INVESTIGATION OF INTERVENTION STRATEGIES FOR Ig-E MEDIATED FOOD ALLERGY IN A ...

168 Pages·2013·1.94 MB·English

INVESTIGATION OF INTERVENTION STRATEGIES FOR
Ig-E MEDIATED FOOD ALLERGY IN A MURINE MODEL
OF COW’S MILK ALLERGY.

by

CIN LAM THANG

Department of Animal Science
McGill University, Montreal, Canada

April 2013

A thesis submitted to McGill University in partial fulfilment of the requirements of the
degree of

DOCTOR OF PHILOSOPHY

© Cin Lam Thang, 2013

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ABSTRACT

Food allergy, an immune-mediated adverse hypersensitivity reaction to ingested food, is
an emerging global health problem that not only causes the disease to susceptible
individuals but also causes psychological impacts and financial damage to their families.
Food allergy incidence is significantly increased in the past decade and currently
estimated at 6-8% in 1 year old children, and 2-4% in older children and adults. Food
allergy is the leading cause of anaphylaxis in children and severe cases may lead to fatal
anaphylaxis reactions. Although continuous efforts have been made to better understand
the nature of allergens, predisposing environmental factors, and the host’s immune
response mechanisms, there is no cure available for food allergy today. In this study, we
have investigated three food allergy reducing strategies by supplementing probiotics, low
doses of allergens and omega-3 poly unsaturated fatty acids (ω-3 PUFAs) in cow’s milk
protein sensitized Balb/c mice. Lactobacillus rhamnosus GG (LGG) supplementation had
tendency to promote Th1 response while VSL#3 provided more potent allergy reducing
effects via inducing intestinal secretory IgA (sIgA). Low doses of allergen administration
offered suppression of allergen-specific immune responses via Treg-mediated active
suppression, indicated by suppressing both allergen-specific Th1 response [reduced BLG-
specific serum IgG2a and elevated IL-12(p40)], and Th2 response [lower BLG-specific
serum IgE and IgG]. Interestingly, mice received both VSL#3 and low doses of allergen
exhibited both allergen-specific active suppression effects and higher sIgA production.
We then investigated the effects of different levels of ω-3 and ω-6 PUFAs in the energy
and fat rich Western-style diet on food allergy development. Observation of elevated
BLG-specific serum immunoglobulins in all experimental mice indicated that both ω-3
and ω-6 PUFAs failed to prevent the development of allergen-specific immune response.
However, ω-3 PUFAs alleviated anaphylactic reactions and the severity of allergic
reaction as indicated by the unchanged rectal temperature, lower hypersensitivity scores,
and Th1-favoured immune responses in BLG-sensitized O3H mice. In general, this study
revealed the promising strategies for treatments and prevention against food allergy in the
near future.
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RÉSUMÉ
Les allergies alimentaires, réactions dues à une hypersensibilité du système immunitaire
après ingestion d’un certain type d’aliments, constituent un problème de santé publique
croissant qui, en plus de dégrader la santé des individus susceptibles, engendre un impact
psychologique et financier pour les familles touchées. L’incidence des allergies
alimentaires a significativement augmenté ces dernières années et le taux est actuellement
estimé être de 6-8% chez les jeunes enfants d’1 an et de 2-4% chez les autres enfants et
les adultes. Les allergies alimentaires représentent la première cause d’anaphylaxie chez
l’enfant et plusieurs cas d’allergies peuvent causer une réaction anaphylactique fatale.
Bien que des efforts continus aient été faits pour tenter de mieux comprendre la nature
des allergènes, les facteurs environnementaux prédisposant aux allergies et les
mécanismes de la réponse immunitaire, aucun traitement contre les allergies alimentaires
n’est disponible à l’heure actuelle. Dans cette étude, nous avons testé trois différentes
stratégies visant à réduire l’allergie alimentaire, à savoir l’utilisation de probiotiques, de
faibles doses d’allergènes et d’acides gras polyinsaturés oméga-3 chez des souris Balb/c
sensibilisées aux protéines de lait de vache. L’administration de Lactobacillus rhamnosus
GG (LGG) a montré une tendance à promouvoir une activation des cellules Th1 alors que
VSL#3 a engendré une plus forte réduction de l’allergie via l’induction de la sécrétion
intestinale d’IgA (sIgA). L’administration de faibles doses d’allergènes a provoqué une
suppression de la réponse immunitaire dirigée contre les allergènes via l’activation des
cellules Treg, ceci étant suggéré par la suppression des réponses immunitaires Th1
spécifique [diminution des IgG2a BLG-spécifiques et augmentation des IL-12(p40)] et
Th2 spécifique [diminution des IgE et IgG BLG-spécifiques]. De plus, les souris ayant
reçu à la fois VSL#3 et des faibles doses d’allergènes ont montré une suppression des
effets de la réponse allergénique et une production plus élevée de sIgA. Nous avons
ensuite étudié les effets de différents niveaux d’oméga-3 et d’oméga-6 dans un régime
alimentaire de type occidental riche en énergie et en matières grasses sur le
développement des allergies alimentaires. L’observation d’une élévation des
immunoglobulines BLG-spécifiques chez toutes les souris expérimentales ont indiqué
que les oméga-3 et -6 n’ont pas réussi à empêcher le développement de la réponse
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immunitaire dirigée contre les allergènes. Cependant, les oméga-3 ont diminué les
réactions anaphylactiques et le degré de sévérité de la réaction allergique, ceci étant
suggéré par un score d’hypersensibilité plus faible, une absence de changement de la
température rectale et une réponse immunitaire favorisée par les cellules Th1. Pour
conclure, cette étude a révélé des stratégies prometteuses pour le traitement et la
prévention des allergies alimentaires dans les années à venir.

