Cow`s milk allergy

Transcription

Cow`s milk allergy

Cow’s milk allergy
immune modulation by
dietary intervention
Bastiaan Schouten
ISBN/EAN: 978-90-393-5210-6
Cover photo, design and thesis lay-out: Bastiaan Schouten
Printed by: GVO | Ponsen & Looijen B.V., Ede, The Netherlands
The printing of this thesis was financially supported by:
Danone Research – Centre for Specialised Nutrition, J.E. Jurriaanse Stichting,
Utrecht Institute for Pharmaceutical Sciences, TNO / Utrecht Center for Food
Allergy, Infection & Immunity Center Utrecht, BD Biosciences, FrieslandCampina
Domo
© 2009 Bastiaan Schouten
All rights reserved. No part of this thesis may be reproduced or transmitted in any
form, by any means, electronic or mechanical, without prior written permission of
the author.
immune modulation by dietary intervention
Koemelkallergie
immuunmodulatie door dieetinterventie
(met een samenvatting in het Nederlands)
Proefschrift
ter verkrijging van de graad van doctor aan de Universiteit Utrecht
op gezag van de rector magnificus, prof.dr. J.C. Stoof,
ingevolge het besluit van het college voor promoties,
in het openbaar te verdedigen op
woensdag 25 november 2009 des middags te 2.30 uur
door
Bastiaan Schouten
geboren op 27 februari 1981 te Utrecht
Promotor:
Prof.dr. J. Garssen
Co-promotoren:
Dr. L.E.M. Willemsen
Dr. L.M.J. Knippels
Contents
Chapter 1
General introduction
9
Chapter 2
Nutrition and food allergy
25
Chapter 3
Acute allergic skin reactions and intestinal contractility
changes in mice orally sensitized against casein or whey
55
Chapter 4
Contribution of IgE and immunoglobulin free light chain in the allergic reaction to cow’s milk proteins
77
Chapter 5
Non-digestible oligosaccharides reduce immunoglobulin free light-chain levels in infants at risk for allergies
95
Chapter 6
Cow’s milk allergy symptoms are reduced in mice fed dietary synbiotics during oral sensitization with whey
103
Chapter 7
Oligosaccharide induced whey-specific CD25+ regulatory T-cells are crucial in the suppression of cow’s milk allergy
in mice
119
Chapter 8
Dietary non-digestible carbohydrates inhibit the allergic effector response via induction of casein-specific CD25+
regulatory T-cells in orally sensitized mice
141
Chapter 9
iNKT cells contribute to the allergic response in cow’s milk protein sensitized mice
163
Chapter 10
General discussion
181
Miscellaneous
Summary
198
Nederlandse samenvatting
202
Dankwoord
207
Curriculum Vitae
212
List of publications
213
Abbreviations
217
197
1
General introduction
General Introduction
1
The adaptive immune response and regulatory T-cells
The human body is continuously under attack by all kinds of microbes like bacteria, fungi,
parasites and viruses and has at least three different parts to overcome these invaders.
The first is a physicochemical barrier, the epithelial lining of the skin, respiratory and
intestinal mucosa forming intrinsic tight junctions which prevent paracellular passage
of antigenic particles. In the intestinal and respiratory mucosa the mucus layer forms an
extrinsic barrier, preventing direct contact of the microbes with the epithelial lining. The
second line of defense is the innate immune system, which comprises the complement
system, natural killer cells, basophils, neutrophils and eosinophils, they are rigid and attack
pathogens non-specific. The adaptive immune system forms the third immune defense and
can be divided in a humoral and a cellular part which are able to recognize and remember
specific antigens. In allergic individuals an adaptive immune response is generated towards
a harmless antigen. In food allergic patients immunity instead of oral tolerance is raised
towards food proteins. This results in a TH2 type effector immune response and, more
chronically, involves an allergen specific TH1 response [1]. Other effector cells like TH17, TH3
and others have not been implicated in the pathology of cow’s milk allergy. Tolerance or
immunity to food antigens is generated in the gut associated lymphoid tissue (GALT) (Figure
1). The Peyer’s patches (PP) and mesenteric lymph nodes (MLN) are the inductive sites
of the GALT, here immunity or oral tolerance is induced. Food proteins can induce clonal
deletion or anergy (high dose tolerance), rendering cells non-responsive for the antigen,
or active suppression by regulator T-cells (Treg cells) (low dose tolerance) [2]. Antigens are
taken up by dendritic cells (DC) in the PP or lamina propria and travel to the MLN [3]. In the
MLN and PP, DC present antigens to naïve T-cells and induce tolerance (anergy/deletion or
suppression by Treg cells) or an effector response against the antigen. Antigen-responsive
T-cells (CD4+) leave the MLN, travel subsequently through the bloodstream and home back
into the intestinal mucosa (lamina propria) and transfer to the peripheral immune system
(e.g. spleen) [2, 3]. Different Treg cell subsets are able to actively suppress effector T-cell
responses and regulate immune homeostasis of the GALT. Antigen specific T-cells or Treg
cells from the MLN in this respect can relocate local induced immune responses to the
periphery. Cytokines produced by allergic TH2 effector cells instruct isotype switching and
maturation of B cells resulting in immunoglobulin (Ig)E production in human and in mice
both IgE and IgG1 antibodies were produced against this allergen. These antibodies bind to
Fc receptors on mast cells and after a second allergen exposure, receptor crosslinking results
in mast cell activation and degranulation. Mast cell mediator release such as histamine and
leukotriens cause the acute allergic symptoms like eczema, diarrhea, asthma and in some
cases anaphylaxis.
The humoral response of the murine adaptive immune system is complex and covers both
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Chapter 1
TH1/Treg (IgG2a and IgA), TH2 (IgE, IgG1) and Ig free light chain (Ig-fLC) molecules. Ig-fLC
are present in serum and their production is augmented under inflammatory conditions
including allergic as well as some autoimmune diseases [4-6]. For example, in patients
suffering from multiple sclerosis free kappa light chains correlate with disability prognosis
[7] and in rheumatoid arthritis there is a significant correlation between kappa and lambda
fLC and the disease activity score [8]. There is another subset of T-cells that regulates TH1
and TH2, these are the Treg cells. The Treg cells are a heterogeneous group of cells that
act via cytokine secretion or via direct cell-cell contact. In food allergy the delicate balance
between oral tolerance and hypersensitivity is regulated by these cells [2, 3]. Cells with
regulatory properties include TGF-β-producing TH3 cells, IL-10-producing Tr1 cells, adaptive
and natural Foxp3 regulatory T (Treg)-cells and natural killer (NK) T cells [9-11]. In addition,
the TGF-β and IL-10 producing naturally occurring CD4+CD25+Foxp3+ Treg cells were described
to be important for maintaining peripheral tolerance and for suppression of CD4+ and CD8+
T cell proliferation [12, 13]. Treg represent 5-10% of the CD4+ T lymphocyte population and
express the transcription factor forkhead box p3 (Foxp3), which is confirmed as a key gene
for generation and maintenance of Treg cells. Adaptive Treg cells are educated in the GALT
in particular and cells have similar surface markers; however their inhibitory capacities
act via cell-cell contact instead of cytokines [14]. Treg cells are believed to alter mast cell
degranulation via IL-10 or TGF-β production or cell-cell contact [15, 16]. In addition, Treg
cells have been shown to suppress Fc-receptor expression on mast cells which would reduce
the capacity of these cells to degranulate and induce an acute skin response [12]. NKT cells
express a T cell receptor, but recognize glycolipids presented by CD1d molecules [17, 18]. In
allergic asthma α-galactosylceramide induced NKT cells have been shown to suppress the
airway hyper responsiveness, hence also NKT cells can modulate the allergic response [19].
Food allergy is a growing problem in Western Europe and the USA. Clinical symptoms may
involve the skin, respiratory tract and gastrointestinal tract, which can even lead to a systemic
anaphylactic reaction [20, 21]. Cow’s milk allergy (CMA) is one of the leading causes of food
allergy in adults [22]. In developed countries approximately 2 to 3% of infants exhibit CMA.
Although most infants outgrow CMA before their fifth year, IgE-mediated CMA predisposes
the development of other (food) allergies and even asthma later in life [23, 24]. Cow’s milk
contains two main protein classes, which are the caseins (30 g/L) and whey proteins (5 g/L).
The caseins consist mainly of αS1-, αS2-, κ- and β-casein, whereas whey proteins comprise
of β-lactoglobulin, α-lactalbumin, bovine serum albumin, serum immunoglobulins and
lactoferrin [25]. Large population studies with cow’s milk allergic infants have shown that
the major allergens are β-lactoglobulin and αS1-casein [26, 27].
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12
Figure 2. Working hypothesis of this thesis. a Dietary cow’s milk proteins such as casein and whey can cause sensitization by breaking
tolerance in the intestinal mucosa; hydrolysates and amino acid formulas are developed to reduce the risk of sensitization. b If these food
components (and other substances) are taken up by M-cells, antigen presenting dendritic cells (DC) in the Peyer’s patches (PP) present
these proteins to naïve T cells (TH0) , which differentiate, dependent on the cytokine milieu, into TH1 or TH2 effector cells (immunity) or
into regulatory T (Treg)-cells when tolerance develops. In case of high dose tolerance anergy or clonic deletion occurs. c DC’s can also
traffic from the PP to the MLN or d sample antigen from the lumen and effector sites and than traffic to the MLN were antigens are
presented to naive T-cells. e Generated Treg cells or effector cells enter the bloodstream and home back to the intestinal mucosa were they
will become resident in the lamina propria. f Treg cells or effector T-cells generated in the MLN can also traffic to the peripheral immune
system and transfer tolerance or immunity. g/h B-cells expanse and mature in spleen and traffic back to the effectors site where they
produce specific IgE, IgG1, IgG2a, Ig-fLC or IgA. i These Ig’s bind to mast cells, which resides and wait for another antigen exposure. j This
immune mechanism can be modulated by food components e.g. prebiotics, probiotics, the combination (synbiotics) and omega-3 fatty acids.
TH: T helper cells; Treg: Regulatory T cells; NKT: Natural Killer T cells; DC: dentritic cells; pCH: partial casein hydrolysate; eCH: extensive casein
hydrolysate; pWH: partial whey hydrolysate; eWH: extensive whey hydrolysate; AA: amino acids
Chapter 1
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1
Development of a mouse model for orally induced CMA
Animal models of CMA provide a tool to unravel mechanisms involved in CMA and may
explore new therapeutic and preventive approaches. However, in most existing food allergy
models the animals are not sensitized via the oral route (e.g. intra peritoneal), while in
reality most humans are sensitized orally [28-36]. To further assess mechanisms underlying
cow’s milk allergy and/or test new concepts for prevention and/or treatment of CMA, the
major goal of the present thesis was to introduce and develop new tools to address the
pathological changes upon oral sensitization with casein and whey in mice. In this model
humoral Ig and Ig-fLC responses were determined and the involvement of Treg cells in
the allergic response was evaluated. Furthermore measurement of the acute allergic skin
response, as a potential equivalent of the human skin prick test, was introduced as a new
readout of systemic sensitization. In this thesis the preventive effects of dietary intervention
with prebiotic non-digestible oligosaccharides and a candidate probiotic bacterial strain the
Bifidobacterium Breve M-16V were studied.
Prebiotics
Non-digestible carbohydrates form a major constituent of breast milk. Human milk contains
approximately 7-12 g/L oligosaccharides [37]. At least 130 different oligosaccharides
have been isolated from human milk and the two main categories are neutral and acidic
oligosaccharides [38]. These human milk oligosaccharides, but also specific dietary fibers,
such as inulin derived from chicory, can selectively support growth of health promoting
commensal bacteria in the gut and are therefore called prebiotics [39-42]. Prebiotics were
defined as “food ingredients that beneficially affect the host by selectively stimulating the
growth and/or activity of one or a limited number of bacterial species already resident in
the colon, and thus attempt to improve host health” [39]. Recently this definition is adapted
to: “A prebiotic is a selectively fermented ingredient that allows specific changes, both in
the composition and/or activity in the gastrointestinal microflora that confers benefits upon
host wellbeing and health” [43]. These bacterial species comprise among others the Grampositive Lactobacillus genus and the Bifidobacterium genus. Besides their effects on the
intestinal microbiota non-digestible carbohydrates may improve absorption of minerals and
water holding capacity of the fecal bulk due to their physicochemical properties [44]. Shortchain fatty acids, released by for example lactic acid producing Bifidobacteria and Lactobacilli
upon fermentation of prebiotics, are essential nutrients for intestinal epithelial cells and
support gut function [45]. Besides the physicochemical properties of prebiotics, these nondigestible carbohydrates can enhance non-specific defense mechanisms [46]. There are
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Chapter 1
different types of dietary fibers e.g. (hemi)cellulose, lignin, β-glucans, pectins, gums, inulin
and oligosaccharides [47]. Different non-digestible oligosaccharides with specific properties
are obtained or manufactured from natural sources and used as dietary supplement.
Dietary fibers are extracted from plants and/or produced enzymatically or chemically [48].
Short-chain galacto-oligosaccharides (scGOS), long-chain fructo-oligosaccarides (lcFOS)
and pectin-derived acidic oligosaccharides (pAOS) are some examples of non-digestible
oligosaccharides that mimic the functionality and molecular size distribution of human milk
oligosaccharides and have been investigated in this thesis (Figure 2). Prebiotic scGOS are
produced by enzymatic elongation of galactose (derived from lactose) by β-galactosidase,
with a degree of polymerization (dp) of 3–8. Chicory inulin is a polymer from D-fructose
residues linked by β(2-1) bonds with a terminal α(1-2) linked D-glucose (dp 2-60). lcFOS are
produced from inulin and have a mean dp >23, whereas scFOS have mainly a dp <23. lcFOS
are added to infant formulas. pAOS is a mixture of linear oligomers and small polymers of
A
B
CH2OH
CH2OH
CH2OH
O
OH
O
O
CH2OH
OH
OH
O
O
OH
OH
O
OH
OH
OH
CH2OH
OH
OH
O
O
OH
OH
CH2
n<6
OH
COOR
CH2OH
O
mean
n>21
O
O
OH
COOR
CH2OH
OH
O
O
COOR
OH
O
O
OH
C
OH
OH
OH
OH
n<18
OH
Figure 2. Structures representing non-digestible carbohydrates used in this thesis. a Enzymatic
elongation of galactose using β-galactosidase results in short-chain galacto-oligosaccharides
(dp of 3-8). Structure represents two β(1-4) linked galactose molecules connected to a
terminal glucose monomer. b Long-chain fructo-oligosaccharides are produced from chicory
inulin (dp of 7-60 and mean dp > 23). Structure represents one glucose molecule and two
fructose molecules connected with a β(2-1) linkage. c Basic chemical structure of pectin
derived acidic oligosaccharides consisting of α(1-4) linked galacturonic acid molecules (dp of
1-20) (R=H or R=CH3). Adapted from Vos et al. [49].
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galacturonic acid produced from from food grade pectin by pectin cleaving enzymes and have
a dp of 1-20. Currently, it is not feasible to produce the precise human milk oligosaccharides.
However, the used specific oligosaccharides have proven efficacy in clinical trials since they
reduce the incidence of allergic disease and have been shown to enhance fecal Bifidobacteria
counts [50-54]. Another prebiotic fructo-oligosaccharide, kestose, showed to be effective in
reducing the incidence of atopic dermatitis in infants as well [55].
The intestinal microbiota and probiotics
The human gastrointestinal tract is composed of several compartments. The majority of
macro- and micronutrients from food is absorbed in the duodenum and jejunum of the
small intestine, while the ileum has residual absorptive capacities. After the ileum the chyme
enters the large intestine which is mainly involved in water and electrolyte absorption.
However, the colon is also the main reservoir for the intestinal microbiota which comprises a
multitude of different bacterial strains. The human gastrointestinal tract contains ten times
more bacteria than the total number of cells in the human body [56]. The colonization of
the gastrointestinal tract by bacteria starts immediately after birth [57, 58]. Most of the
bacteria in the gastrointestinal tract are commensals, bacteria that do not negatively affect
human health. A well balanced intestinal microbiota consists of less than one percentage
of pathogens, e.g. Klebsiella pneumoniae and Salmonella [59, 60]. A particular class of
bacteria, i.e. probiotic bacteria, has been shown to be beneficial for the host. Probiotics can
be defined as: “live microbial food ingredients that alter the enteric microflora and have a
beneficial effect on health” [61]. Bifidobacteria together with Lactobacilli are well known for
their health promoting functions. Bifidobacteria alone comprise of more than 60% in fecal
samples of children [62]. Evidence is accumulating that both Bifidobacteria and Lactobacilli
may account for as much as 90% of the total flora of breast fed infants [63]. Some of
these are selected species and used as probiotics [20, 64, 65]. Probiotics stabilize the gut
microflora hereby preventing pathogenic infection and pathogen induced diarrhea. Ongoing
studies are being performed to unravel the immune modulatory effects of probiotics and
their efficacy to reduce the incidence or relieve symptoms of various diseases caused by
dysregulated immune responses [66].
