Allergy
BRIEF COMMUNICATION
Effects of a pre-existing food allergy on the oral introduction of food proteins: findings from a murine model M. Kulis & A. W. Burks Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
To cite this article: Kulis M, Burks AW. Effects of a pre-existing food allergy on the oral introduction of food proteins: findings from a murine model. Allergy 2015; 70: 120–123.
Keywords cashew; egg; food allergy; peanut; walnut. Correspondence Mike Kulis, PhD, UNC-Chapel Hill, Campus Box # 7231, Chapel Hill, NC 27599, USA. Tel.: 919-962-4403 Fax: 919-962-4421 E-mail: [email protected]
Abstract
Cashew-allergic mice develop elevated walnut-specific IgE upon oral feeding of walnut proteins. Ingestion of tree nuts in the presence of a known nut allergy could lead to additional sensitizations and anaphylaxis following subsequent exposure.
Accepted for publication 20 August 2014 DOI:10.1111/all.12519 Edited by: Antonella Muraro
An estimated 3.9% of children in the United States have food allergy (1), and it is not uncommon for patients to be allergic to multiple foods (2). For example, the majority of 196 foodallergic children in one study reacted to more than one food during double-blind, placebo-controlled challenges (3). One possible explanation for co-sensitization to several foods in the same patient is that food allergy may arise from a breakdown in oral tolerance mechanisms, such that the immune system is primed to develop deleterious immune responses (i.e., allergy) to newly introduced foods (4). Other co-sensitizations may be explained by cross-reactivity of homologous allergens in various foods, such as the seed storage proteins found in tree nuts (5). Using a mouse model, we aimed to study the effects of a single-food allergy on the oral introduction of new food proteins into the diet. Four common food allergen sources were used: egg, peanut, cashew, and walnut. The overall strategy for this study conducted in a mouse model of food allergy was to (i) establish a primary food allergy using the food allergen and cholera toxin as an adjuvant (ii) and then, after a resting period, to introduce a new food into the diet without any adjuvant. The protocol is designed to mimic a person with an allergy being exposed to a new source of food proteins, with the aim of investigating the effects a primary allergy has on the development of a
120
secondary allergy. Four-week-old female C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME, USA) were used in the protocol, which was approved by the IACUC at Duke University Medical Center (Durham, NC, USA). During the protocol, mice were kept on a chow that was free of egg, peanut, cashew, and walnuts. Mice were sensitized to egg, peanut, or cashew by oral gavage of 2 mg food protein extract plus 10 lg cholera toxin on days 1, 8, and 15 and 5 mg of food protein extract plus 10 lg cholera toxin on day 22, to establish the primary allergy. Then, on days 43, 50, and 57, mice were fed 2 mg of peanut, egg, walnut, or PBS alone by oral gavage without any adjuvant. Specifically, mice sensitized to egg were then fed peanut or PBS in the absence of adjuvant; mice sensitized to peanut were then fed egg or PBS in the absence of adjuvant; and mice sensitized to cashew were then fed egg, peanut, walnut, or PBS in the absence of adjuvant. These combinations were chosen to test whether initial food allergies would lead to subsequent food allergies when the allergens are homologous (e.g., cashew proteins share homology with walnut proteins) or nonhomologous (e.g., egg and peanut proteins are not homologous). Mice were bled on days 40 (after the primary sensitization) and 71 (after the introduction of a new food) to quantify specific IgE, following a method described previously (6).