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ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to Professor Xin Zhao, my thesis supervisor,
for providing guidance and tireless support in academic, financial, and real life situations
during my study. I was extremely humbled and blessed to have Dr. Zhao as my mentor at
the most important segment of my life.
I am greatly thankful to Dr. Joyce I. Boye, thesis committee member and research
collaborator, of Food Research and Development Centre, Agriculture and Agri-Food
Canada for her support and supervision in my experiments. I am also grateful to Dr.
Benjamin K. Simpson, thesis committee member, for his valuable advice and assistance.
I am indebted to Dr. Kevin M. Wade (Chair), other Faculty members, and administrative
staff, Ms. Barbara Stewart and Ms. Cinthya Horvath, of the Department of Animal
Science for their supports throughout my study.
My special appreciation goes to Ms. Sabine Ribereau of Agriculture and Agri-Food
Canada for arranging material supply and all staff of Small Animal Research Unit
(SARU) for providing a good care to the mice for my experiments.
I also wish to extend my gratitude to all my laboratory mates and fellow graduate
students for their support and friendship. Specifically, I would like to thanks Drs.
Aloysius Ibeagha-Awemu and Baurhoo Bushansingh for their assistance and friendship
that go beyond the laboratory.
Finally, I am very grateful to my parents, family, wife, and daughter Rachel Ciim for
their inspiration, support, encouragement, and understanding.

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CONTRIBUTION TO KNOWLEDGE
Chapter II: This experiment observed the effects of LGG probiotics on CMA
development in Balb/c mice that were sensitized via gavage or i.p. route. While gavage
sensitization of CMP with cholera toxin-B subunit (CTB) failed to induce allergic
reaction, i.p. sensitization with CMP alone (adjuvant free) induced CMP-specific
immediate immune responses. We here report that mice with adjuvant-free i.p.
sensitization followed by oral challenge with CMP were suitable to be used as a model
for food allergy study. In addition, we revealed that LGG probiotic contributed an
immunomodulation effect in CMP-sensitized mice by promoting Th-shifting toward Th1-
dominated responses.
Chapter III: We have examined the effects of single or co-administration of VSL#3 and
low doses of allergen in BLG-sensitized mice. To our best knowledge, this is the first
experiment that determined the combined effects of supplementing VSL#3 and low doses
of allergens in BLG-sensitized mice. We found that VSL#3, unlike LGG, significantly
alleviated allergic reaction by producing sIgA while low doses of allergen
supplementation suppressed the allergen-specific immune responses by Treg mediated
active suppression. Interestingly, mice received the combined treatments (VSL#3 and low
doses of allergen) suppressed allergen-specific immune responses and demonstrated a
higher sIgA production.
Chapter IV: This experiment, for the first time, investigated the effects of different
ratios of ω-6 and ω-3 fatty acids in Western-style diets on BLG-sensitized mice.
Observation of BLG-specific immunoglobulins in all groups suggested that inclusion of
both ω-3 and ω-6 PUFAs in Western-style diet did not avert development of food
allergy. We further revealed that ω-3 had prevented development of anaphylaxis reaction
and reduced the severity of allergic symptom in BLG-sensitized mice.
Overall, this study has examined the effects of probiotics, low doses of allergen,
and supplementation of different ratios of ω-6 and ω-3 fatty acids to diets on cow’s milk
protein allergy development and results from three experiments have potential
implication for development of novel treatments and prevention of food allergy.