Breast fed neonates have higher stool counts of Bifidobacteria compared with babies fed
standard infant formula. When added to standard milk formula, the prebiotic mixture scGOS/
lcFOS was found to enhance Bifidobacteria fecal counts in bottle fed infants to levels almost
similar to those found in breast fed infants [67]. High Bifidobacteria counts were found to
associate with a reduced risk of development of atopic disease in infants of westernized
countries [20, 68]. Therefore probiotics have been investigated in multiple clinical studies for
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Chapter 1
the prevention and/or treatment of allergic diseases [65, 69-78]. However, there is still no
consensus regarding the efficacy of these bacteria in having immune-modulatory effects and
their clinical relevance [79]. In two Cochrane meta-analysis of randomized controlled trials
the effectiveness of probiotics in the prevention or treatment of eczema is not confirmed
[80, 81]. Several factors are suggested which influence the outcome of the studies, e.g.
probiotic strain or mixture, environmental factors, intervention duration and timing and
bacterial load [82, 83]. Most of the clinical studies are performed using Lactobacillus strains,
however in breast fed infants in particular Bifidobacteria have been found in the feces [84].
In a bacterial strain comparison study with six different Bifidobacterium and Lactobacillus
strains, the Bifidobacterium breve M-16V was identified as being the most potent antiallergic strain in the treatment of ovalbumin-induced allergic asthma in mice [85]. Based
on these results the Bifidobacterium breve M-16V was the strain of choice in the studies
conducted in this thesis.
Synbiotics
To improve human health and reduce the development of allergies the combination of
prebiotics and probiotics, called synbiotics, gain increased scientific attention. The hypothesis
that pre- and probiotics together are more effective than the use of single preparations is
very promising, although not many studies have been performed in relation to allergy [8689]. The (preclinical) studies that have been carried out show potential for synbiotics to
reduce the incidence and severity of allergic disease, since synbiotics were found to increase
total serum (soluble) IgA antibodies and to reduce dermatitis-like skin lesions [86, 88].
Aim and scope of this thesis
This thesis provides preclinical data concerning prevention of orally induced cow’s milk allergy
using dietary intervention with non-digestible carbohydrates and/or Bifidobacterium breve
M-16V. Furthermore some of the underlying mechanisms of the pathophysiology of cow’s
milk allergy are studied and in particular the function of Treg cells is addressed. Chapter 2
provides a literature study on food allergy and nutrition, its scope is wider than that of cow’s
milk allergy solely, and multiple routes of intervention in the process of sensitization and
allergy are described. Chapter 3 describes the establishment of a murine model of orally
induced sensitization with casein or whey protein adapted from Li et al. [35] and Frossard
et al. [36]. These groups have introduced an oral mouse model for whole cow’s milk protein
or β-lactoglobulin, whereas in this thesis mice were sensitized with the complete whey or
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casein protein mixture. In Europe most of the infant formulas are based on whey proteins,
whereas in the USA the majority of infant formulas are based on casein proteins, however
infant formulas with a mix of both casein and whey are on the market as well. We choose
to develop a casein and a whey model. Surprisingly, the underlying mechanisms responsible
for either casein- or whey-sensitization were found to be different. Chapter 4 describes the
involvement of immunoglobulin free light chain (Ig-fLC) in the allergic effector response in
casein sensitized mice using Ig-fLC blocker F991 in the active model and in passive transfer
studies with spleen supernatants. The potential contribution of Ig-fLC in human allergic
disease was evaluated as well. Ig-fLC (λ and κ) concentrations were determined in plasma
samples from high risk children who developed atopic dermatitis (AD) and compared to
children who did not develop AD. Chapter 5 describes the results of Ig-fLC measurement
in sera of children at risk who were supplemented with the prebiotic mixture scGOS/lcFOS
(Immunofortis®) for six months. The infants in this trial had fewer infections and fewer
children suffered from atopic dermatitis [49-51]. In chapter 6 a dietary intervention study,
with the same prebiotic mixture as tested in the clinical trial (scGOS/lcFOS), the candidate
probiotic strain (Bifidobacterium breve M-16V) and the combination of both (synbiotics) was
performed in whey sensitized mice in a preventive setup. The effectiveness of the diets in
reducing the acute allergic skin response, anaphylaxis and multiple serological parameters
was studied. In chapter 7 a third potential effective non-digestible carbohydrate, pAOS, was
added to the already potent scGOS/lcFOS mixture and fed to the mice during oral sensitization
with whey. In these mice the contribution of CD25+ regulatory T cells in the reduction of
the allergic effector response caused by the diet was explored using adoptive transfer
experiments. Chapter 8 describes in vivo depletion studies to explore CD25+ regulatory T
cell function and in addition, an adoptive transfer experiment was performed to study the
role of CD4+CD25+ Treg cells in more detail. Chapter 9 describes the contribution of another
subset of regulatory T cells, the NKT cells, in allergic sensitization with whey using NKT cell
agonist α-galactosylceramide. Chapter 10 provides an overall discussion of the results of
this thesis putting together a small part of the cow’s milk allergy puzzle.
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23
1
2
Bastiaan Schouten1
Betty C.A.M. van Esch1,2
Léon M.J. Knippels2
Linette E.M.Willemsen1
Johan Garssen1,2
1
Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical
Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands
2
Danone Research – Centre for Specialised Nutrition, Wageningen, The Netherlands
To be submitted for publication
Content
2
Abstract
27
1.
Introduction
28
2.
Food Allergens
29
3 Diagnosis 31
3.1 Food Challenges 31
3.2 Skin Prick Test 31
3.3 Specific Immunoglobulins 31
4. Dietary intervention 32
4.1 Prevention 34
4.1.1 Breast Milk 34
4.1.2 Hypoallergenic Infant Formulas 34
4.1.3 Introduction of Solid Foods 35
4.1.4 Prebiotics 35
4.1.5 Probiotics 37
4.1.6 Synbiotics 38
4.1.7 Postbiotics 39
4.1.8 Polyunsaturated Fatty Acids (PUFA) 39
4.1.9 Imprinting During Pregnancy 40
4.2 Treatment 40
4.2.1 Epitope Exclusion 41
4.2.2 Hydrolysates 41
4.2.3 Pre-, Pro-, Synbiotics and PUFA 41
4.2.4 Traditional Chinese Herbal Medicine 42
4.2.5 Immune Therapy and Induction of Oral Tolerance 42
5. Conclusions
43
6. References
44
26
Chapter 2
Abstract
The increased incidence of food allergies gives rise to higher medical costs, but also warrants
more intensive preclinical and clinical research in order to prevent and/or treat food allergy.
In general, immunoglobulin (Ig)E and non-IgE mediated food allergies can be distinguished
for which major food allergens have been identified. Standard operating procedures in
diagnoses of food allergies have been developed and perfected; of which double-blind
placebo controlled food challenges are the internationally accepted and recommended
golden standard. Many dietary factors have been identified and tested for their potential to
alter the outcome of food allergy in both pre-clinical as well as clinical studies. Among these
components are prebiotics, probiotics, synbiotics, postbiotics, traditional Chinese herbal
medicine, poly unsaturated fatty acids and peptides. There are several concepts that might
inhibit the onset and severity of food allergic disorders. The timing of the intervention is a
crucial aspect. Intervention during early life and even during pregnancy might interfere with
immune development and the consequence in terms of allergy in the offspring. This can be
recognized as the so-called immune imprinting hypothesis. This review aims to provide an
overview regarding different dietary procedures for either prevention and/or treatment of
food allergy.
27
2
1. Introduction
2
There are five different types of immunological hypersensitivity reactions, type I-VI. Type
I is immunoglobulin (Ig)E dependent and results in acute (<1 h) symptoms due to mast
cell activation upon antibody (especially IgE) receptor crosslinking. Type II, III and IV
hypersensitivity reactions result in delayed or intermediate delayed symptoms, 6-92 h, upon
allergen challenge. Type II is IgG mediated and directed against cell-surface antigens. Type III
is antibody mediated also and is directed against soluble antigens. Type IV hypersensitivity
reactions are T-cell mediated and different mechanistic steps are involved in this complex
type of hypersensitivity. An important feature is orchestrated by TH1 cells and results in
macrophage activation. Additionally, TH2 cells and eosinophils are involved as well in some
subtypes. Finally, cytotoxic T-cells seem to play a role in this inflammatory type IV process
as well, although this highly depends on the type of antigen or hapten the cells encounter.
Type V uses a stimulating antibody, IgG antibodies react with tissue receptors like in Graves
disease and also via antibody dependent cell mediated cytotoxicity. Target cells coated with
antibodies are destroyed by specialized killer cells (e.g. NK cells and macrophages), which
bear receptors for the Fc portion of the coated antibodies. Food allergy (FA) is the leading
cause of anaphylaxis (type I hypersensitivity) treated in hospital emergency facilities in
Western Europe and the USA and will be the major focus of this review.
Adverse food reactions include any abnormal reaction resulting from ingestion of a food, or
food additive, and might be the result of food intolerances (e.g. lactase deficiency) or food
hypersensitivity/allergy [1]. Food hypersensitivity, as stated by the European Academy of
Allergy and Clinical Immunology nomenclature task force [2], can be divided in non-allergic
food hypersensitivity, in which an immunological mechanism is excluded, and FA, in which
an immunological mechanism is defined or strongly suspected. The latter can be divided into
IgE-mediated FA (type I) and non-IgE-mediated FA (Figure 1). However, it should be realized
that a third category exists as well which is a mixed IgE- and non-IgE mediated group. In the
first few years of life the prevalence of food hypersensitivities is highest, affecting about 6%
of infants under the age of 3 years [3]. In westernized countries the prevalence of allergic
disorders has increased the last decades, however the underlying ethiology is still unclear.
Decline in breast-feeding, early introduction of solid foods, construction of air-tight highlyinsulated homes and changes in diet (for example reduced intake of n-3 polyunsaturated
fatty acids) are probably part of the explanation [4]. In addition, the ‘hygiene’ or ‘microbial
deprivation’ hypothesis is suggested, which states that a lower exposure to infectious
agents during early childhood might cause a rapid rise in atopic disorders due to defective
development of the adaptive immune system [5].
28
Chapter 2
Adverse food reactions
Intolerance
Food hypersensitivity
e.g. lactose intolerance
Food allergy
2
Non allergic food hypersensitivity
immune system not involved
IgE‐mediated food allergy
Non‐IgE‐mediated food allergy
e.g. immediate type of food allergy
e.g. delayed type of food allergy
Figure 1. Nomenclature of food hypersensitivity (adapted from [2]).
2. Food Allergens
Among hundreds of different proteins in the human diet numerous are identified as
allergens. Only a small part of them is responsible for the majority of hypersensitive adverse
food reactions. The eight most common food allergies are cow’s milk, egg, peanut, tree
nuts, fish, shellfish, soy and wheat allergy. The ‘big eight’ food allergens are well described
and characterized (Table I).
Cow’s milk allergy has been regarded by pediatricians as a typical FA for several centuries
[6]. It is the first allergy that children can develop in life, usually within their first year of life,
and affects 2-3% of the children; 4 out of 5 children develop clinical tolerance by their fifth
year of life. About 60% of these patients have cow’s milk specific IgE-mediated reactions [7,
8].
Chicken egg protein allergy is the second most common cause of FA in children, with an
estimated prevalence of 2.5% [3, 9]. The majority of children outgrow their food allergy by
school age [10].
Peanut allergy is regarded as the most severe, most dangerous type of food allergy. Most
reported cases of anaphylaxis are caused by peanut, often induced by almost deniable
doses. In contrast to cow’s milk allergy this allergy remains for life. The prevalence rates
excess 1% [11, 12].
Nuts are known for their beneficial health effects since they may reduce the risk of
cardiovascular disease and diabetes. Nuts have many favorable ingredients such as vitamins,
29
polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids [13]. However, many
tree nuts also can cause severe or less severe allergic reactions; the most common allergic
nuts are hazelnut, walnut, cashew and almond. Other nuts like Pecan, Brazil, macadamia,
pistachio, chestnut and coconut cause less severe allergic reactions [14, 15].
2
Fish and shellfish (seafood) allergy comprises 3 divisions of sea organisms, the Chordata,
Mollusca and the Arthropoda, these include different kinds of fish (vertebrates), shrimps, crab
(Crustacea) and mollusks [16, 17]. Europe, Spain, Portugal and obviously the Scandinavian
countries have the highest fish consumption and prevalence of fish allergy. [18].
Soybean is a member of the legume family and an important nutrient, in particular in formula
feeding as a substitute for cow’s milk [19, 20]. Soy allergy has been described primarily in
young children with atopic dermatitis (AD) [21]. The immunological and the clinical basis is
highly complex, which complicates the diagnosis of soy allergy and the advice in regard to
risk management.
Table I. Major food allergens of the ‘big eight’ that have been identified.
Food allergy
Major allergens (protein class)
References
β-lactoglobulin, α-Lactalbumin, Bovine serum
albumin (BSA), αS1-casein, αS2-casein, β-casein,
κ-casein
[1, 25]
Chicken egg white
allergy
Gal d 1 (ovomucoid), Gal d 2 (ovalbumin), Gal
d 3 (ovotransferrin), Gal d 4 (lysozyme), Gal d 5
(α-Livetin)
[1, 26, 27]
Peanut allergy
Ara h 1 (vicilin); Ara h 2, Arg h 6, Arg h 7 (conglutin);
Ara h 3, Arg h 4 (glycinin); Ara h 5 (profilin); Ara h
8 (nonfusion protein); peanut oleosin
[28-33]
Tree nut allergy
Cas s (chestnut), Cor a (hazelnut), Ber e (brazil
nut), Jug n (black walnut), Jug r (English walnut),
Ana o (cashew nut)
[1, 14, 15]
Seafood allergy
(fish and shellfish
allergy)
Met e 1 (shrimp), Hom a 1 (American lobster),Pan
s 1 (spiny lobster), Cha f 1 (crab), Sal s 1 (Atlantic
salmon), Gad c 1 (cod), Sco s 1 (mackerel)
[1, 16, 17,
34]
Soy allergy
Gly m 1.0101 (hydrophobic protein), Gly m 2 (hull
protein), Gly m 3 (profilin), Gly m 4, Gly m BD 30 k
(vacuolar protein), glycinin, vicilin, β-conglycinin,
Kunitz trypsin inhibitor
[1, 35, 3638]
Wheat allergy
Serpin, α-amylase inhibitor,
molecular weight glutenin
[39, 40]
30
γ-gliadin,
low
Chapter 2
Wheat allergy and celiac disease are two different disorders. Celiac disease is caused by
gluten intolerance which is believed to be in part non-immune mediated. However, gluten
reactive T-cells have also been implicated in the pathophysiology of the disease [22]. In
contrast, wheat allergy is an IgE mediated allergy. There are two forms of wheat allergy,
bakers’ asthma (occupational asthma caused by flour inhalation) and food allergy to wheat
proteins [23, 24].
3. Diagnosis
3.1 Food Challenges
The most effective approach to determine whether a person is allergic to food ingredients
is by an oral challenge with the particular food. The risk of an anaphylactic reaction is
always present and caution is needed when performing such a diagnostic food challenge.
The oral food challenge can be performed open, single-blind placebo controlled or doubleblind placebo controlled (DBPCFC) [41]. The latter is the golden standard for research and
clinical purposes. Subjective symptoms are eliminated with the DBPCFC test since both the
clinician and patient are unaware whether the placebo or food of interest is tested [42, 43].
Difficulties with the single-blind and the DBPCFC is to mask the food and flavor. Often this is
performed by using another food or capsules.
3.2 Skin Prick Test
The skin prick puncture test (SPT) is commonly used to detect sensitization to certain foods.
This SPT is performed to screen patients with suspected IgE-mediated FA. Food extracts are
diluted and applied and the skin is punctured with a needle through the epidermis [44]. A
positive (histamine) and negative (saline) control are applied as well. A patient is sensitized
for a certain food when the wheel diameter is 3 mm or larger compared to the negative
control. When compared to the DBPCFC the positive predictive accuracy of the SPT is less
than 50% [45].
3.3 Specific Immunoglobulins
There are several analytical tools to determine allergen specific serum IgE levels. However
31
2
2
the most recent, best validated, common one is the Pharmacia CAP-System FEIA. The
detection limit of this system is 0.35 kU/L, therefore patients with lower levels are regarded
as non-sensitized and patients with IgE levels above 0.35 kU/L are regarded as sensitized [4649]. Although allergen specific IgE is typically involved in induction of the allergic symptoms
other isotypes of allergen specific immunoglobulins (Ig) such as IgG have the properties to
block the binding of IgE and therefore inhibit mast cell degranulation [50]. Ruiter et al. [51]
demonstrated that the maintenance of oral tolerance to cow’s milk is characterized by high
levels of cow’s milk specific IgG4 in combination with low levels of cow’s milk specific IgE.