Allergy 70 (2015) 120–123 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Kulis and Burks
Introducing foods in the presence of an allergy
Mice were challenged by i.p. injection of 200 lg of the newly introduced food and assessed for allergic symptoms and body temperatures, as described previously (7). Mice sensitized to egg and then fed peanut did not have any differences in egg-specific IgE on day 71 compared with egg-sensitized mice fed vehicle alone as determined by a Mann–Whitney U-test (Fig. 1A). In the egg-sensitized mice, feeding peanut induced a nonsignificant increase in peanutspecific IgE (Fig. 1B). Peanut-sensitized mice fed either egg or vehicle alone did not have differences in peanut-specific IgE (Fig. 1C) or egg-specific IgE (Fig. 1D). Mice sensitized to egg that had peanut introduced into their diet did not react on challenge to peanut, whereas mice that were sensitized to peanut and then fed egg proteins had allergic symptoms and decreased body temperature following the peanut challenge (Fig. 2A,B). Groups of mice sensitized to cashew proteins were fed egg, peanut, walnut, or a saline solution. There were no differences in cashew-specific IgE between the groups of mice
on day 71 as determined by Mann–Whitney U-test (Fig. 1E). Interestingly, mice fed walnut after the cashew allergy was established had significantly elevated walnut-specific IgE from day 40 to day 71 (P = 0.0164 using a paired t-test), which was not observed in the other groups (Fig. 1F). All five mice in the walnut group had walnut-specific IgE levels increase after being fed walnut, with four of the five mice having walnut-specific IgE higher than any mice from the other groups (see dashed line in Fig. 1F). The elevated walnut-specific IgE levels had in vivo implications, as the mice with elevated walnut-specific IgE had allergic reactions that did not occur in the other groups during the walnut challenge (Fig. 2C,D). Importantly, the four mice in the walnut group with walnut-specific IgE above the dashed line in Fig. 1F were the same four mice that developed allergic symptoms (Fig. 2C) and experienced decreased body temperatures below 37.0°C (see dashed line in Fig. 2D). Introduction of new foods into the diet of mice with an existing peanut or egg allergy does not seem to increase the
A
B
C
D
E
F
Figure 1 Food-protein-specific IgE in mouse sera. Egg-specific (A) and peanut-specific IgE (B) in mice sensitized to egg and then fed peanut (Egg/PN) or vehicle alone (Egg/PBS). Peanut-specific (C) and egg-specific IgE (D) in mice sensitized to peanut and then fed egg (PN/Egg) or vehicle (PN/PBS). Cashew-specific (E) and walnut-spe-
cific IgE (F) in mice sensitized to cashew and then fed egg (CSH/ Egg), peanut (CSH/PN), walnut (CSH/WN), or vehicle (CSH/PBS). Individual data are shown with lines connecting IgE levels postsensitization (day 40) and postintroduction of new food (day 71). * Represents P < 0.05 as determined by a paired t-test.
Allergy 70 (2015) 120–123 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Introducing foods in the presence of an allergy
Kulis and Burks
B
A
D C
Figure 2 Food challenge outcomes. Symptom scores (A) and body temperatures (B) following peanut challenge in mice sensitized to egg and then fed peanut (Egg/PN) and in mice sensitized to peanut and then fed egg (PN/Egg). Symptom scores (C) and body temperatures (D) following walnut challenges in mice sensitized to
cashew and then fed egg (CSH/Egg), peanut (CSH/PN), or walnut (CSH/WN). Individual data are shown with lines connecting prechallenge body temperatures and body temperatures 30 min after challenge in (B) and (D). * represents P < 0.05; ** represents P < 0.01 as determined by a Mann–Whitney U-test.
risk of developing additional food allergies, as was demonstrated in mice sensitized to egg or peanut proteins and then fed the other food without developing significantly increased specific IgE. Even though there was a modest, nonsignificant, increase in the peanut-specific IgE of egg-sensitized mice later fed peanut proteins, these mice did not have allergic reactions during a peanut challenge. The most interesting finding of our study was that mice with a cashew allergy later fed walnut (in the absence of any Th2-skewing adjuvant) had significant increases in walnut-specific IgE that was not found in sera of cashew-allergic mice fed egg or peanut. A challenge to walnut provided direct evidence that the mice with increased walnut-specific IgE, but not the other groups of mice, would experience anaphylaxis upon exposure. We have previously demonstrated that T-cell cross-reactivity exists between the two tree nuts, cashew and walnut (6). Thus, we hypothesize that cashew-specific Th2 cells were activated by oral feedings of walnut antigens, which led to the increased walnut-specific IgE. It is not likely that T-cell cross-reactivity exists between egg and peanut, for example, and this may explain why those allergies do not have an effect on subsequent introduction of nonhomologous food proteins.