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CONTRIBUTION OF AUTHORS
In this thesis, three co-authored manuscripts are presented.

Authors of Manuscript 1 (Chapter II): C. L. Thang, B. Baurhoo, J. I. Boye, B. K.
Simpson, and X. Zhao.
C. L. Thang designed and carried out all experiments. C.L. Thang and X. Zhao performed
data analysis and wrote the manuscript. B. Baurhoo assisted with microbiological and
statistical analyses. J. I. Boye assisted in milk protein purification. B. K. Simpson and J.
I. Boye reviewed the manuscript. This manuscript has been published in Allergy, Asthma
& Clinical Immunology journal. Allergy Asthma Clin Immunol. 2011 Dec 6;7:20.

Authors of Manuscript 2 (Chapter III): C. L. Thang, J. I. Boye, and X. Zhao.
C. L. Thang designed and carried out experiments. C.L. Thang and X. Zhao analyzed
data, interpreted results and wrote the manuscript. J. I. Boye reviewed the manuscript.
This manuscript has been published in Journal of Nutrition. J Nutr. 2013 Feb;143(2):136-
41.

Authors of Manuscript 3 (Chapter IV): C. L. Thang, J. I. Boye, H. N. Shi and X.
Zhao. C. L. Thang designed and carried out experiments. . C.L. Thang and X. Zhao
analyzed data, interpreted results and wrote the manuscript. J. I. Boye and X. Zhao
assisted in experimental design. H. N. Shi assisted in cytokines analysis from
lymphocytes culture supernatants. J. I. Boye and H. N. Shi reviewed the manuscript. The
revised manuscript has been submitted to Molecular Nutrition and Food Research.