Therefore, measurements of specific immunoglobulins will be more useful if all isotypes are
determined and taken into account. Especially ratios between IgE and IgG isotypes might be
predictive for sensitization linked to allergic symptoms and to tolerance.
A subgroup of patients do have the symptoms of immediate (mast cell mediated) type of
hypersensitivity, however they lack the presence of specific IgE. There are indications that
immunoglobulin free light chains are involved in some hypersensitivity disorders [52]. In
particular preclinical data are available [53-55], however there are a few clinical studies
in relation to immunoglobulin free light chains as well [56] (Schouten et al. submitted).
Complete antibodies have light chains integrated in their structure, however immunoglobulin
light chains are released as singular (free) molecules also. Immunoglobulin light chains
are produced and secreted by B lymphocytes or plasma cells. Also Immunoglobulin light
chains bind to mast cells and upon encounter with the specific allergen the mast cell
degranulates and a type I hypersensitivity reaction is induced [57, 58]. More data regarding
immunoglobulin free light chains are needed to address their utility as a predictive and/or
diagnostic biomarker.
4. Dietary intervention
One of the major focus areas in this review is the possibility of dietary intervention to
prevent or treat FA (Figure 2). Preventive intervention protocols preferably start early in
life or importantly already during pregnancy since this might affect the immune system of
the offspring. This phenomenon is called immune imprinting and as a consequence of the
mother’s dietary restrictions or additives the offspring will develop less, or less severe, allergy.
Currently, large cohort studies are being conducted to find evidence for this hypothesis.
32
Chapter 2
2
Figure 2. Schematic representation of the sensitization and effector phase occurring in food
allergic patients. These are respectively targets of prevention (4.1) and treatment (4.2) using
dietary intervention to control food allergy. Instead of oral tolerance, food allergic patients
develop an IgE mediated allergic reaction towards harmless food antigens. Prevention of
development of allergy should take place before or during the sensitization phase. Dietary
intervention may target allergen presentation to naïve T-cells in the GALT (TH1 instead of
allergic TH2 polarization), enhance regulatory T-cell function (suppresses effector response)
and/or reduce allergen specific IgE production by B-cells. Possible preventive approaches
involve breast milk (4.1.1), hypoallergenic infant formulas (4.1.2), timing of introduction of
solid foods (4.1.3), prebiotics (4.1.4), probiotics (4.1.5), synbiotics (4.1.6), postbiotics (4.1.7),
polyunsaturated fatty acids (PUFA) (4.1.8) and imprinting during pregnancy (4.1.9). There
might be overlap between the preventive and the treatment strategies. Treatment protocols
focus on reduction of mast cell degranulation, which may occur direct or indirect via altered
or reduced antibody production by B-cells or support of regulatory T-cell function. Ultimately
oral tolerance is induced (desensitization). Possible treatment approaches include epitope
exclusion (4.2.1), the use of hydrolysates (4.2.2), pre-, pro, synbiotics and PUFA (4.2.3) and
traditional Chinese herbal medicine (4.2.4). PP: Peyer’s Patch; MLN: mesenteric lymph node;
apc: antigen presenting cell; Treg: regulatory T-cell; TH1: T helper 1 cell; TH2: T helper 2 cell.
33
4.1 Prevention
2
Strategies to prevent or reduce sensitization for food allergens include dietary restriction
(allergen avoidance) or dietary intervention to actively support the process of naturally
occurring oral tolerance induction via improved immune development.
4.1.1 Breast Milk
The best nutritional intervention in early life is breastfeeding. It provides a unique
combination of lipids, proteins, carbohydrates, vitamins, and minerals. Furthermore, there
are numerous bioactive components in it with immunological properties like soluble IgA,
antioxidants, oligosaccharides, bacterial fragments, Toll-like receptor ligands, cytokines,
hormones, fatty acids and many more. Each component can individually, additionally or
synergistically act on the immune system of the neonate. The breast milk content changes
over time to ensure optimal passive and active protection and growth for the child [59-61].
However, the quality of the breast milk might vary between mothers and therefore also
the effectiveness in the induction of oral tolerance. Little is known about the influence of
dietary intervention on the quality of breast milk and more preclinical as well as clinical data
are needed. Improvement of the quality of breast milk via a healthy balanced diet of the
mother is a promising approach. It is known, however, that food antigens as well as IgE can
be transferred to breast milk and therefore potentially can act as a risk factor for children
genetically more susceptible for developing allergic disease [60]. Usually, the advantage of
breast feeding is larger than the potential risk of sensitization.
4.1.2 Hypoallergenic Infant Formulas
Hypoallergenic infant formulas play a crucial role in the prevention of allergies in high risk
children as well as in the treatment (4.2.2). Prevention might occur either by avoidance of
the allergen early in life or by the induction of oral tolerance. It is believed that avoidance of
the allergen might be a good approach for the prevention of allergy. However, for example
cow’s milk allergy might predispose the development of other allergies later in life. Therefore
active induction of immunological oral tolerance may be a preferable approach, since it
may also positively affect the atopic constitution of the child. Hypoallergenic infant formulas
or hydrolysates are categorized into partial and extensive hydrolysates, based upon the
molecular weight and length of the remaining polypeptides which determines the degree
of allergenicity. The allergenicity of proteins can be reduced using enzymatic hydrolysis,
34
Chapter 2
heat treatment and/or ultra filtration [62, 63]. Ultimately a third formula can be used
consisting single amino acids only and cannot cause any hypersensitivity response, which
is allergen avoidance. The main disadvantage of this formula is that it cannot induce any
immunological oral tolerance [64]. A T cell epitope consists of 8-25 amino acids, depending
on the MHC class. Single amino acids are therefore not capable of inducing any immune
responsiveness and thus cannot induce immunological (oral) tolerance. Most of the (pre-)
clinical studies that are performed to prevent allergic sensitization in infants at risk focus on
the hydrolysates with low allergenic potential which may still poses tolerogenic capacities
[65-67]. A difficulty in interpreting clinical results of different formulas is the heterogeneity
in family history of allergies [68]. A formula, partial or extensive hydrolysate based, that
shows no allergenic potential, but can still induce oral tolerance is the ultimate goal in this
field of nutritional research.
4.1.3 Introduction of Solid Foods
Another factor that might influence the onset of allergies is the timing of introduction of
solid foods. Initially much debate was going on regarding the introduction of solid foods,
however there is consensus to avoid solid food before 17 weeks of age and to start no
later than 26 weeks [69-71]. With the introduction of complementary feeding (e.g. nutrition
other than derived from breast milk and infant formula) the risk for allergies is rising.
Therefore caution is necessary, for children at risk for allergies, with the introduction of
potential clinical significant allergens which can be found in peanuts, egg white, whole milk
and (shell)fish. The late introduction of complementary foods cannot fully prevent the onset
of allergies [71].
4.1.4 Prebiotics
Human breast milk contains approximately 7-12 g/L oligosaccharides [72]. At least
130 different oligosaccharides have been isolated from human milk and the two main
categories are neutral and acidic oligosaccharides [73]. These oligosaccharides are nondigestible carbohydrates, or dietary fibers as they are frequently named, that have many
different properties and are believed to act on the microbiotia in the gut [74-77]. Due to
physicochemical properties of non-digestible carbohydrates the absorption of minerals and
fecal consistency improves [78]. There are different types of dietary fibers e.g. (hemi)cellulose,
lignin, β-glucans, pectins, gums, inulin and oligosaccharides [79]. In addition, different nondigestible oligosaccharides with specific properties are obtained or manufactured from
35
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2
natural sources [80]. Some of them have specific properties and can be used as prebiotics
in a dietary supplement. Prebiotics are defined as “food ingredients that beneficially affect
the host by selectively stimulating the growth and/or activity of one or a limited number of
bacterial species already resident in the colon, and thus attempt to improve host health”
[74]. Recently this definition is adapted to: “A prebiotic is a selectively fermented ingredient
that allows specific changes, both in the composition and/or activity in the gastrointestinal
microflora that confers benefits upon host wellbeing and health” [81]. Prebiotics enhance
defense mechanisms of the host by stimulation of growth of Bifidobacteria and Lactobacilli
[82] Short-chain fatty acids, released by these bacteria upon fermentation of prebiotics, are
essential nutrients for intestinal epithelial cells and support gut function [83]. In vivo and in
vitro studies have shown beneficial effects of prebiotics on the innate as well as the adaptive
immune system [84].
Short-chain galacto-oligosaccharides (scGOS), long-chain fructo-oligosaccarides (lcFOS)
and pectin-derived acidic oligosaccharides (pAOS) are some examples of non-digestible
oligosaccharides that mimic the functionality and molecular size distribution of human
milk oligosaccharides. Prebiotic scGOS are produced by enzymatic elongation of galactose
(derived from lactose) by β-galactosidase, with a degree of polymerization (dp) of 3–8. FOS
is derived from chicory inulin which is a polymer from D-fructose residues linked by β(2-1)
bonds with a terminal α(1-2) linked D-glucose (dp 2-60). lcFOS are produced from inulin and
have a mean dp >23, whereas scFOS have a mean dp <23. lcFOS are added to infant formula.
pAOS is a mixture of linear oligomers and small polymers of galacturonic acid produced from
from food grade pectin by pectin cleaving enzymes and have a dp of 1-20. Currently, it is
not feasible to produce oligosaccharides identical to human milk oligosaccharides present
in breast milk, however the used specific oligosaccharides (scGOS and lcFOS) have proven
efficacy in clinical trials since they reduce the incidence of allergic disease and have been
shown to enhance fecal Bifidobacteria counts [85-88].
The effects of scGOS and lcFOS (Immunofortis®) have been studied in a murine vaccination
model [89, 90], in an allergic asthma model [91] and a cow’s milk allergy model [92, 93].
The response to vaccination of mice fed the scGOS/lcFOS diet was significant enhanced,
as well as the fecal Bifidobacteria and Lactobacilli proportions in colon samples [90]. In
addition, pAOS enhanced the murine vaccination response and the combination (scGOS/
lcFOS/pAOS) was even more effective [89]. In infants ,pAOS is considered to be safe with
regard to intestinal flora, stool characteristics, pH, growth, crying, vomiting and regurgitation
patterns as compared to control infants [94]. Furthermore, systemic TH1 dependent immune
responses were enhanced using the prebiotics without inducing autoimmunity, as TH1 is low
in newborn infants. On the other hand in an ovalbumin model for allergic asthma, which
is TH2 dependent, the identical oligosaccharides reduced airway hyperresponsiveness and
36
Chapter 2
lowered influx of cells in the broncho-alveolar lavage fluid [91]. In addition, in a murine
model for cow’s milk allergy dietary intervention with scGOS/lcFOS showed a significant
decrease of the allergic response and increased specific IgG2a levels [93]. Another prebiotic
fructo-oligosaccharide, kestose, showed to be effective in reducing the incidence of AD in
infants as well [95].
Recently it was found that dietary intervention with scGOS/lcFOS/pAOS reduces the
development of an acute allergic response upon antigen challenge, although specific
immunoglobulins levels remain high. Both in vivo as well as ex vivo depletion of CD25+
Treg cell abrogated the diminished acute allergic response and imply crucial involvement
of antigen specific CD25+ Treg cells in the suppression of the allergic effector response
(Schouten et al., submitted).
Furthermore, clinical studies have been performed with the scGOS/lcFOS mixture in children
at high risk for allergies. A reduction in the incidence of AD [85] and the incidence of
allergic manifestations during the first 6 months of life [87] was observed, furthermore this
reduction lasted at least until 2 years of age [86]. In another clinical study, using the scGOS/
lcFOS mixture, fecal secretory IgA was increased in healthy infants [96]. There is cumulative
evidence that prebiotic mixtures might beneficially affect the host in both TH1 as well as TH2
prone settings as it might prevent food allergy (TH2) and enhances the vaccination response
(TH1). Although it is believed that prebiotics exert their effect via stimulation of growth of
selective bacterial species that beneficially improve host health, there is debate about this
mechanism and there are potentially microbiota-independent mechanisms as well [84].
4.1.5 Probiotics
Probiotic means ‘for life’ and probiotics can be defined as: “live microbial food ingredients
that alter the enteric microflora and have a beneficial effect on health” [97]. For the
prevention of allergic disease probiotics are widely investigated in multiple clinical studies
[98-108]. However, there is still no consensus regarding the efficacy of these bacteria in
having immune-modulatory effects and their clinical relevance [109]. Several factors are
able to influence the outcome of the studies, e.g. candidate probiotic strain or mixture,
environmental factors, intervention duration, and timing and bacterial load [110, 111].
Among young infants 10% will develop AD, which can objectively be scored and approximately
one-third of these children are affected with FA [112, 113]. When pregnant mothers with
a family history of allergic disease and their babies were supplemented, for 6 mo, with
Lactobacillus GG, the cumulative incidence of atopic disease was reduced after 2 y compared
37
2
with placebo controls [103]. The supplementation was still effective after 4 y and even 7 y
[101, 107]. In a similar study, Lactobacillus reuteri reduced the incidence of IgE-associated
eczema and the occurrence of a positive skin prick test for almost 50% [105].
2
In germ-free mice the fundamental contribution of the gastrointestinal microbiota in the
induction of oral tolerance was confirmed. Furthermore, the gastrointestinal microbiota
composition may be a prerequisite for the development of allergy [114-116].
To gather more data regarding underlying mechanisms of immune modulation by probiotics
animal models are widely used [93, 117-121]. The candidate probiotic strain B. breve
M-16V effectively inhibited the development of the acute allergic skin response in mice
orally sensitized with whey and increased serum levels of whey-specific IgG2a, whereas
no decrease in IgE and IgG1 levels was observed [93]. In addition, it was shown that
supplementation of UV-killed bacteria of the same strain during the sensitization phase had
similar effects (Schouten et al., unpublished data). In an ovalbumin induced mouse model
oral supplementation of L. acidophilus, B. lactis, B. bifidum as well as L. casei, but not E.
coli decreased ovalbumin-specific IgE levels [118, 119]. Furthermore, Feleszko et al. [121]
showed that L. rhamnosus GG and B. lactis decreased the antigen specific recall proliferation
of splenocytes as well as TH2 type cytokine production of mesenteric lymph node cells in a
mouse model of asthma.
These preclinical models can bridge the gap between in vitro selection of specific candidate
probiotics and use in clinical studies. Furthermore, they can provide more insight in the
underlying mechanisms of action.
4.1.6 Synbiotics
To improve human health and reduce the incidence of allergies in a natural way the
combination of prebiotics and probiotics, called synbiotics, is being investigated as well.
The hypothesis that pre- and probiotics together are more effective than the sum of single
preparations is very promising, however not many studies have been performed in relation
to allergy [93, 122-124]. Recently, in a mouse model of orally induced cow’s milk allergy
indeed preventive dietary intervention with a synbiotic mixture of scGOS/lcFOS and B. breve
MV-16 was found to be significantly more effective in reducing several allergic parameters
when compared to intervention with pro- or prebiotics alone [93]. Hence, the (preclinical)
studies that have been carried out show potential in reducing the incidence and severity of
allergic disorders. Timing and dosage of both the prebiotics as well as the probiotics need
further attention. In general, more research is needed to confirm the additional effects of
38
Chapter 2
combining prebiotics and probiotics.
4.1.7 Postbiotics
An emerging field in nutritional sciences is the supplementation of isolated bacterial
products called postbiotics (ferments). These products include short-chain fatty acids, like
butyrate, however other more unknown products (e.g. cytokines, toll like receptor ligands,
carbohydrates and bacterial glycans) may also have important immunomodulatory effects
[125]. So far, to our knowledge, there is no literature available regarding the use postbiotics in
FA. However, in a stress-induced gut permeability rat model a probiotic strain in combination
with its bacterial (or fermentation) products synergistically counteracted the stress-induced
gut permeability and visceral sensitivity [126]. These data may open new avenues in the
research concerning prevention or treatment of food allergies with postbiotics whether or
not in combination with pre- pro- or synbiotics.
4.1.8 Polyunsaturated Fatty Acids (PUFA)
The increased prevalence of childhood allergic diseases coincides with altered dietary fatty
acid intake in westernized countries. N-3 (alpha-linolenic acid) and n-6 PUFA (linoleic acid)
are essential for humans and have to be taken up via the diet. PUFA are incorporated into the
cellular membrane and are eicosanoid precursors hereby affecting the immune response.
In this regard in particular long chain n-3 PUFA (EPA and DHA), which are obtained via diet
or synthesized from alpha-linolenic acid, are regarded to be anti-inflammatory while n-6
PUFA are able to boost inflammatory responses. In westernized countries the n-3 fatty acid
ingestion is no longer favored over omega-6 PUFA which may have implications for immune
homeostasis. It has been stated that the original diet of the human race consists of a n-6 :
n-3 ratio of approximately 1:1 and that this has changed over the past decades to a ratio of
at least 15:1 [127]. This shift is suggested to be explanatory for the increased incidence of
cardiovascular diseases, chronic inflammatory diseases and also allergic diseases. Therefore
pre-clinical as well as clinical studies are performed in order to investigate this hypothesis.