These data indicate that feeding tree nuts to someone with an existing tree nut allergy could lead to the production of specific IgE to the newly fed tree nut, which may then lead to clinical reactions upon subsequent exposure. These data in this mouse model would support physicians’ advice to avoid all tree nuts once a reaction has occurred to any tree nut (8). Interestingly, it is not simply IgE cross-reactivity which could provoke allergic reactions that should be of concern, but also the production of IgE to the newly introduced tree nut that may lead to clinical allergic reactions following future exposures. More longitudinal studies in food-allergic children will help to understand this issue better. Funding Food Allergy Initiative (FAI) – prior to merging with Food Allergy and Anaphylaxis Network (FAAN) to form Food Allergy Research and Education (FARE). Conflicts of interest The authors declare that they have no conflicts of interest.
References 1. Branum AM, Lukacs SL. Food allergy among children in the United States. Pediatrics 2009;124:1549–1555.
122
2. Wang J. Management of the patient with multiple food allergies. Curr Allergy Asthma Rep 2010;10:271–277.
3. Sampson HA, Ho DG. Relationship between food-specific IgE concentrations and the risk of positive food challenges in children and
Allergy 70 (2015) 120–123 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Kulis and Burks
adolescents. J Allergy Clin Immunol 1997;100:444–451. 4. Chehade M, Mayer L. Oral tolerance and its relation to food hypersensitivities. J Allergy Clin Immunol 2005;115: 3–12. 5. de Leon MP, Glaspole IN, Drew AC, Rolland JM, O’Hehir RE, Suphioglu C. Immunological analysis of allergenic cross-
Introducing foods in the presence of an allergy
reactivity between peanut and tree nuts. Clin Exp Allergy 2003;33:1273–1280. 6. Kulis M, Pons L, Burks AW. In vivo and T cell cross-reactivity between walnut, cashew and peanut. Int Arch Allergy Immunol 2009;148:109–117. 7. Kulis M, Chen X, Lew J, Wang Q, Patel OP, Zhuang Y et al. The 2S albumin allergens of Arachis hypogaea, Ara h 2 and
Allergy 70 (2015) 120–123 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanutallergic mice. Clin Exp Allergy 2012;42: 326–336. 8. Ball H, Luyt D, Bravin K, Kirk K. Single nut or total nut avoidance in nut allergic children: outcome of nut challenges to guide exclusion diets. Pediatr Allergy Immunol 2011;22:808– 812.
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BRIEF COMMUNICATION
Effects of a pre-existing food allergy on the oral introduction of food proteins: findings from a murine model M. Kulis & A. W. Burks Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
To cite this article: Kulis M, Burks AW. Effects of a pre-existing food allergy on the oral introduction of food proteins: findings from a murine model. Allergy 2015; 70: 120–123.
Keywords cashew; egg; food allergy; peanut; walnut. Correspondence Mike Kulis, PhD, UNC-Chapel Hill, Campus Box # 7231, Chapel Hill, NC 27599, USA. Tel.: 919-962-4403 Fax: 919-962-4421 E-mail: [email protected]
Abstract
Cashew-allergic mice develop elevated walnut-specific IgE upon oral feeding of walnut proteins. Ingestion of tree nuts in the presence of a known nut allergy could lead to additional sensitizations and anaphylaxis following subsequent exposure.
