuvant-free mouse model that had been developed in our laboratory. In the first part, Type-2 cytokine responses in hazelnut and sesame allergy models were characterized. There are four novel and important findings from this study: (1) transdermal exposure to hazelnut and sesame allergens was sufficient to activate Type-2 cytokine response by spleen cells; (2) recall allergen-driven Type-2 cytokine response was largely CD4 dependent in both hazelnut and sesame allergy models; (3) hazelnut allergen-driven IL-4 but not IL-S/IL-13 was STAT6 dependent; and (4) IL-4 is required for allergic and systemic anaphylaxis to hazelnut in this mouse model. In the second part, a novel approach of examining the impact of herbal supplements on ex vivo allergen-driven Type-2 cytokine responses by spleen cells from hazelnut and sesame allergy mouse models was identified. This study found that among the herbal supplements tested, only licorice fiom all three sources, inhibited allergen-driven ex vivo Type-2 cytokine responses in both hazelnut and sesame allergy models. Future directions Part-I Further characterization of Type-2 cytokine response need to be done in this model. Mainly one has to study the upstream regulation of this response by 106 studying the role of transcription factors such as GATA-3 and c-MAF that control IL-4 gene expression. This can be tested either by using gene knock out mouse or by using techniques like RNA interference. One has to study the heritability of IL-4 control of food allergy in this model. One way to test this hypothesis was using the crossing and back crossing between the strains that produces IL-4 (ASW) and the strains that don’t produce IL-4 (SJ L). Pail; This study used the ex-vivo model to study the effect of different herbal supplements on Type-2 cytokine responses in hazelnut and sesame allergy models. To further validate the effects that were shown by the licorice in ex- vivo model one need to do the in-vivo studies such as feeding licorice and study the IL-4 responses and on disease readouts (study both preventive and therapeutic approaches). This study found that the whole licorice extract was responsible for inhibiting the Type-2 cytokine responses in ex-vivo model. This study also tested glycyrrhizin one of the active ingredient is not responsible for the effects observed in the ex-vivo model. One need to isolate and identify the active ingredient of licorice responsible for decreasing the Type-2 cytokine responses in this model. 107 10. 11. 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