Several clinical studies have shown that enhanced n-3 PUFA intake reduces the incidence
of allergic disorders, e.g. AD, less sensitization to egg (reviewed by [128]). Two studies by
Dunstan et al. [129, 130] show reduced sensitization to egg and less AD when pregnant
woman were supplemented with fish oil. Recently, Furuhjelm et al. showed that maternal n-3
PUFA intake decreased the risk of FA and IgE-associated eczema in children at risk for allergy
[131]. In contrast, in a clinical study by Almqvist et al. [132] it is shown that supplementation
39
2
2
of n-3 PUFA, starting at a maximum of six months of age, did not prevent children with a
family history of asthma from developing atopy, eczema nor asthma at the age of 5 years.
Hence, discrepancy on effects of n-3 PUFA in prevention of allergic disease exists. However,
in a review by Prescott and Calder [133] it was put forward that such dietary factors are
under-explored and that there is a need for novel synthetic PUFA which may be potent in
the reduction of the allergic burden. One of the strategies that are proposed is the use of
selected PUFA in the formula feeding of young children at high risk for allergies [134].
4.1.9 Imprinting During Pregnancy
In order to prevent an allergic outcome intervention during pregnancy has growing interest.
The prenatal period is crucial in the priming of the immune system and therefore it may
alter the susceptibility for development of allergies later in life. All of the above discussed
dietary components, like pre-, pro-, synbiotics and PUFA have been studied for their
immunomodulatory capacities [98, 128, 130, 135, 136, 137, 138]. Regarding pre- and
probiotics the effects are variable. Kukkonen et al. [98] have shown that supplementation of
pregnant women with a mixture of probiotics and supplementation of their offspring with
prebiotics and the probiotics, decreased the incidence of atopic eczema. However a study
by Boyle et al. [139] with one probiotic strain (Lactobacillus rhamnosus GG) supplemented
only to the mothers (from 36 weeks gestation until delivery) did not result in any beneficial
antigen-specific immune response of the neonate. Also enhanced dietary intake of n-3 PUFA
during pregnancy may be able to alter the immune status of the neonate. PUFA can pass the
placental barrier and are incorporated in the membranes of cells of the fetus [140]. Dietary
intervention during pregnancy shows promising results, however these results are highly
variable depending on the experimental setup. Therefore more studies with larger cohorts
are necessary and if possible with dietary mixtures consisting of both synbiotics and PUFA.
4.2 Treatment
Besides prevention of the development of allergic disease treatment is essential as well.
However, the immunological network is highly complex and although there are numerous
pathways to intervene FA; e.g. anti-IgE therapy, corticosteroids, mast cell stabilizers,
leukotriene inhibitors, TGF-β administration and anti-IL-5 therapy [141, 142], those
interventions are still experimental. Here, the focus will be on the methods already used
in the clinic, daily practice and dietary intervention studies with the objective to treat or
reduce symptoms of allergic disease and in particular FA. For treatment both avoidance
(epitope exclusion) as well as desensitization strategies are important.
40
Chapter 2
4.2.1 Epitope Exclusion
The single most effective strategy to prevent an adverse allergic reaction in food allergic
individuals is by exclusion diets, which is avoidance of the allergen(s) [143]. If there is a
suspicion of FA it is advised to eliminate several major food allergens from the diet. After
several days there should be relief of symptoms and if there is relief one by one the different
foods are introduced again until an allergic reaction occurs. Exclusion of possible allergens
may be used for the management of FA. However, it should be ensured that during a multiple
food exclusion diet the actual diet is still nutritionally adequate.
4.2.2 Hydrolysates
In general there are two different types of milk (infant formula) hydrolysates. In the USA, in
particular casein protein based formulas are available, whereas in Europe most formulas are
whey protein based. Partial hydrolysates (see also 4.1.2) contain peptides with a molecular
weight ranging between 3.000 and 10.000 Da, while the extensive formula have generally
peptide lengths under 3.000 Da [144]. Both partial as well as extensive hydrolysates can be
whey or casein based. In some cases these small proteins are still able to trigger an allergic
reaction [62, 63], Besides the two hydrolysate formula feedings, soy-based and amino
acid-based formulas can be used [64]. The main advantage of soy- and amino acid-based
formulas is that no cow’s milk allergenic potential is present, though oral tolerance to cow’s
milk proteins cannot develop either, since there are no T-cell epitopes present.
4.2.3 Pre-, Pro-, Synbiotics and PUFA
To stimulate the immune system with the intention to prevent the onset of an allergic
TH2 phenotype pre-, pro-, synbiotic and PUFA are under investigation. However, similar
strategies are used for treatment with the intention to skew the immune response towards
a more TH1/Treg polarized phenotype after the onset of allergy resulting in desensitization
and ultimately oral tolerance induction. In parallel, modulation of the immune system using
dietary components is highly interesting and might be helpful in order to induce a tolerogenic
immune environment. In spite of the common belief that the intervention should take place
as early in life as possible, several studies have shown successful treatment of AD in children
above 2 years of age, suggesting that the immune system can still be influenced later in life
[145-147]. In contrast several other studies show no positive effect of a probiotic intervention
on AD [100, 106, 148]. Brouwer et al. [106] tested two different strains (Lactobacillus
41
2
2
rhamnosus and Lactobacillus GG) and found no effect on AD in infants enrolled before the
age of 5 years. Sistek et al. [100] tested the combination of Lactobacillus rhamnosus and
Bifidobacteria lactis and found only a decrease in AD in a subgroup of children enrolled
between age 1 and 10 years. Not much is known about the possible curative properties of
PUFA in allergic disease however studies are being conducted to investigate this [135]. One
study showed that female adults with high fish and DHA intake have a lower rate in allergic
sensitization [149]. Use of pre-, pro-, synbiotics and PUFA to ameliorate or treat allergic
disease may be advantageous, however the need for more and comprehensive (pre-)clinical
data is essential.
4.2.4 Traditional Chinese Herbal Medicine
Complementary to regular medicine, alternative medicine have growing interest in the
western world especially in the field of allergic asthma. Additionally scientific evidence for
the treatment of FA is increasing [150, 151]. A few studies in preclinical models for FA using
a traditional Chinese herbal formula have been published [152-154]. Two different formulas
have been tested; the FA herbal formula (FAHF)-1 and FAHF-2, which are mixtures of 9-11
different herbs. Classically, these herbs were prescribed for gastrointestinal disorders, as
diarrhea and vomiting and therefore thought to be effective in FA [151]. In the preclinical
model these herbs were highly effective in protecting the animals from anaphylaxis after
oral peanut challenge and reducing peanut-specific IgE levels [152, 153]. Currently, a phase
I clinical trial is ongoing to test the safety of FAHF-2 in humans and phase II is planned to
determine its efficacy in peanut allergic patients [141, 150].
4.2.5 Immune Therapy and Induction of Oral Tolerance
A more clinical approach in the treatment of FA is immune therapy. Several different
procedures are under investigation. Subcutaneous injections of increasing doses of food
allergen extracts have been shown to induce unacceptable side-effects, in contrast with
other non-food allergens [155]. Other disadvantages are the introduction of sensitization to
new allergens present in the used extracts and it is not effective in all patient groups [156]. To
circumvent this major drawback, two alternative immunotherapies are being developed. In
oral immunotherapy (OIT) also known as specific oral tolerance induction (SOTI) the allergen
is being swallowed directly and in sublingual immunotherapy (SLIT) the allergen dose is held
under the tongue for a few minutes before swallowing [142]. The dosages are increasing
over time and the duration of these therapies is several months upto two years. Besides
42
Chapter 2
the effects on the clinical outcome of a provocation test, also some studies indicate that
allergen specific IgE levels are altered, mostly decreased, which indicates desensitization
[157-159]. In particular SOTI has been investigated with interesting results. There are two
different mechanisms with regard to oral tolerance induction; low dose and high dose.
Low dose tolerance is, in the rat, induced by orally applied multiple doses (1 mg) of the
specific allergen, while high dose oral tolerance (20 mg) is induced after a single dosage
[160]. Active suppression by means of regulatory T cells underlies the mechanism of action
of low dose oral tolerance [161, 162]. Allergies develop as a consequence of a disparity in
the TH1/TH2 balance towards TH2 [163, 164]. Different Treg cell subsets (CD4+CD25+FOXP3+,
TH3 and Tr1) actively regulate this balance locally in the gastro-intestinal tract via excretion
of immunosuppressive cytokines IL-10 and TGF-β and via cell-cell contact. Antigens, or
dendritic cells loaded with antigen, travel from the Peyer’s patches to the mesenteric
lymph nodes (MLN) and are presented to naïve T-cells. Antigen-responsive T-cells (CD4+)
travel subsequently through the bloodstream and home back into the intestinal mucosa
or traffic to the peripheral immune system (e.g. spleen) [165, 166]. Antigen specific T-cells
from the MLN in this respect can relocate systemic tolerance. There are several host factors
contributing to the success of oral tolerance induction e.g. genetics, age and microbiotic flora
[167]. For SOTI the allergen of interest is administered orally, from a very low dose which
is daily increased. After a few months the amount of food that is tolerated is equivalent to
a usual daily quantity. However a daily maintenance dose is required to remain tolerant
[168]. The mechanism by which SOTI exerts its effect is not clear yet and it remains to be
investigated whether specific immunological mechanisms underlie the increase in threshold
dose. Hence, both oral and sublingual immunotherapy may be usefull for food allergic
patients, however the protocols are time consuming and sometimes with transient efficacy.
Although oral immunotherapy could be an interesting approach, more studies are needed
to understand mechanisms of action.
5. Conclusions
Food allergies are of growing health concern in the Westernized world. Together with the
rising incidence of food allergies the medical costs rise. If oral tolerance to a specific allergen
is not induced, a subject becomes sensitized. After a recurrent encounter with the same
allergen a clinical reaction is provoked, since mast cells degranulate and even anaphylaxis
may occur. FA can be diagnosed, for example, by analyzing serum specific IgE [46-48].
Furthermore, a DBPCFC or a SPT may be performed to establish a reliable diagnose [41, 44].
In addition, Ig-fLC measurements may add to the diagnosis of food allergies [58], Schouten et
al., submitted). Pre-clinical and clinical research on FA is expanding. Currently, no medicines
to prevent or cure food allergies are available, however dietary interventions are safe and
43
2
2
may prevent or ultimately cure food allergies as well. Therefore multiple approaches are
studied, in particular for the prevention of allergies in children, using prebiotics, probiotics,
synbiotics, postbiotics and PUFA. These food supplements are sometimes administered
before birth, to the pregnant women, and are believed to induce beneficial immune
imprinting in the neonate [98, 139].
Promising for the prevention of AD, which is related to food allergies, are the prebiotic
mixtures of non-digestible carbohydrates and/or the use of specific probiotic strains. Future
studies will have to reveal whether combined preparations of multiple dietary supplements,
e.g. synbiotics and PUFA will further improve the efficacy.
FA is most probably caused by an imbalance between TH1, TH2 and Treg cell activity. The
mechanisms by which the different dietary interventions act are currently unclear, however
enhanced CD25+ regulatory T cell function may be one of possibilities (Schouten et al.,
submitted). Research on food allergies should not only focus on testing more potential
dietary components, but on the working mechanism also. If there is more insight in the
mechanism, selection of food components might be easier. Multidisciplinary approaches
of immunologists, nutritionists and allergists would help in a better understanding and
approach to prevent and treat food allergy.
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2
3
Acute allergic skin reactions and intestinal
contractility changes in mice orally sensitized
against casein or whey
Bastiaan Schouten1
Gerard A. Hofman1
Lieke W.J. van den Elsen1
Johan Garssen1,2
1
2
International Archives of Allergy and Immunology 2008;147:125-134
S. Karger AG, Basel
Acute allergic skin reactions and intestinal contractility changes in mice orally
sensitized against casein or whey
Abstract
3
Background: Cow’s milk allergy (CMA) is characterized by hypersensitivity against casein
or whey, affecting 2.5% of young infants. The pathogenesis of CMA involves IgE as well
as non-IgE-mediated reactions and clinical symptoms are found in the skin, lungs and
gastrointestinal tract. In this study, local and systemic immunopathology was determined in
whey- or casein-allergic mice.
Methods: Mice were orally sensitized with casein or whey using cholera toxin as an
adjuvant. Serum immunoglobulins and the acute allergic skin reaction (ear swelling 1 h
after intradermal allergen challenge) were determined to reveal systemic hypersensitivity.
Furthermore, pathophysiological changes were assessed within the intestine.
Results: An acute allergic skin reaction was induced in both whey- and casein-sensitized
mice. In these mice, whey-specific IgE, IgG1, IgG2a and casein-specific IgG1 levels were found
to be increased. In addition, the serum mouse mast cell protease-1 (mMCP-1) concentration
was enhanced, reflecting mast cell degranulation. Indeed, the number of mMCP-1-positive
mast cells within the colon was diminished in both whey- and casein-sensitized mice. Only
in casein-sensitized mice isometric contraction of the colon was reduced, reflecting motility
alterations.
Conclusion: Mice, orally sensitized against casein or whey, revealed an allergen-specific
acute allergic skin reaction. In casein-sensitized mice, hypocontractility of the colon reflected
pathophysiological changes within the intestine. Allergen induced ear swelling and intestinal
contractility changes are novel parameters in animal models of CMA which may add to the
search for new therapeutic strategies to relieve symptoms of CMA.
56
Chapter 3
Introduction
Cow’s milk allergy (CMA) is one of the leading causes of food allergy in adults [1]. In developed
countries, approximately 2–3% of infants exhibit CMA. Although most infants outgrow CMA
before their fifth year, IgE mediated CMA predisposes the development of other (food)
allergies and even asthma [2, 3]. Clinical features due to IgE-mediated reactions are expressed
as immediate symptoms mostly. Clinical symptoms may involve the skin, respiratory tract and
gastrointestinal tract, and can even lead to a systemic anaphylactic reaction [4, 5]. However,
it should be realized that a particular group of patients, up to 40%, exhibit clinical features
of CMA without detectable cow’s milk-specific serum IgE [6-8]. So far, the only therapeutic
approach has been the elimination of cow’s milk proteins from the diet. Cow’s milk contains 2
main protein classes, caseins (30 g/l) and whey proteins (5 g/l). The caseins consist mainly of
αS1-, αS2- and β-casein, whereas whey proteins comprise of β-lactoglobulin, α-lactalbumin,
bovine serum albumin, serum immunoglobulins and lactoferrin. Large population studies
with cow’s milk-allergic infants have shown that the major allergens are β-lactoglobulin,
caseins and α-lactalbumin [9, 10]. The pathogenesis and development of CMA probably
involves a partial dysfunction in immunological tolerance induction during early life combined
with enhanced intestinal permeability [5]. There is growing evidence that intestinal allergic
responses can initiate motility changes through attraction and activation of mast cells and
production of TH2 type of cytokines [11]. Motility changes of the intestine are a clinical
problem in CMA patients, which manifests as either diarrhea or even constipation [12-14].
Animal models of CMA provide a tool to reveal mechanisms involved in CMA and may explore
new therapeutic and preventive approaches in those models. However, in most existing
food allergy models, the animals are not sensitized via the oral route (but, for example,
intraperitoneally), while in reality humans are sensitized orally. Although numerous animal
models for food allergy are available using an intraperitoneal sensitization protocol, only a
few models use oral sensitization [15-23]. Li et al. [22] were the first to introduce a model
in which mice were sensitized orally against complete cow’s milk, while Frossart et al. [23]
showed oral sensitization to β-lactoglobulin. In these models, mice were sensitized by
means of intragastric (i.g.) gavage using cholera toxin (CT) as an adjuvant. The primary read
out parameters were IgA and IgE, and in particular cow’s milk-specific IgE. Furthermore, Li et
al. [22] described systemic anaphylaxis upon i.g. challenge with cow’s milk. They described
vascular leakage, mast cell degranulation and enhanced serum histamine levels in CMA
animals. The systemic allergic responses to food allergens that are described in these models
resemble features found in the clinic; however, the mechanisms behind the development of
CMA are still only partially understood.
57
3
To further assess mechanisms underlying CMA and/or test new concepts for prevention
and/or treatment for CMA, in the present study new tools to address the pathological
changes in casein and whey allergy in mice have been developed. Measurement of the
acute allergic skin response, as an equivalent of the human skin prick test, was introduced
to reflect a systemic sensitization. Intestinal contractility changes were measured to address
local pathophysiological changes after oral challenge with the specific allergens.
3
Methods
Chemicals
Casein and whey were obtained from DMV International (Veghel, The Netherlands). Cholera
toxin was purchased from Quadratech Diagnostics (Epsom, UK) and PBS from Cambrex
Bio Science (Verviers, Belgium). Biotin-labeled rat anti-mouse IgE, IgG1 and IgG2a, and
unlabeled rat anti mouse IgE were obtained from BD Biosciences (Alphen aan den Rijn, The
Netherlands). All other chemicals were obtained from Sigma-Aldrich-Chemie (Zwijndrecht,
The Netherlands).