Accepted for publication 20 August 2014 DOI:10.1111/all.12519 Edited by: Antonella Muraro
An estimated 3.9% of children in the United States have food allergy (1), and it is not uncommon for patients to be allergic to multiple foods (2). For example, the majority of 196 foodallergic children in one study reacted to more than one food during double-blind, placebo-controlled challenges (3). One possible explanation for co-sensitization to several foods in the same patient is that food allergy may arise from a breakdown in oral tolerance mechanisms, such that the immune system is primed to develop deleterious immune responses (i.e., allergy) to newly introduced foods (4). Other co-sensitizations may be explained by cross-reactivity of homologous allergens in various foods, such as the seed storage proteins found in tree nuts (5). Using a mouse model, we aimed to study the effects of a single-food allergy on the oral introduction of new food proteins into the diet. Four common food allergen sources were used: egg, peanut, cashew, and walnut. The overall strategy for this study conducted in a mouse model of food allergy was to (i) establish a primary food allergy using the food allergen and cholera toxin as an adjuvant (ii) and then, after a resting period, to introduce a new food into the diet without any adjuvant. The protocol is designed to mimic a person with an allergy being exposed to a new source of food proteins, with the aim of investigating the effects a primary allergy has on the development of a
120
secondary allergy. Four-week-old female C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME, USA) were used in the protocol, which was approved by the IACUC at Duke University Medical Center (Durham, NC, USA). During the protocol, mice were kept on a chow that was free of egg, peanut, cashew, and walnuts. Mice were sensitized to egg, peanut, or cashew by oral gavage of 2 mg food protein extract plus 10 lg cholera toxin on days 1, 8, and 15 and 5 mg of food protein extract plus 10 lg cholera toxin on day 22, to establish the primary allergy. Then, on days 43, 50, and 57, mice were fed 2 mg of peanut, egg, walnut, or PBS alone by oral gavage without any adjuvant. Specifically, mice sensitized to egg were then fed peanut or PBS in the absence of adjuvant; mice sensitized to peanut were then fed egg or PBS in the absence of adjuvant; and mice sensitized to cashew were then fed egg, peanut, walnut, or PBS in the absence of adjuvant. These combinations were chosen to test whether initial food allergies would lead to subsequent food allergies when the allergens are homologous (e.g., cashew proteins share homology with walnut proteins) or nonhomologous (e.g., egg and peanut proteins are not homologous). Mice were bled on days 40 (after the primary sensitization) and 71 (after the introduction of a new food) to quantify specific IgE, following a method described previously (6).
Allergy 70 (2015) 120–123 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Kulis and Burks
Introducing foods in the presence of an allergy
Mice were challenged by i.p. injection of 200 lg of the newly introduced food and assessed for allergic symptoms and body temperatures, as described previously (7). Mice sensitized to egg and then fed peanut did not have any differences in egg-specific IgE on day 71 compared with egg-sensitized mice fed vehicle alone as determined by a Mann–Whitney U-test (Fig. 1A). In the egg-sensitized mice, feeding peanut induced a nonsignificant increase in peanutspecific IgE (Fig. 1B). Peanut-sensitized mice fed either egg or vehicle alone did not have differences in peanut-specific IgE (Fig. 1C) or egg-specific IgE (Fig. 1D). Mice sensitized to egg that had peanut introduced into their diet did not react on challenge to peanut, whereas mice that were sensitized to peanut and then fed egg proteins had allergic symptoms and decreased body temperature following the peanut challenge (Fig. 2A,B). Groups of mice sensitized to cashew proteins were fed egg, peanut, walnut, or a saline solution. There were no differences in cashew-specific IgE between the groups of mice
on day 71 as determined by Mann–Whitney U-test (Fig. 1E). Interestingly, mice fed walnut after the cashew allergy was established had significantly elevated walnut-specific IgE from day 40 to day 71 (P = 0.0164 using a paired t-test), which was not observed in the other groups (Fig. 1F). All five mice in the walnut group had walnut-specific IgE levels increase after being fed walnut, with four of the five mice having walnut-specific IgE higher than any mice from the other groups (see dashed line in Fig. 1F). The elevated walnut-specific IgE levels had in vivo implications, as the mice with elevated walnut-specific IgE had allergic reactions that did not occur in the other groups during the walnut challenge (Fig. 2C,D). Importantly, the four mice in the walnut group with walnut-specific IgE above the dashed line in Fig. 1F were the same four mice that developed allergic symptoms (Fig. 2C) and experienced decreased body temperatures below 37.0°C (see dashed line in Fig. 2D). Introduction of new foods into the diet of mice with an existing peanut or egg allergy does not seem to increase the
A
B
C
D
E
F
Figure 1 Food-protein-specific IgE in mouse sera. Egg-specific (A) and peanut-specific IgE (B) in mice sensitized to egg and then fed peanut (Egg/PN) or vehicle alone (Egg/PBS). Peanut-specific (C) and egg-specific IgE (D) in mice sensitized to peanut and then fed egg (PN/Egg) or vehicle (PN/PBS). Cashew-specific (E) and walnut-spe-
cific IgE (F) in mice sensitized to cashew and then fed egg (CSH/ Egg), peanut (CSH/PN), walnut (CSH/WN), or vehicle (CSH/PBS). Individual data are shown with lines connecting IgE levels postsensitization (day 40) and postintroduction of new food (day 71). * Represents P < 0.05 as determined by a paired t-test.