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TABLE OF CONTENTS
Page
ABSTRACT...………………………………………..…………………………… ..ii
RESUME …………………………………………….…………………………….. iii
ACKNOWLEDGEMENTS ……………………….………………………............ v
CONTRIBUTION TO KNOWLEDGE ………………………………………..... vi
CONTRIBUTION OF AUTHORS …………………………………………….....vii
TABLE OF CONTENTS ………………………….…….……………………….. viii
LIST OF TABLES ……………………………….………………………………..xiii
LIST OF FIGURES …………………………………….…………………………xiv
LIST OF ABBREVIATIONS ……………………………………………………..xv
CHAPTER I. GENERAL INTRODUCTION AND LITERATURE REVIEW..1
GENERAL INTRODUCTION.……………..……………………………………….1
LITERATURE REVIEW ………. …………….……………………………………..3
1.1 Food allergy ………………………………………………………………………3
1.1.1 Classification of food allergy………………………………………………….5
1.1.1.1 IgE-mediated food allergy (Immediate hypersensitivity)……………..5
1.1.1.2 Non-IgE-mediated food allergy (Delayed hypersensitivity)…………..6
1.1.1.3 Mixed IgE- and non-IgE-mediated food allergy………………………6
1.1.2 Symptoms and diagnosis………………………………………………………7
1.1.3 Major food allergens…………………………………………………………..10
1.1.4 Predisposing factors …………………………………………………………..11
1.1.5 Attempts to reduce severity of food allergy…………………………………...12
1.1.5.1 Attempts to reduce allergenicity of major allergens…………………..13
1.1.5.2 Medications for food allergy symptoms relief ………………………..13
1.1.5.3 Newly developed therapeutic strategies………………………………14
1.1.5.3.1 Allergen-specific therapy…………………………………………..14
1.1.5.3.2 Allergen-nonspecific therapy………………………………………14
1.2 Cow’s milk allergy………………………………………………………………...17
1.2.1 Cow’s Milk in general…………………………………………………………18
1.2.2 Milk proteins/allergens………………………………………………………...18
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1.3 Overview of immune system………………………………………………………21
1.3.1 The role of T cells in immunity………………………………………………...21
1.3.2 Immune response to infectious microorganisms……………………………….23
1.3.3 Immune response to infectious macroparasites and non-infectious stimuli……24
1.4 Immune response to gut microbiota………………………………………………..24
1.4.1 Establishment of gut microbiota……………………………………………….24
1.4.2 The role of intestinal epithelium……………………………………………….25
1.4.3 Host-microbes cross-talk ………………………………………………………27
1.5 Effects of Probiotics in allergy development………………………………………29
1.5.1 General overview of probiotic………………………………………………….29
1.5.2 Beneficial role of probiotics on hosts ………………………………………….30
1.5.3 Rationale for interest of probiotic in allergic disease ………………………….30
1.5.4 Antiallergic mechanisms of probiotics in animal models………………………31
1.6 Oral tolerance development………………………………………………………...32
1.6.1 General mechanism of oral tolerance …………………………………………..32
1.6.2 Development in oral tolerance induction……………………………………….34
1.7 Dietary fatty acids…………………………………………………………………..35
1.7.1 Physiological roles of dietary fatty acids……………………………………….37
1.7.1.1 Energy reservoir…………………………………………………………37
1.7.1.2 Vital components for cell membranes…………………………………..37
1.7.1.3 Precursor for signaling molecules and inflammatory mediators………..37
1.7.2 Changes in Dietary fatty acids intake and allergy development………………..39
1.7.3 Potential immunomodulatory effects of ω-3 PUFAs on allergy………………..41
1.7.3.1 By incorporating into the phospholipids of inflammatory
cell membranes…………………………………………………………42
1.7.3.2 By acting directly to cell surface or intracellular receptors…………….43
1.7.3. 3 Precursors for molecules that have anti-inflammatory
and resolving properties………………………………………………..44
1.8 The use of mouse models in the food allergy study………………………………..44
WORKING HYPOTHESES AND RESEARCH OBJECTIVES ……………………..47
1. Working hypotheses ………………………………………………………....47
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2. Overall research objectives …………………………………………………48
REFERENCES …………………………………………………………………..........48
CHAPTER II. EFFECTS OF LACTOBACILLUS RHAMNOSUS GG
SUPPLEMENTATION ON COW’S MILK ALLERGY IN A MOUSE
MODEL……………………………………………………………………………….67
2.1 ABSTRACT ……………………………………..……….……………………….68
2.2 BACKGROUND ………………………………..…….…………………………..69
2.3 MATERIALS AND METHODS ………………..………………………………..70
Cow’s Milk Proteins …………………………………………………………………………70
Mice…………………………………………………………………………………………….71
Sensitization and Challenge Procedures…………………………………………………..71
Evaluation of Hypersensitivity Symptoms………………………………………………...72
Determination of Serum CMP-specific (IgG1, IgG2a and IgG) and Total IgE……..72
Cytokine Measurements from Spleen Lysates…………………………………………….73
PCR Identification of Fecal Lactobacillus………………………………………………..73
Enumeration of Fecal Lactobacilli…………………………………………………………74
Statistical Analysis……………………………………………………………………………74
2.4 RESULTS …………….………………………………….……………………….75
Hypersensitivity Responses………………………………………………………………….75
CMP-specific Immunoglobulin Levels in Serum…………………………………………75
Cytokine Levels in Spleen Lysates………………………………………………………….75
Fecal Counts and PCR Analysis of Lactobacilli…………………………………………76
2.5 DISCUSSION …………………………………………….……………………...76
2.6 CONCLUSIONS………………………………………………………………....78
2.7 ACKNOWLEDGEMENTS ……………………………………………………...79
2.8 TABLES AND FIRURES………………………………………………………..80
2.9 REFERENCES ………………………………..…….…………………………...87

CONNECTING STATEMENT 1………………………………………………………….92
x