Oral Sensitization and Challenge of Mice
Three- to five-week-old specific pathogen-free female C3H/HeOuJ mice (4–6 mice per group)
C3H/HeOuJ
Female 3/5 wks
0
7
14
Challenge i.d. 10 µg
21
28
35
Challenge i.g. 100 mg
40 42
Sensitization by gavage with casein or whey + CT or CT alone (control)
Skin response 1, 4 and 24 hours post challenge
days
‐ Serum parameters
‐ Functional changes intestine
‐ Histology
Figure 1. A schematic overview of the sensitization and challenge protocol and the parameters
that are analyzed.
58
Chapter 3
were purchased from Charles River Laboratories (Maastricht, The Netherlands), maintained
on cow’s milk protein-free mouse chow (Special Diets Services, Witham, UK) and housed
in the animal facility at the Utrecht University. Animal care and use were performed in
accordance with the guidelines of the Dutch Committee of Animal Experiments. Mice were
sensitized i.g. with 0.5 ml homogenized casein or whey (40 mg/ml PBS) with CT (20 μg/
ml PBS) as an adjuvant, using a blunt needle. Control mice received CT alone or PBS. Mice
were boosted weekly for a period of 4–6 weeks, 1 week after the last sensitization mice
were challenged i.g. with 100 mg casein or whey in 1 ml PBS (Figure 1). Blood samples were
collected and centrifuged (15 min at 13,500 rpm). Sera were stored at –70° C. Mice were
sacrificed by cervical dislocation 30 min after i.g. challenge.
Allergen-Specific Skin Response
The acute allergen-specific skin response was measured after injection of the specific protein
in the ear pinnae intradermally (i.d.). Before i.g. challenge (t = 0), the control, casein- and
whey-sensitized mice were injected i.d. in the left ear with 20 μl homogenized casein or
whey (0.5 mg/ml in PBS), respectively. In the right ear, 20 μl PBS was injected as a vehicle
control. Also, the CT and PBS sham-sensitized mice received a casein or whey ear challenge
using PBS injections as control. Ear thickness was measured in duplicate using a digital
micrometer (Mitutoyo, Veenendaal, The Netherlands), at t = 0 as well as 1, 4 and 24 h after
challenge. The allergen-specific net ear swelling was calculated by subtracting the basal
thickness (0 h) from the thickness measured at 1, 4 and 24 h after injection in the left ear. In
addition, the control (right ear) swelling measured at the same time points was subtracted.
The ear swelling is expressed as delta μm.
Measurement of Serum Immunoglobulin and Mouse Mast Cell Protease-1
Concentrations of total IgE and levels of casein- or whey-specific IgE, IgG1 and IgG2a were
determined in serum of sacrificed mice by means of ELISA. Microlon plates (Greiner, Alphen
aan den Rijn, The Netherlands) were coated with 100 μl whey or casein (20 μg/ml) in coating
buffer or rat anti-mouse IgE (1 μg/ml; carbonate-bicarbonate buffer, 0.05 M , pH = 9.6) for
18 h at 4°C. Plates were washed and blocked for 1 h with 5% BSA. Serum samples were
applied in several dilutions (4–100 times) and incubated for 2 h at room temperature. Plates
were washed and incubated with biotin-labeled rat anti-mouse IgE, IgG1 or IgG2a (1 μg/ml)
for 90 min at room temperature and washed. The plates were incubated with streptavidinhorseradish peroxidase for 1 h, washed and developed with o-phenylendiamine. After 5
59
3
min, the reaction was stopped with 4 M H2SO4 and absorbance was measured at 490 nm
on a Benchmark microplate reader (Bio-Rad, Hercules, Calif., USA). Serum concentrations
of mouse mast cell protease-1 (mMCP-1) were determined as described previously using a
commercially available ELISA kit (Moredun Scientific Ltd., Penicuik, UK) [24].
3
Immunohistology of the Colon
For determination of histopathological alterations, Swiss rolls of 4 cm colon were prepared
[25]. The colon was carefully dissected, opened longitudinally over the mesenteric line and
luminal contents were removed by gently washing in saline. The colon was placed with the
mucosal side down. Colons were rolled from the distal to the proximal end, fixed in ice-cold
10% formaldehyde in PBS (for 24–48 h) and embedded in paraffin (Leica EG1150c; Leica
Microsystems, Rijswijk, The Netherlands). Sections of 5 μm were cut using a microtome (Leica
RM2165; Leica Microsystems), stretched on water (Tissue Flotation Bath TFB45; Medite,
Nunningen, Switzerland) and placed on poly-L-lysine-coated slides. Deparaffinized Swiss roll
sections were stained with mMCP-1 to detect mucosal mast cells [26]. In short, after fixation
in acetone, the sections were blocked with 10% normal goat serum, followed by incubation
with rat anti-mouse mMCP-1 (kindly donated by Dr. H.R.P. Miller, Edinburgh, UK). The primary
antibody was detected with a biotinylated polyclonal goat anti-rat antibody (Pharmingen,
Aalst, Belgium), followed by incubation with streptavidin-horseradish peroxidase (Sanquin,
Utrecht, The Netherlands). Color was developed with AEC chromogen staining kit (Sigma
Chemical Co., St. Louis, Mo., USA) and sections were counterstained with hematoxylin. Mast
cell counts were performed by counting 1 complete section of every animal.
Isometric Contraction of the Colon
The colon, caudal from the cecum, was dissected free of connective tissue and mesenterium.
Colon parts of 1 cm length were mounted in an organ bath, using 2 small clamps, containing
10 ml tyrode buffer (in mM: NaCl 136.89, KCl 2.68, MgCl 2 1.05, CaCl 2 1.77, NaH2 PO4 0.42,
NaHCO3 11.9 and glucose 5.55) [27-29]. The tissue was kept at 32°C, to prevent spontaneous
contraction, and continuously gassed with a 5% CO2 and 95% O2 gas mixture. One clamp
was attached to a fixed point in the organ bath and the other clamp was connected to
an isometric transducer (Harvard Apparatus Ltd., Edenbridge, UK) with an analog recorder
(BD40; Kipp & Zn., Delft, The Netherlands). Contractions were measured under a constant
preload of 1.00 g. The preparations were equilibrated for 1 h in the organ bath before starting
a log dose-response curve for carbachol (10–8 until 10–3 M). After every dosage, the organ
60
Chapter 3
bath was flushed twice and the tissue was allowed to recover for 10 min before addition of
the next concentration. Also, basal activity and spontaneous contraction frequencies of the
tissue were recorded.
Water Percentage in the Stool
Twenty-four hours after oral challenge, feces were collected from every animal. These
samples were weighted, dried for several days and weighted again. The difference in weight
is the evaporated water. The relative amount of water in the feces (relative to the total
weight of the feces) was calculated and statistically evaluated.
Statistics
All data except for the isometric contractions were analyzed using one-way ANOVA and
post hoc Dunnett’s test. Isometric contraction data were analyzed using repeated measures
ANOVA and post hoc Dunnett’s test. Statistical analyses were conducted using GraphPad
Prism software (version 4.0). For correlations, the Spearman rank order correlation coefficient
test was used in SPSS 13.0 for Windows. Data are represented as means ± SEM.
Results
Acute Allergen-Specific Skin Reaction
In order to study allergic skin responses, mice were challenged i.d. in the ear pinnae with
casein or whey 1 week after the last oral sensitization. One hour after dermal challenge,
the allergen-specific acute ear swelling response was maximal when compared to PBS and
CT sham-sensitized control mice. The delta ear swelling in the casein- and whey-sensitized
animals was 71.2 ± 8.4 and 137.9 ± 21.7 μm, respectively, while this was neglectable in PBS
and CT sham-treated mice (8.1 ± 9.4 and –4.6 ± 4.7 μm, respectively; Figure 2, p<0.01, n=4
in PBS group, n=6 in all other groups). All sensitized mice reacted positively to the allergen,
indicating that there are no non-responders. Four hours after challenge, the ear swelling
turned to basal levels in the casein group, while in the whey-sensitized group the swelling
remained slightly enhanced up to 24 h after challenge (37.5 ± 12.6 μm in whey-sensitized vs.
–3.9 ± 11.4 μm in CT-sensitized animals, p<0.05). In additional experiments (data not shown)
61
3
3
Figure 2. Induction of an acute ear swelling in casein- and whey-sensitized mice in
comparison with sham-sensitized (CT and PBS) mice. Delta ear swelling (µm) is calculated
by subtracting the specific ear swelling induced by the corresponding antigen with vehicle
(PBS) induced swelling at 1, 4 and 24 hours. * p<0.05, ** p<0.01, n=4 in PBS group, n=6 in
all other groups.
it was found that the marginal 24 h response was not followed by a response at later time
points. In sham-sensitized animals, no differences were found between casein and whey
challenge at any time points. Furthermore, whey challenge in casein-sensitized mice or vice
versa did not result in any significant swelling response, indicating the antigen specificity of
the skin reaction (data not shown).
Increased Total IgE and Antigen-Specific Serum IgE, IgG1 and IgG2a Levels
Half an hour after oral challenge, serum was obtained from both sensitized and non-sensitized
animals. Total IgE concentrations were increased in casein- and whey-sensitized mice (400 ±
32 and 364 ± 19 ng/ml, respectively) in comparison with CT and PBS sham-sensitized mice
(243 ± 37 and 218 ± 31 ng/ml, respectively; data not shown, p<0.01 or p<0.05, n=4 in PBS
group and n=6 in all other groups). Casein-specific IgE as well as casein-specific IgG2a levels
were not increased (Figure 3a and c). In contrast, casein-specific IgG1 levels were enhanced
in the casein-sensitized mice (0.537 ± 0.135 OD A490) when compared to CT and PBS shamsensitized animals (0.087 ± 0.017 and 0.062 ± 0.013, respectively; Figure 3b, p < 0.01). In
62
Chapter 3
3
Figure 3. Enhanced serum immunoglobulins in casein- and whey-sensitized mice a, b,c In
the casein sensitized mice only casein specific IgG1 levels were enhanced, no specific IgE and
IgG2a was measured. d, e, f In the whey-sensitized mice specific IgE, IgG1, and IgG2a serum
levels were enhanced. ** p<0.01, n=4 for PBS and n=6 for all other groups.
63
3
Figure 4. Regression analyses between ear swelling and Ig’s for casein- and whey-sensitized
mice. Correlation between ear swelling and a casein-specific IgE (p=0.342; r2=0.033; n=29) b
casein-specific IgG1 (p=0.521; r2=0.015; n=29) c whey-specific IgE (p=0.015; r2=0.276; n=21)
and d whey-specific IgG1 (p=0.043; r2=0.154; n=27).
the whey-sensitized animals, the whey-specific IgE (1.662 ± 0.136), IgG1 (0.988 ± 0.069) and
IgG2a (0.587 ± 0.081) levels were augmented when compared to the CT sham-sensitized
controls (0.000–0.311 ± 0.032; Figure 3d–f, p < 0.01).
Correlation Serum Immunoglobulins and the Acute Allergic Skin Reaction
To investigate the correlation between serum immunoglobulins and the ear swelling
response, linear regression analyses were performed using data from 4–5 independent
experiments. The serum dilutions used to calculate the correlations were: 10 times for IgE,
100 times for IgG1 and 50 times for IgG2a. In whey-sensitized mice the acute ear swelling
was found to correlate positively with whey-specific IgE (Figure 4c; p=0.015, r2= .276, n=21),
whey-specific IgG1 (Figure 4d; p=0.043, r2=0.154, n=27), but not with whey-specific IgG2a
(data not shown; p=0.314, r2=0.038, n=29). Ear swelling of casein-sensitized mice was not
64
Chapter 3
3
Figure 5. Serum mMCP-1 concentrations are increased in casein- and whey-sensitized mice
in comparison to sham-sensitized (CT and PBS) mice. ** p<0.01, n=4 for PBS, n=5 for CT and
n=6 for all other groups.
Figure 6. Decreased mast cell counts in colon of casein- and whey-sensitized mice.
Immunohistochemistry staining of mMCP-1 in 5 µm sections. The mean number of mMCP-1
positive cells was declined in casein and whey mice when compared to CT mice. * p<0.05,
** p<0.01, n=6 for all groups.
found to correlate with casein-specific IgE (Figure 4a; p=0.342, r2=0.033, n=29), caseinspecific IgG1 (Figure 4b; p=0.521, r2=0.015, n=29) and casein-specific IgG2a (data not shown;
p=0.488, r2=0.18, n=29).
65
Increased mMCP-1 Levels in Serum
3
mMCP-1 is a protease specific for mouse mucosal mast cells and will appear in the
bloodstream after mast cell degranulation. To assess mast cell degranulation, mMCP-1 levels
were determined in the serum 30 min after oral challenge. mMCP-1 serum concentrations
of casein-and whey-sensitized mice were enhanced when compared to CT and PBS controls
(3.890 ± 0.769 and 6.472 ± 2.341 vs. 0.357 ± 0.139 and 0.878 ± 0.338 ng/ml, respectively;
Figure 5; n=4–6, p<0.01).
Figure 7. Contractility changes in the colon of casein-sensitized mice after carbachol
stimulation. Contractility of the colon of casein mice was decreased when compared to CT
and whey treated mice. ** p<0.01, n=6 in all groups.
Figure 8. Water percentage water in the stool of casein- and whey-sensitized mice compared
to CT controls. The stool water content was found to be increased in both casein and whey
mice, 24 hours after oral challenge. * p<0.05, ** p<0.01, n=6 in all groups.
66
Chapter 3
Decreased Number of mMCP-1-Positive Mast Cells in the Colon
After oral challenge, mice were sacrificed and the colon was obtained for histological
examination. mMCP-1-positive cells were counted after immunohistochemical staining. A
decreased number of mucosal mast cells was counted in the colon of casein- (7.33 ± 5.13;
p<0.05) and whey-sensitized animals (6.33 ± 5.32; p<0.01) when compared to non-sensitized
control mice (20.67 ± 10.27; Figure 6; n=6).
3
Altered Isometric Contraction of the Colon
Isometric contraction of the colon was determined by assessing contractility upon
exposure to muscarinic receptor agonist carbachol (noncumulative dose response, 10–8
– 10–3 M). Reduced contractility for all carbachol concentrations was found for the colon
of casein-sensitized mice when compared to sham-treated mice (Figure 7; p<0.01, basal
contractility did not differ between groups). This hyporesponsiveness of the colon cannot
be characterized by a right movement of the EC50 concentration, but only by a reduction in
maximal contraction force (Emax ) (4,975 ± 1,046, 2,242 ± 296 and 5,933 ± 1,071 mg in control,
casein and whey groups). The hyporesponsiveness of the colon in the casein-sensitized mice
was found to be consistently present in all experiments, while none of these effects were
seen in the whey-sensitized animals.
Table 1. Overview of major parameters tested in both models. + : increased compared to
control (CT) mice; - : decreased compared to control (CT) mice; ND : no difference observed
regarding to control (CT) mice.
Parameter
Casein
Whey
IgE
ND
+
IgG1
+
+
ear swelling
+
+
Mast cell degranulation mMCP-1
+
+
Physiology
water percentage
+
+
motility
-
ND
Sensitization
67
Water Percentage in the Stool
The relative amount of water in the feces was increased in the casein- (Figure 8; 61.97 ±
0.97%, p<0.05, n=6) and whey-sensitized animals (63.22 ± 1.78%, p<0.01, n=6) when
compared to the control mice (56.68 ± 0.62%, n=6).
3
Discussion
One of the most common food allergies in childhood is CMA. CMA is diagnosed when
symptomatic patients have enhanced serum levels of cow’s milk protein-specific IgE
(radioallergosorbent test) and/or a positive skin prick test (SPT). In addition, a double-blind
placebo-controlled oral challenge can be performed, which is the most reliable test for
food allergy [9, 30, 31]. Currently, no immunotherapy is available for CMA patients, hence
patients need to avoid cow’s milk allergens in the diet and use hydrolyzed milk formulas [9].
In the present study, oral sensitization against the cow’s milk proteins whey and casein is
investigated in mice that were sensitized orally. Specific immunoglobulin levels were induced
and local and systemic symptoms were evaluated by studying effects in the gastrointestinal
tract and skin. The investigated parameters closely resemble diagnostic tools that are used
in the clinic; they are summarized in table 1.
Besides screening for cow’s milk-specific serum IgE, the SPT is used for the diagnoses of CMA
in humans [30]. In the current study, the allergen-induced ear swelling is introduced as a
possible equivalent for the SPT. It is a new tool to determine systemic sensitization for casein
and whey in mice. Oral sensitization with casein or whey consistently resulted in a positive
acute allergic ear swelling response upon local allergen challenge. Furthermore, it can be
concluded that sensitization via the oral route was able to induce systemic sensitization
in mice treated either with casein or whey. In other models for skin hypersensitivity,
comparative values for ear swelling responses have been found [32].