Allergy 70 (2015) 120–123 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
121
Introducing foods in the presence of an allergy
Kulis and Burks
B
A
D C
Figure 2 Food challenge outcomes. Symptom scores (A) and body temperatures (B) following peanut challenge in mice sensitized to egg and then fed peanut (Egg/PN) and in mice sensitized to peanut and then fed egg (PN/Egg). Symptom scores (C) and body temperatures (D) following walnut challenges in mice sensitized to
cashew and then fed egg (CSH/Egg), peanut (CSH/PN), or walnut (CSH/WN). Individual data are shown with lines connecting prechallenge body temperatures and body temperatures 30 min after challenge in (B) and (D). * represents P < 0.05; ** represents P < 0.01 as determined by a Mann–Whitney U-test.
risk of developing additional food allergies, as was demonstrated in mice sensitized to egg or peanut proteins and then fed the other food without developing significantly increased specific IgE. Even though there was a modest, nonsignificant, increase in the peanut-specific IgE of egg-sensitized mice later fed peanut proteins, these mice did not have allergic reactions during a peanut challenge. The most interesting finding of our study was that mice with a cashew allergy later fed walnut (in the absence of any Th2-skewing adjuvant) had significant increases in walnut-specific IgE that was not found in sera of cashew-allergic mice fed egg or peanut. A challenge to walnut provided direct evidence that the mice with increased walnut-specific IgE, but not the other groups of mice, would experience anaphylaxis upon exposure. We have previously demonstrated that T-cell cross-reactivity exists between the two tree nuts, cashew and walnut (6). Thus, we hypothesize that cashew-specific Th2 cells were activated by oral feedings of walnut antigens, which led to the increased walnut-specific IgE. It is not likely that T-cell cross-reactivity exists between egg and peanut, for example, and this may explain why those allergies do not have an effect on subsequent introduction of nonhomologous food proteins.
These data indicate that feeding tree nuts to someone with an existing tree nut allergy could lead to the production of specific IgE to the newly fed tree nut, which may then lead to clinical reactions upon subsequent exposure. These data in this mouse model would support physicians’ advice to avoid all tree nuts once a reaction has occurred to any tree nut (8). Interestingly, it is not simply IgE cross-reactivity which could provoke allergic reactions that should be of concern, but also the production of IgE to the newly introduced tree nut that may lead to clinical allergic reactions following future exposures. More longitudinal studies in food-allergic children will help to understand this issue better. Funding Food Allergy Initiative (FAI) – prior to merging with Food Allergy and Anaphylaxis Network (FAAN) to form Food Allergy Research and Education (FARE). Conflicts of interest The authors declare that they have no conflicts of interest.
References 1. Branum AM, Lukacs SL. Food allergy among children in the United States. Pediatrics 2009;124:1549–1555.
122
2. Wang J. Management of the patient with multiple food allergies. Curr Allergy Asthma Rep 2010;10:271–277.
3. Sampson HA, Ho DG. Relationship between food-specific IgE concentrations and the risk of positive food challenges in children and
Allergy 70 (2015) 120–123 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Kulis and Burks
adolescents. J Allergy Clin Immunol 1997;100:444–451. 4. Chehade M, Mayer L. Oral tolerance and its relation to food hypersensitivities. J Allergy Clin Immunol 2005;115: 3–12. 5. de Leon MP, Glaspole IN, Drew AC, Rolland JM, O’Hehir RE, Suphioglu C. Immunological analysis of allergenic cross-
Introducing foods in the presence of an allergy
reactivity between peanut and tree nuts. Clin Exp Allergy 2003;33:1273–1280. 6. Kulis M, Pons L, Burks AW. In vivo and T cell cross-reactivity between walnut, cashew and peanut. Int Arch Allergy Immunol 2009;148:109–117. 7. Kulis M, Chen X, Lew J, Wang Q, Patel OP, Zhuang Y et al. The 2S albumin allergens of Arachis hypogaea, Ara h 2 and
Allergy 70 (2015) 120–123 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanutallergic mice. Clin Exp Allergy 2012;42: 326–336. 8. Ball H, Luyt D, Bravin K, Kirk K. Single nut or total nut avoidance in nut allergic children: outcome of nut challenges to guide exclusion diets. Pediatr Allergy Immunol 2011;22:808– 812.
123