Although both whey- and casein-sensitized animals showed evidence of systemic allergy
according to the ear swelling response, differences were found in serum immunoglobulin
responses between the groups. Although total IgE concentrations and specific IgG1 levels
were enhanced in both the casein- and whey-sensitized mice, specific IgE and IgG2a were
found to be increased only in the whey-sensitized mice. In the clinic, 40% of CMA patients
have a negative radioallergosorbent test for cow’s milk proteins; however, they reveal to be
positive in the SPT [6-8]. In a recent study, the SPT was found to correlate better with the
double-blind food provocation test than serum immunoglobulins, although larger studies
are necessary to confirm these findings [33]. Typically, in the casein-sensitized mice, no
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Chapter 3
specific IgE was detected, while the mice indeed showed systemic allergy as seen in the
acute allergen-induced ear swelling response. In contrast, the whey-sensitized mice showed
the classical characteristics of type I IgE-mediated allergy, scoring positive for allergenspecific IgE and acute ear swelling, underlining the validity of this tool [34-36]. In wheysensitized mice, the validity of the acute skin reaction test as a tool to determine systemic
sensitization was supported by the finding that challenge-induced ear swelling correlated
with whey-specific IgE and IgG1. The acute allergic ear swelling in casein-sensitized mice
was not associated with enhanced specific IgE levels, which may imply that there is no role
for IgE in this model under the current protocol. In CMA patients, casein-specific IgE can
be detected in the serum, but levels vary. It is known that patients with persistent CMA
over the age of 9 years had elevated levels of casein-specific IgE compared to younger
children with CMA. Therefore, low levels of casein-specific IgE early in life could indicate a
non-persistent form of CMA [31]. Instead of IgE, the acute ear swelling in mice might have
been triggered by IgG1, which is generally known to play a crucial role in mast cell activation
in rodents. It has been shown that immunoglobulins can significantly interfere, positively
and negatively, with mast cell responses (reviewed by Bruhns et al. [37]). In addition, it is
known from the literature [38] that even in FcεRI-deficient mice, anaphylactic reactions are
possible, indicating that IgE is not always a prerequisite for anaphylaxis. Although, in caseinsensitized mice no significant correlation was found between ear swelling and levels of IgG1,
the scatter plot indeed shows a subgroup of mice in which the ear swelling is positively
associated with the level of IgG1. Hence, although casein-allergic mice lack specific IgE,
enhanced IgG1 levels in these mice may, at least partly, reflect allergic sensitization. Apart
from IgE and IgG1, Redegeld et al. [39] have found that mast cell degranulation can occur
with immunoglobulin light chain. Immunoglobulin light chain is produced in excess during
the formation of immunoglobulins. Hence, casein-specific immunoglobulin light chain may
have caused the ear swelling in casein mice with an acute allergic skin response that could
not be explained by the presence of IgE nor IgG1. However, this remains speculative and
further research is necessary.
Whey-sensitized mice show a slight but significant ear swelling at 24 h. Together with the
higher allergen-specific IgG2a levels in these animals, this might reflect a late-phase response
after whey sensitization. Frossard et al. [40] also found enhanced serum levels of IgE/IgG1
and IgG2a, characteristic for a mouse TH2 and TH1 type of immune response, respectively,
against β-lactoglobulin in a C3H/HeOuJ mice model for β-lactoglobulin. These levels of IgG2a
are in accordance with enhanced levels of IgG2a that have been found in a similar mouse
model for peanut allergy [22, 23, 41].
Clinical features of CMA are generally known to be elicited by mucosal mast cells [4, 22,
42]. Those mast cells are present in the intestinal mucosa and additionally drawn to the
site of allergen challenge. In casein- and whey-sensitized mice, mast cell numbers of the
69
3
3
colon were found to be declined in comparison to sham-sensitized mice. Mucosal mast
cells were stained with mMCP-1, a β-chymase present in the mast cell granules, which end
up in the bloodstream after degranulation [43]. In both whey- and casein-sensitized mice,
serum mMCP-1 concentrations were increased, 30 min after i.g. challenge, reflecting mast
cell degranulation. Upon oral allergen exposure, mucosal mast cells may have degranulated,
resulting in enhanced mMCP-1 serum levels. After mast cell degranulation, the mast cell
itself is not visible anymore with mMCP-1 staining, which might explain the drop in mast cell
numbers in the colon of casein- and whey-sensitized mice [44].
One of the most prominent symptoms of food allergy is discomfort in the gastrointestinal
tract which can be abdominal pain, diarrhea or sometimes constipation [4, 5]. The
collected stool samples of casein- and whey-sensitized animals contained a higher water
percentage than the controls, which is suggestive of diarrhea. Besides diarrhea, dysmotility
of the intestine is a problem in CMA patients [12, 13]. The casein-sensitized mice showed
contractility changes in organ bath studies, while whey-, CT and PBS-sensitized mice did not.
Isometric contractions of colon segments of casein-sensitized animals were hyporeactive
in comparison to CT controls. It is known that allergy has adverse effects on the gut health,
causing dysmotility [45, 46]. In this study, smooth muscle contractility differences between
control and casein-sensitized animals may reflect changes in motility, which is supported by
findings of Kobayashi et al. [47] in a model for diarrhea. Motility measurements are often
performed when studying inflammatory bowel diseases, in which similar intestinal symptoms
can be found. In these studies, local intestinal inflammation is indicated as causative factor
for motility changes reflecting alterations in smooth muscle contractions [48]. Hence,
local intestinal inflammation may have induced hypomotility occurring in the casein mice.
However, histological evaluation did not reveal any obvious signs of inflammatory cell
infiltrates after oral challenge. Possibly during sensitization, casein might have caused local
inflammation, hereby reducing sensitivity of cholinergic neurons for carbachol stimulations,
known to alter colonic motility [49]. In addition, local mast cell degranulation may have
induced motility changes [11]. Mule et al. [29, 50] have shown that activation of proteaseactivated receptors (PAR) can cause colon smooth muscle relaxation as well as contraction.
PAR agonists such as mast cell-derived tryptase may have caused PAR activation in both
casein- and whey-sensitized mice. It remains speculative why hypomotility of the colon
was only observed in casein-sensitized mice; however, local levels of tryptase and PAR
expression may differ between casein- and whey-sensitized mice and will be the focus of
future investigation.
Additionally, casein and whey proteins differ with regard to physical, physiological and
dietary properties and therefore may cause differential effects within the intestine. For
example, it is known that there is a difference in digestive speed of casein and whey [51].
Casein protein is a slowly digested dietary protein, while whey protein is a fast one [52].
70
Chapter 3
In addition, both proteins have different effects on satiety and gastrointestinal hormone
response [53]. Hence, these proteins differentially influence the intestinal physiology.
Furthermore, it is known that casein by itself can cause DNA damage in the colon, which is
associated with a thinner mucus barrier. In the same study, whey did not cause these effects
[54]. Two studies have shown hypomotility of the intestine after ingestion of casein, which
might relate to the presence of casomorphins that were found to reduce motility [55, 56].
Hence, casein may also have intrinsic properties that can cause hypomotility. At this stage,
the exact mechanism behind the hypocontractility found in the casein-sensitized mice is not
known and will be the topic of future studies.
Taken together, altered motility suggests subtle local changes in intestinal discomfort and
provides a new tool to measure local intestinal alterations as a consequence of allergic
sensitization in mice.
Both casein and whey sensitization consistently resulted in an acute allergic skin reaction
after allergen challenge, which was associated with specific IgE and IgG1 serum levels in
whey-sensitized mice and specific IgG1 serum levels in casein-sensitized mice. Decreased
numbers of mMCP-1-positive mucosal mast cells within the colon and enhanced mMCP-1
levels in the serum suggest a pathophysiological role for mucosal mast cells in this model.
In addition, the stool water content of allergic mice was enhanced, reflecting occurrence of
diarrhea 24 h after oral allergen challenge. In the casein-sensitized mice, reduced intestinal
smooth muscle contraction was observed. This suggests dysmotility of the colon shortly
after oral exposure to the allergen. Indeed, constipation and/or diarrhea are among the
symptoms in patients affected with CMA. Overall, the tools described in this study might
open new avenues to unravel underlying mechanisms of whey and casein allergy in mice.
Acknowledgement
We would like to thank Dr. Anneke Rijnierse for assistance with mMCP-1 immunohistochemistry
and helpfull discussions.
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75
4
Contribution of IgE and Immunoglobulin free light
chain in the allergic reaction to cow’s milk proteins
Bastiaan Schouten1
Bart R.J. Blokhuis1
Tom Groot Kormelink1
Gerard A. Hofman1
Guido E. Moro3
Günther Boehm4,5
Sertac Arslanoglu3
Frank A. Redegeld1
Johan Garssen1,2
1
2
3
Center for Infant Nutrition, Macedonio Melloni Maternity Hospital, Milan, Italy
4
Danone Research – Centre for Specialised Nutrition, Friedrichsdorf, Germany
5
Sophia Children’s Hospital, Erasmus University, Rotterdam, The Netherlands
Submitted for publication
Contribution of IgE and Immunoglobulin free light chain in the allergic reaction to cow’s milk proteins
Abstract
4
Background: Cow’s milk allergy (CMA) is affecting 2-5% of young infants. A group of
CMA patients has no detectable cow’s milk specific serum immunoglobulin (Ig)E. In
previous studies it was observed that allergic sensitization of mice to the major cow’s milk
allergens, casein and whey, led respectively to IgE-independent and IgE-dependent clinical
responses.
Objectives: In this study, the mechanism of the IgE-independent hypersensitivity response
to casein was further explored.
Methods: Mice were orally sham-, casein- or whey-sensitized. Acute allergen specific skin
responses were determined, serum immunoglobulins and immunoglobulin free light chains
(Ig-fLC) were measured. Ig-fLC dependency was investigated using a specific blocker and
passive transfer studies of spleen supernatants.
Results: After sensitization, no specific IgE was detectable in serum of casein-sensitized
mice, while in whey-sensitized mice specific IgE was enhanced. Instead, Ig-fLC levels were
increased in serum from casein-sensitized mice. Furthermore, blocking Ig-fLC strongly
diminished allergic skin responses in casein-sensitized mice. In addition, allergic sensitization
could be transferred using spleen cell culture supernatant from casein-sensitized mice and
was found to be Ig-fLC dependent.
Conclusions: This study indicates that sensitization with cow’s milk proteins can lead to
both IgE-dependent and Ig free light chain-dependent allergic hypersensitivity responses
and suggests that measurement of Ig-fLC may be important in the diagnosis of allergy. The
possible clinical relevance of these results was further substantiated by the finding that
kappa free light chains were significantly increased in mild atopic dermatitis patients.
78
Chapter 4
Introduction
Cow milk allergy (CMA) is a complex disorder, arising early in life and affecting 2-5% of the
children of the western world. CMA is also associated with an increased risk to develop
asthma later in life [1, 2]. The majority of CMA patients is allergic to the major milk proteins
casein and/or whey [3, 4]. Usually, reactions to a skin prick test (SPT) are measured and/
or titers of cow’s milk specific immunoglobulin (Ig)E are analyzed in the serum of patients.
However, there are CMA patients which exhibit acute clinical features of CMA without
detectable titers of specific IgE [3, 5, 6]. Therefore the ‘golden standard’ for diagnosis is still
an orally induced double blind food challenge [7-10]. The main disadvantage of an oral food
challenge is the time consuming character and the potential of inducing an anaphylactic
shock. Although the incidence of anaphylaxis is low, its potential risk is quite stressful for
the patient [7]. Therefore the search for another clinical tool to diagnose CMA is ongoing.
So far, no (immuno)therapy is available and merely avoidance of cow’s milk and the use of
hydrolyzed formulas are the only effective strategies to prevent symptoms of CMA [10].
Previously, two preclinical models for CMA have been introduced in which mice were
sensitized orally for whey or casein [11]. In these models the acute allergic skin reaction was
monitored as a possible equivalent of the SPT. In both models all mice exhibit an enhanced
ear swelling upon intra dermal (i.d.) allergen challenge, which reflects systemic sensitization.
The whey model resembles a typical type I allergy with high levels of whey-specific IgE and
IgG1. However, despite developing a pronounced acute allergic skin reaction upon local
allergen challenge, the response to casein was not associated with detectable levels of
casein-specific IgE. Although the casein-sensitized mice did have enhanced specific titers of
IgG1 this was found not to correlate quantitatively with the skin reaction [11]. Therefore, an
alternative explanation for the induction of the acute allergic skin reaction was explored. In
previous studies, it was demonstrated that antigen-induced acute allergic responses could
be elicited via immunoglobulin free light chains (Ig-fLC) [12-14]. For instance, transfer of
trinitrophenol-specific Ig-fLC into naïve mice sensitized them to the respective antigen.
Local challenge of the passively sensitized animals with the appropriate antigen resulted in
the induction of mast cell degranulation, leading to a local inflammation. This Ig-fLC-elicited
hypersensitivity response can be inhibited by local or systemic application of a specific
antagonist, a 9-mer peptide F991. Acute allergic responses induced by IgG or IgE are not
inhibited by F991 [12, 14]. Immunoglobulin free light chains (Ig-fLC) are present in serum
and their production is augmented under inflammatory conditions including allergic as well
as some autoimmune diseases [12, 15, 16]. For example, in patients suffering from multiple
sclerosis, free kappa light chains correlate with disability prognosis [17] and in rheumatoid
arthritis there is a significant correlation between kappa and lambda fLC and the disease
activity score [18].
79
4
In this study it is demonstrated that circulatory Ig-fLC are increased after oral sensitization
with casein. Moreover, acute allergic skin reactions to casein are inhibited by F991, which
indicates that Ig-fLC plays a pivotal role in the allergic sensitization. Preliminary clinical data
indicate that Ig-fLC concentrations are increased in children at risk for allergy with mild
atopic dermatitis (AD).
Methods
Chemicals
4
Casein and whey were obtained from DMV international, Veghel, The Netherlands. Cholera
toxin is purchased from Quadratech Diagnostics, Epsom, UK. PBS from Cambrex Bio Science,
Verviers, Belgium. Biotin-labeled rat anti-mouse IgE and IgG1 from BD Biosciences, Alphen
aan den Rijn, The Netherlands. The Ig-fLC antagonist F991 (AHWSGHCCL) was synthesized by
Fmoc chemistry, Ansynth, Roosendaal, The Netherlands. All other chemicals were obtained
from Sigma-Aldrich-Chemie, Zwijndrecht, The Netherlands.
Oral sensitization and challenge of mice
Three- to 5-week-old specific pathogen free female C3H/HeOuJ mice (n = 6 per group) were
purchased from Charles River Laboratories (Maastricht, The Netherlands), maintained on
cow’s milk protein free mouse chow (Special Diets Services, Witham, Essex, UK) and housed
in the animal facility at the Utrecht University. Animal care and use were approved by and
performed in accordance with the guidelines of the Animal Ethics Committee of the Utrecht
University. Mice were sensitized i.g. with 0.5 mL homogenized casein or whey (40 mg/mL
PBS) with cholera toxin (CT, 20 μg/mL PBS) as an adjuvant, using a blunt needle. Control
mice received CT alone or PBS. Mice were boosted weekly for a period of 5 weeks. One week
after last sensitization an i.d. ear challenge was performed. After 24 hours blood samples
were collected and centrifuged (15 min at 16000 x g). Sera were stored at – 70oC. Mice were
sacrificed by cervical dislocation. Cells of spleen and mesenteric lymph nodes of sensitized
mice were collected and cultured in serum free RPMI (1 x 106 cells/mL) for 24 hours [14].
Supernatants were collected in order to measure Ig and/or Ig-fLC levels.
80
Chapter 4
Measurement of specific serum immunoglobulins
Levels of casein- or whey-specific IgE and IgG1 were determined in serum by means of ELISA.
Microlon plates (Greiner, Alphen aan den Rijn, The Netherlands) were coated with 100 µL
whey or casein (20 µg/mL) in coating buffer (carbonate-bicarbonate buffer, 0.05 M, pH=9.6)
for 18 hours at 4oC. Plates were washed and blocked for 1 hour with 5% BSA. Serum samples
were applied in several dilutions and incubated for 2 hours at room temperature. Plates were
washed and incubated with biotin-labeled rat anti-mouse IgE or IgG1 (1 μg/mL) for one and
a half hour at room temperature and washed. The plates were incubated with streptavidinhorseradish peroxidase (HRP) for one hour, washed and developed with o-phenylendiamine.
After 5 min the reaction was stopped with 4M H2SO4 and absorbance was measured at 490
nm on a Benchmark microplate reader (Biorad, California, USA). Results were expressed as
arbitrary units (AU), with pooled sera from casein and whey alum-i.p. immunized mice used
as a positive reference serum to make the titration curve, as an internal standard.
Immunoblotting Ig-fLC
Prior to use, serum samples were precipitated to deplete high amounts of albumin using
trichloroacetic acid/acetone, as previously described [19]. Samples were fractionated by
SDS-PAGE under non-reducing conditions, electroblotted to PVDF membrane (Bio-Rad
Laboratories, Veenendaal, The Netherlands) overnight and probed with anti-mouse kappa
HRP Ab (Southern biotech Birmingham, Alabama USA). Immunoreactive bands were visualized
using enhanced chemiluminescence. Optical density (OD) of the immunoreactive bands was
quantified with a calibrated densitometer (BioRad, Veenendaal, The Netherlands).
Acute allergic skin reaction
The allergen-specific skin reaction was measured after injection of the specific protein in
the ear pinnae. Non-sensitized mice were injected i.d. in the left ear with 20 μL casein (0.5
mg/mL in PBS) and in the right ear whey (0.5 mg/mL in PBS) to determine non-specific skin
reactions. The casein- and whey-sensitized mice received in both ears either casein or whey,
respectively. Ear thickness of mice was measured in duplicate using a digital micrometer
(Mitutoyo, Veenendaal, The Netherlands), at t=0 and 1 hour after challenge. The ear swelling
is expressed as μm.
81
4
Passive transfer with spleen supernatants
Spleen supernatants were concentrated using vivaspin 20 spin filters (Millipore). Concentrated
fractions were 0.2 µm filtered and transferred i.v. (100 µL, approximately 400 µg protein/
mL) in naïve recipient mice. Sham mice received PBS as a control. After half an hour skin
reaction tests were performed as described above.
Antagonist studies
4
In the oral sensitized mice as well as in mice passively transferred with spleen supernatants
of sensitized mice, F991, an Ig-fLC inhibitor [13, 20], or PBS as a control was administered i.v.
(100 μL, 0.2 mg/mL F991) to determine whether the ear swelling was Ig-fLC dependent or
not. Half an hour after F991 administration the ear swelling test was performed as described
above.
Infants at high risk for allergy; serum Immunoglobulin kappa- and lambdafLC levels
Term born infants with a parental history of atopic eczema, allergic rhinitis or asthma in
either mother or father were eligible for a prebiotic intervention study, which was described
before [21, 22]. The study protocol was approved by the Ethical Committee of the Macedonio
Melloni Maternity Hospital in Italy. In a subpopulation plasma immunoglobulin levels were
measured as described by Van Hoffen et al. (CMP-IgE, IgG1, total- IgE, IgG1 and IgG2) [22].
In total, 25 infants had mild symptoms of AD (SCORAD 5.2 – 24.7) and 49 infants did not
develop AD at the age of 6 months. For this study total levels of Ig-fLC kappa and lambda
were determined in plasma as described previously [12, 23].
Statistical analysis
Ear swelling data and murine Ig-fLC data were analyzed using one way ANOVA and post hoc
Dunnett’s test and all other serological data were analyzed using two-tailed Mann-Whitney
tests. Statistical analyses were conducted using GraphPad Prism software (version 4.03). *
means a p-value of <0.05, ** p<0.01 and *** p<0.001. Murine data are represented as mean
± SEM, whereas clinical data are represented as median with interquartile range (IQR).
82
Chapter 4
Results
Serum specific immunoglobulin levels
Serum levels of casein-specific IgE were not increased in mice orally sensitized with casein
when compared to control mice (4.7 ± 1.6 vs 6.0 ± 3.5 AU, respectively) (Figure 1a). In
contrast, casein-specific IgG1 levels were enhanced (404.8 ± 217.5 vs 0 ± 0 AU), although this
was not significant due to two non-responders (Figure 1b). In whey-sensitized mice both
specific IgE (239.4 ± 123.6 AU; p<0.05) and IgG1 (2671 ± 910 AU; p<0.001) were significantly
increased compared to sham-sensitized mice (2.5 ± 1.1 and 14.0 ± 11.2 AU, respectively)
(Figure 1c and d).
Ig-fLC levels in sensitized mice
Next, it was examined if sensitized mice had enhanced total levels of Ig-fLC. Sera of non-,
Figure 1. Specific serum immunoglobulin levels in casein- and whey-sensitized mice. In
the casein-sensitized mice no increase in casein-specific IgE was found a, but IgG1 b was
increased in 4 out of 6 animals. Whey-sensitization induced levels of specific IgE c and IgG1
d. * p<0.05, ** p<0.01 and n=6.
83
4
Igs
Casein
Control
b
Whey
kDa
250
150
100
75
(Lc)2
50
37
Ig‐fLC
4
25
20
15
Figure 2. Densitometric analysis of kappa Ig-fLC in serum and supernatant from local
mesenteric lymph nodes (MLN) revealed Ig-fLC levels to be increased. a Analysis of kappa IgfLC in serum from individual mice. In casein-sensitized mice, levels of Ig-fLC were significantly
increased in comparison to control- or whey-sensitized mice. b Representative Western-blot
of Ig-fLC levels in supernatants of MLN of control-, casein- and whey-sensitized mice. Igs are
total immunoglobulins (IgA, IgD, IgE, IgG and IgM), (Lc)2 are Ig-fLC dimers and Ig-fLC are the
monomers. * p<0.05 and n=6.
Figure 3. Effect of F991 on the induction of an acute allergen-specific skin reaction in (a) caseinand (b) whey-sensitized mice. F991 decreased casein-induced ear swelling significantly but
whey-induced ear swelling was unaffected. Ear swelling (µm) is calculated as the increase in
ear thickness induced by the corresponding antigen at one hour after challenge. ** p<0.01,
*** p<0.001 and n=6.
84
Chapter 4
casein- and whey-sensitized mice were analyzed by immunoblotting for the presence of
kappa Ig-fLC. The concentrations of kappa Ig-fLC in casein-sensitized mice were significantly
increased compared to non- and whey-sensitized animals (p<0.05; non-sensitized: 66.6 ±
5.5; whey 64.2 ± 8.7 and casein 92.3 ± 9.5 OD\mm2, Figure 2a).
To analyze production of Ig-fLC in local lymph nodes, cells from mesenteric lymph nodes
(MLN, n=6 pooled) were cultured for 24 hours and supernatants were analyzed for kappa
Ig-fLC. Samples fractionated by SDS-PAGE under non-reducing conditions showed complete
immunoglobulins (about 180-200 kDa), Ig-fLC dimers (about 45 kDa) and Ig-fLC monomers
(20 kDa). The MLN of casein-sensitized mice produced higher levels of Ig-fLC compared to
the control and whey-sensitized animals (Figure 2b).
4
Ig-fLC inhibitor F991 blocks the acute allergic skin reaction in casein-sensitized mice
To analyze whether the increase in Ig-fLC in casein-sensitized animals was functional relevant
regarding the allergic reaction, the effect of the antagonist F991 was tested on the allergeninduced ear swelling. F991 greatly reduced the skin reaction in the casein-sensitized mice
(147.4 ± 16.0 µm without and 83.3 ± 8.3 µm with F991, p<0.05, Figure 3a). As expected, the
Ig-fLC antagonist did not affect the ear swelling of whey-sensitized mice (Figure 3b, 145.0 ±
9.66 and 148.5 ± 13.9 µm). Also non-specific ear swelling (irritant reaction) in control mice
was not affected by the F991. Casein did not provoke an ear swelling in whey-allergic mice
and vice versa (data not shown).
Figure 4. Effect of F991 on the induction of an acute allergic skin reaction in naïve mice
injected (i.v.) with spleen supernatants or PBS as a control. Passive transfer with spleen
supernatants of a casein and b whey allergic mice resulted in acute allergic skin reaction
upon i.d. allergen challenge. Only in casein recipients the acute allergic skin reaction was
abrogated by F991. *** p<0.001 and n=6.
85
Ig-fLC responsible for passive transfer of allergic sensitization to casein
In the previous experiments, it was shown that local lymphoid organs from caseinsensitized mice produced significantly more Ig-fLC compared to sham- and whey-sensitized
animals. To investigate the involvement of Ig-fLC as a humoral factor in the induction of
the acute allergic skin reaction, naïve recipient mice were injected intravenously (i.v.) with
concentrated spleen supernatants (or PBS as a control), obtained from allergic mice, in
presence or absence of F991 as previously described [14]. Subsequently, the cutaneous
4
Table I. Immunoglobulin levels in infants at high risk for allergy, divided in healthy (SCORAD
-) and AD (SCORAD +) groups.
Atopic dermatitis
SCORAD -
SCORAD +
Number of infants
n=49
n=25
Median (IQR)
0.0 (0)
6.9 (6.5)
Serum antibody
Isotype
Median (IQR)
Median (IQR)
p-value
Total
IgE (kU/L)
4.50 (9)
6.00 (22)
p=0.30
IgG1 (g/L)
2.69 (1.9)
2.95 (2.5)
p=0.15
IgG2 (g/L)
0.78 (0.4)
0.91 (0.6)
p=0.41
IgE (ng/mL)
1.95 (3.1)
2.90 (3.2)
p=0.10
IgG1 (AU/mL)
1.50 (6.5)
3.25 (7.7)
p=0.27
CMP-specific
IQR, interquartile range
Figure 5. Infants at risk for allergy with mild symptoms of AD (SCORAD +, n=25) had enhanced
levels of a kappa Ig-fLC compared to infants without AD (SCORAD -, p=0.0075, n=49). b
Lambda Ig-fLC showed a similar tendency (p=0.14). Medians are presented.
86
Chapter 4
swelling was measured at one hour after intradermal (i.d.) allergen challenge. For recipient
mice receiving spleen supernatants of casein-sensitized mice the allergen challenge induced
a significant ear swelling (110.9 ± 10.2 µm). F991 inhibited the casein-induced ear swelling
highly significantly (57.1 ± 5.7 µm, p<0.001, Figure 4a). The Ig-fLC antagonist did not affect
the ear swelling of recipient mice receiving spleen supernatants of whey-sensitized mice
(105.9 ± 6.6 and 105.0 ± 5.1 µm, Figure 4b), confirming that Ig-fLC did not play a significant
role in the induction of the acute allergic skin reaction against whey protein. Casein did not
provoke an ear swelling in whey spleen supernatant transferred mice and vice versa (data
not shown).
4
Immunoglobulin levels and Ig-fLC levels in infants at high risk for allergy
In order to test the clinical relevance of the current findings, immunoglobulin levels and
Ig-fLC levels were measured in plasma from children at risk of developing allergic disease
from a double-blind, randomized, placebo controlled prebiotic intervention study [21, 22].
These infants were six months of age and developed AD (SCORAD between 5.2 and 24.7) or
remained negative for AD (SCORAD -). No differences were found between the two groups
for total levels of IgE, IgG1, IgG2, cow’s milk-specific IgE and IgG1 (Table I).
In support to our findings, in the preclinical studies Ig-fLC kappa levels were found to be
increased in the infants that developed AD (n=25) when compared to infants without AD
(n=49) (median (IQR) 7.4 (3.0) vs 6.1 (4.0) µg/mL, p=0.0075, Figure 5a) Ig-fLC lambda levels
showed the same tendency (7.7 (5.6) vs 6.1 (4.1) µg/mL, p=0.14, Figure 5b).
Discussion
In previous studies, it was shown that the allergic skin response in mice sensitized with
casein or whey, both major allergens responsible for CMA, is substantially different [10,
11, 24]. Whey-sensitized mice show a classical type I allergic skin reaction combined with
high levels of specific IgE. However, in casein-sensitized mice, acute allergic responses were
elicited in the absence of casein-specific IgE. Here, new data provide evidence that caseinsensitization of mice results in the induction of an immunoglobulin free light chain (Ig-fLC)
dependent hypersensitivity response.
Food allergy is thought to be mediated by IgE and non-IgE dependent mechanisms. IgE
mediated food allergy is a classical type I allergy which manifests itself within a few hours.
87
Non-IgE mediated food allergy usually is defined as a delayed-type hypersensitivity with
enhanced T-helper 1 activity and the symptoms are noticeable after one or more days
while cellular mechanisms are involved [25-29]. However, of interest is a substantial group
of patients which have symptoms of an immediate type hypersensitivity response, while
lacking detectable levels of IgE [3, 5, 6]. In contrast to the murine model, in humans casein
can elicit strong IgE responses [3]. The mice were sensitized for either casein or whey, while
in infants hypersensitivity is raised against whey and casein derived from whole cow’s milk.
Direct comparison between mice and human with regard to the sensitizing capacities for
casein and whey are therefore difficult to perform. However, the concept of Ig-fLC being
involved in the pathophysiology of human disease has been described for inflammatory as
well as allergic disease [12, 14, 17, 18, 30].
4
Several lines of evidence suggest the critical involvement of Ig-fLC in the induction of casein
hypersensitivity in mice. First, casein-sensitization results in increased levels of Ig-fLC in
serum, while local lymph nodes of casein-sensitized mice show enhanced production of
Ig-fLC ex vivo. Secondly, inhibition of Ig-fLC induced hypersensitivity using F991 prevented
skin response in casein-sensitized mice. A minor part of the ear swelling in casein-sensitized
mice was not affected by F991, which could suggest a residual role for IgG1 or for IgE. In
several studies it has been shown that F991 is specific for Ig-fLC and does not affect IgE nor
IgG mediated skin responses. Furthermore, F991 prevents local inflammation in different
hypersensitivity models [12, 14, 20, 31]. Thirdly, allergic sensitivity to casein could be
transferred to naïve animals by i.v. injection of culture supernatant from spleens of caseinsensitized mice and was found to be dependent on Ig-fLC. Together, these data indicate a
prominent role for Ig-fLC in the induction of casein allergy.
Other studies in murine models for cow’s milk allergy various results have been described.
Oral sensitization with α-casein resulted in an increase of α-casein-specific IgE and IgG1 titers
in C3H mice [32]. On the other hand, Lara-Villoslada et al. [33] only detected whey and
casein specific IgG1, while IgE was not detectable in a CMA model induced by simultaneous
oral sensitization with casein and whey. The differences in route of sensitization,
intraperitoneal vs oral, and variation in allergen fractions e.g. α-casein vs complete casein
fraction or combined whey and casein, might explain the contrasting outcome in these and
the present studies. The fundamental different mechanisms of allergy observed for casein
and whey may be due to the differences in digestive speed, dietary, chemical, physical and
physiological features of the proteins [34-37]. Therefore, these differences between the
proteins influence not only the intestinal physiology, but likely the response of the immune
system as well, which is a topic of further studies. Patients with IgE-mediated CMA have
higher specific IgE concentrations to casein compared with whey proteins [3]. It would be
interesting to analyze the presence of total or (e.g. casein-) specific Ig-fLC in those patients
who suffer from cow´s milk allergy in absence of detectable increases in total or specific
88
Chapter 4
IgE.
In previous studies, it has been shown that Ig-fLC can mediate immediate hypersensitivitylike responses. Mast cells are the primary target cells for Ig-fLC and subsequent crosslinking of cell surface-bound Ig-fLC by cognate antigen results in mast cell activation and
the induction of a local inflammatory response [14]. Polyclonal Ig-fLC levels are enhanced
in chronic inflammatory diseases like inflammatory bowel disease [38], rheumatoid arthritis
(16), Sjögren’s syndrome [18], multiple sclerosis [17, 39] and systemic lupus erythematosus
[30]. Also in allergic diseases, increases in Ig-fLC have been described. For example, in
individuals with allergic and non-allergic asthma, Ig-fLC levels are increased when compared
to healthy controls [12]. It is estimated that in one third of the children suffering from AD the
skin symptoms are triggered by sensitization to food [40, 41]. Notably, children with severe
AD have higher levels of lambda and kappa light chain in serum [42]. Furthermore, in nonIgE mediated rhinitis patients both lambda and kappa Ig-fLC levels are found to be enhanced
in nasal secretions (Powe, Groot Kormelink et al., JACI accepted). The present study confirms
that increased Ig-fLC may be associated with development of allergic disease. In sera from
infants at high risk for allergy neither enhancement of CMP-specific IgE and IgG1 nor levels
of total IgE, IgG1 and IgG2 were found. However, serum levels of kappa Ig-fLC were elevated
significantly and lambda Ig-fLC showed a similar increment. This suggests that measurement
of total and ultimately, specific levels of Ig-fLC may be an interesting parameter in the clinical
diagnosis for AD.
In summary, sensitization with cow’s milk proteins can lead to both IgE-dependent and IgEindependent allergic hypersensitivity responses. In this study, evidence is provided indicating
that the IgE-independent allergic response to casein is mediated, at least in part, by specific
Ig-fLC. An increase in Ig-fLC levels may be of relevance in allergies without noticeable
involvement of IgE. Further research is warranted to determine the clinical significance of
assaying Ig-fLC for the diagnosis of allergic diseases.
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the first 3 years of life. Clinical course in relation to clinical and immunological type of
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Vanto, T., et al., Prediction of the development of tolerance to milk in children with cow’s
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Gabriele, C., et al., Fractional exhaled nitric oxide in infants during cow’s milk food challenge.
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van Houwelingen, A.H., et al., Topical application of F991, an immunoglobulin free light
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van Hoffen, E., et al., A specific mixture of short-chain galacto-oligosaccharides and long-
Chapter 4
chain fructo-oligosaccharides induces a beneficial immunoglobulin profile in infants at high
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Abe, M., et al., Differences in kappa to lambda (kappa:lambda) ratios of serum and urinary
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Lied, G.A., Gastrointestinal food hypersensitivity: Symptoms, diagnosis and provocation
tests. Turk J Gastroenterol, 2007. 18(1): p. 5-13.
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Sletten, G.B., et al., Changes in humoral responses to beta-lactoglobulin in tolerant patients
suggest a particular role for IgG(4) in delayed, non-IgE-mediated cow’s milk allergy. Pediatr
Allergy Immunol, 2006. 17(6): p. 435-43.
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Huang, Z.Q. and P.W. Sanders, Localization of a single binding site for immunoglobulin light
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Lara-Villoslada, F., M. Olivares, and J. Xaus, The balance between caseins and whey proteins
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4
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Chapter 4
4
93
5
Non-digestible oligosaccharides reduce
immunoglobulin free light-chain levels in infants at
risk for allergies
Bastiaan Schouten1
Betty C.A.M. Van Esch1,2
Tom Groot Kormelink1
Guido E. Moro3
Sertac Arslanoglu3
Günther Boehm4,5
Frank A. Redegeld1
Linette E.M. Willemsen1
Johan Garssen1,2
1
2
3
Center for Infant Nutrition, Macedonio Melloni Maternity Hospital, Milan, Italy
4
Danone Research – Centre for Specialised Nutrition, Friedrichsdorf, Germany
5
Sophia Children’s Hospital, Erasmus University, Rotterdam, The Netherlands
Non-digestible oligosaccharides reduce immunoglobulin free light-chain levels
in infants at risk for allergies
Abstract
5
Background: In previous studies it has been shown that a prebiotic mixture of short-chain
galacto-oligosaccharides and long-chain fructo-oligosaccharides (Immunofortis®) reduces
the incidence of atopic dermatitis. In this study the plasma immunoglobulin free light-chain
(Ig-fLC) concentrations were measured in infants at risk for allergic disease.
Methods: In a double-blind, randomized placebo-controlled trial infants received a
hypoallergenic whey formula containing 8 g/L of the prebiotics or maltodextrine as a placebo
for 6 months. After intervention plasma samples were collected. Total plasma concentrations
of lambda and kappa Ig-fLC were analyzed using ELISA.
Results: Total plasma levels of lambda and kappa Ig-fLC were lower in infants fed the
prebiotic mixture (n=34) compared to the placebo (n=40; p<0.001).
Conclusions: These data demonstrate that the specific prebiotic mixture is capable of
preventing the lambda and kappa Ig-fLC plasma levels to increase in infants at high risk
for allergies. This may have contributed to the reduced incidence of the atopic dermatitis
observed.
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Chapter 5
Introduction
The incidence of allergies is increasing in the western world and treatment strategies are
limited. Prebiotics as well as probiotics are intensively studied for a possible preventive
role due to their capacities to positively influence the immune system [1]. Prebiotics are
non-digestible carbohydrates that are fermented by Bifidobacteria and Lactobacilli in the
gastrointestinal tract [2]. Immunofortis® is a specific prebiotic mixture, adapted from the
breast milk oligosaccharide composition, consisting of short chain Galacto-oligosaccharides
(scGOS) and long chain Fructo-oligosaccharides (lcFOS) in a 9:1 ratio. It was shown that oral
supplementation of this scGOS/lcFOS prebiotic mixture reduces the cumulative incidence of
atopic dermatitis (AD) in infants at high risk for allergies in comparison to the placebo bottle
fed infants [3]. In the 6 months follow-up of this study it was shown that total plasma levels
of IgE, IgG1, IgG2 and IgG3, but not IgG4, of the infants who received the prebiotic mixture
were reduced. Furthermore, cow’s milk protein (CMP) specific IgG1 levels were decreased,
whereas no effect on specific IgE was observed at six months of age [4]. In a preclinical mouse
model for orally induced sensitization for cow’s milk protein, specific IgE levels against casein
were almost not detected. A role for immunoglobulin free light-chain (Ig-fLC) in the acute
allergic skin response was observed (Schouten et al., submitted). Ig-fLC are produced by B
cells, bind to mast cells and cause degranulation after second allergen encounter causing
an immediate type of allergic response [5]. Ig-fLC concentrations were measured in the
same infants evaluated by Moro et al. [3] in a double blind placebo controlled study where
neonates at risk for allergies were fed a formula containing a scGOS/lcFOS mixture. This
prebiotic mixture was found to reduce plasma levels of both lambda and kappa Ig-fLC, which
may relate to the reduced incidence of atopic dermatitis found in this study population.
Methods
Subjects and samples
The study setup has been described by Moro et al. [3, 4]. In short, the study was a randomized
double blind placebo controlled trial performed in infants at risk for atopy. Neonates with a
parental history of allergic diseases in either mother or father were enrolled in the study and
started formula feeding within the first two weeks of life. Two formulas were used, based on
extensively hydrolyzed cow’s milk whey protein supplemented either with 0.8 g scGOS/lcFOS
(in a 9:1 ratio) or 0.8 g maltodextrin per 100 mL. AD was scored using the SCORAD figure
for infants under 2 years by two clinicians. Plasma samples of subpopulation representing
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5
the whole population were obtained after 6 months of intervention. The study protocol was
approved by the Ethical Committee of the Macedonio Melloni Maternity Hospital in Italy.
Lambda and kappa immunoglobulin (Ig) free light-chain levels
For this study, total levels of Ig-fLC kappa and lambda were determined in plasma as
described previously [6]. For each sample, four different dilutions were incubated, and at
least three of them within the linear portion of the standard curves were used to calculate
the concentration.
5
Statistics
Kappa Ig-fLC and lambda Ig-fLC date were analyzed using two-tailed Mann Whitney U test.
Statistics were calculated using GraphPad Prism 4.03. Data are represented as medians.
Results
Ig-fLC concentrations were determined in plasma samples that were previously analyzed for
Figure 1. Oral supplementation with short chain Galacto-oligosaccharides (GOS)/ long chain
Fructo-oligosaccharides (FOS) reduces the plasma concentrations of both (a) lambda and
(b) kappa immunoglobulin free light-chains in infants at high risk for allergy at six months of
age. Placebo n=40 and GOS/FOS n=34. *** P < 0.001.
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Chapter 5
immunoglobulin content [4]. Oral supplementation of the prebiotic scGOS/lcFOS mixture
significantly reduced plasma levels of both lambda (placebo 8.17 (6.0-11) µg/mL vs scGOS/
lcFOS 5.62 (4.7-7.9) µg/mL, p<0.001) and kappa (placebo 7.58 (5.8-9.2) µg/mL vs scGOS/
lcFOS 5.81 (4.0-6.7) µg/mL, p<0.001) Ig-fLC in infants at high risk for allergies (Figure 1). In
this subgroup of the original study population, 19 out of 40 infants in the placebo group and
6 out of 34 infants in the GOS/FOS intervention group developed AD. The severity of the AD
was mild (SCORAD 5.2-24.7).
Conclusions
Evidence for non-digestible carbohydrates as immunomodulatory components is increasing.
However, the underlying mechanism of action is still under debate. This study shows that
prebiotics affect the humoral immune response in infants at risk for developing allergic
diseases. Ig-fLC concentrations were measured in plasma samples derived from a clinical
study, described by Moro et al. [3], in which a scGOS/lcFOS mixture was found to reduce
the cumulative incidence of AD. The GOS/FOS diet was found to reduce Ig-fLC in infants at
risk for allergies.
The presented results are in accordance with the findings of Van Hoffen et al. [4]. In plasma
samples of the same study they found GOS/FOS to reduce total levels of immunoglobulins
(IgE, IgG1) and CMP specific levels of IgG1. The prebiotics did not affect CMP specific IgE,
however the plasma levels were very low in both groups.
Up to 40% of CMP allergic patients have almost no detectable CMP specific IgE levels
[7]. Therefore another mechanism might be involved and Ig-fLC is a potential candidate.
Furthermore, there is increasing evidence for the clinical relevance of Ig-fLC in allergic
diseases. For example, it has been shown that in individuals with allergic and non-allergic
asthma Ig-fLC concentrations are increased when compared to healthy controls [6], and in
non-allergic rhinitis with eosinophilic syndrome patients Ig-fLC are enhanced in plasma as
well as in nasal secretions (Powe et al., JACI accepted). In this study, GOS/FOS was found to
reduce Ig-fLC, Moro et al. [3] have shown GOS/FOS to reduce significantly the cumulative
incidence of AD. This indicates a possible role for Ig-fLC in the pathophysiology of AD.
Indeed, Sediva et al. [8] showed that in severe AD patients (n=100; SCORAD 50-80) both
lambda and, especially, kappa Ig-fLC were enhanced in serum when compared to healthy
age matched controls (n=102).
These data demonstrate that dietary intervention with a specific prebiotic GOS/FOS mixture
reduces both lambda and kappa plasma Ig-fLC concentrations in infants at risk for allergies.
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5
The reduced Ig-fLC concentrations may have contributed to the reduced incidence of AD in
infants fed GOS/FOS.
References
1.
Immunol, 2007. 119(1): p. 192-8.
2.
Hoppu, U., et al., Breast milk--immunomodulatory signals against allergic diseases. Allergy,
2001. 56 Suppl 67: p. 23-6.
3.
5
4.
5.
6.
7.
8.
100
Chapter 5
5
101
6
Cow’s milk allergy symptoms are reduced in mice
fed dietary synbiotics during oral sensitization
with whey
Bastiaan Schouten1
Gerard A. Hofman1
Suzan A. C. M. van Doorn2
Jan Knol2
Alma J. Nauta2
Johan Garssen1,2
1
2
The Journal of Nutrition 2009;139(7):1398-1403
Cow’s milk allergy symptoms are reduced in mice fed dietary synbiotics during
oral sensitization with whey
Abstract
6
Background: Cow’s milk allergy is the most common food allergy in children. So far, no
effective treatment is available to prevent or cure food allergy. The purpose of this study was
to compare effects of dietary supplementation with a prebiotic mixture (Immunofortis®),
a probiotic strain (Bifidobacterium breve M-16V), or a synbiotic diet combining both on
the outcome of the allergic response when provided during oral sensitization with whey in
mice.
Methods: Mice were fed diets containing 2% (wt:wt) Immunofortis® and/or the B. breve
M-16V (n = 6/group). The acute allergic skin response was determined by measuring ear
swelling. Antigen-induced anaphylaxis was scored. Furthermore, whey-specific serum
immunoglobulins and mouse mast cell protease-1 (mMCP-1) were determined.
Results: In mice fed the synbiotic mixture, the allergic skin response and the anaphylactic
reaction were strongly reduced compared with whey-sensitized mice fed the control diet
(p<0.01). Immunofortis® or B. breve M-16V alone were significantly less effective in reducing
the allergic skin response than the synbiotic diet and did not reduce the anaphylactic reaction.
The whey-specific IgE and IgG1 responses were not affected; however, IgG2a was greater in all
treated groups than in the control group (p<0.05). Serum mMCP-1 concentrations, reflecting
mucosal mast cell degranulation, were lower in mice fed synbiotics compared with those fed
the control diet (p<0.01).
Conclusions: Dietary supplementation with Immunofortis®, B. breve M-16V, and particularly
the synbiotic mixture, provided during sensitization, reduces the allergic effector response
in a murine model of IgE-mediated hypersensitivity that mimics the human route of
sensitization. This model shows the potential for dietary intervention with synbiotics in
reducing the allergic response to food allergens.
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Chapter 6
Introduction
The incidence of food allergy is rising in the western world [1]. Approximately 2–5% of
children in the western world are affected with cow’s milk allergy (CMA). Many studies
suggest that food allergy in early life is associated with the development of respiratory
allergy and even asthma later in life [2-4]. So far, no effective preventive strategy or
treatment protocol is available for CMA [5]. Avoidance of the affecting food in the diet is
the single effective solution. Some studies suggest that breastfeeding protects the infant
from developing allergic disease. Breast-feeding supports maturation of the immune system
of the young infants and protects the child from infections [6, 7]. In human milk, there
are many components with immunological properties, e.g. non-digestible oligosaccharides,
secretory IgA (sIgA), mucins, cytokines, long-chain PUFA, and hormones. In particular,
oligosaccharides may support immunocompetence, a prerequisite for adequate capacity
to induce oral tolerance, either via direct effects or via stimulation of a beneficial intestinal
microbiota [8-10].
Non-digestible prebiotic oligosaccharides are defined as “food ingredients that beneficially
affect the host by selectively stimulating the growth and/or activity of 1 or a limited number
of bacterial species already resident in the colon, and thus attempt to improve host health”
[11]. Non-digestible carbohydrates, which mimic the oligosaccharides in human milk, are
known to have immune regulatory properties [12]. In a mouse vaccination model, the
vaccine-specific T helper cell (TH1) response was improved when a prebiotic diet was given
starting 1 wk prior to the first vaccination, whereas it failed when the diet was provided after
the first vaccination [13]. This indicated that the prebiotics are especially effective during
the priming phase of an immune response. Therefore, the prebiotic concept was suggested
to be a relevant tool for the prevention of immune disorders such as allergies. In clinical
preventive studies in children, early dietary intervention resulted in reduced incidence of
allergic manifestations and infections [14], improved microbiota [15], increased fecal sIgA
[16], and reduced incidence of atopic dermatitis (AD) [17]. Prebiotic oligosaccharides are
known to stimulate the growth and/or activity of probiotic bacteria. Bifidobacteria together
with Lactobacilli are well known for their health-promoting functions and selected species
are used as probiotics [18-20]. Neonates receiving breastfeeding have higher counts in
Bifidobacteria compared with babies fed standard infant formula. When added to standard
milk formula, the prebiotic mixture Immunofortis® enhanced Bifidobacteria fecal counts in
bottle-fed infants to levels similar to those found in breast-fed infants [21]. In a double-blind,
placebo-controlled study, a mixture of probiotics containing Lactobacillus rhamnosus GG,
Bifidobacterium breve, and Propionobacterium freudenreichii spp. Shermanii JS alleviated
atopic eczema/dermatitis syndrome in IgE-sensitized infants [22]. In a recent review by
Savilahti et al. [23], the positive effects of probiotics on allergy prevention and treatment
105
6
have been evaluated. They concluded the effects are highly variable and that optimal
treatment remains unsettled. Because prebiotic oligosaccharides selectively stimulate the
growth and/or activity of bacterial species such as B. breve, synbiotic concepts are currently
introduced combining pro- and prebiotics enabling optimized function of the pro- and
prebiotics. The potential of selective pre-, pro-, and synbiotics to prevent the development
of allergic disease has been shown in some clinical studies [17, 20, 24-26]. However, the
outcomes of these studies vary and questions remain concerning the mechanisms of action.
In the present study, we used a mouse model of orally induced CMA to assess preventive
effects of dietary intervention with Immunofortis®, a probiotic diet, or a synbiotic diet [27,
28].
Methods
Chemicals
6
Whey proteins were obtained from DMV International. Cholera toxin (CT) was purchased
from Quadratech Diagnostics. PBS was from Cambrex Bio Science, and biotin-labeled rat
anti-mouse IgE, IgG1, IgG2a, and IgA were from BD Biosciences. All other chemicals were
obtained from Sigma-Aldrich-Chemie.
Diets
Semi purified cow milk protein-free, AIN-93G-based diets were composed and mixed with
pro-, pre-, or synbiotics by Research Diet Services [29]. The probiotic strain, B. breve M-16V
(freeze-dried on maltodextrin carrier and conserved at -20oC), was obtained from Morinaga
(Morinaga Milk Industry) and 2% (wt:wt) 2 X 109 colony forming units/g was mixed through
the diets and pressed into pellets. Viability of B. breve M-16V after diet manufacturing was
confirmed by plating on MRS agar. The prebiotic diet contained 2% (wt:wt) Immunofortis®,
a 9:1 (wt:wt) mixture of short-chain galacto-oligosaccharides (GOS; obtained by enzymatic
elongation of lactose with galactose by β-galactosidase, with a degree of polymerization of
3–8) and long-chain fructo-oligosaccharides (FOS; derived from chicory inulin with a mean
degree of polymerization of >23). Immunofortis® is a spray-dried powder of GOS (Vivinal
GOS, FrieslandCampina Domo, Zwolle, The Netherlands) and long-chain FOS (Raftiline HP).
Immunofortis® consists of ~50% GOS and FOS, 19% is maltodextrin (glucidex 2, Roquette),
16% lactose, 14% glucose, and 1% galactose (13). Immunofortis® was exchanged against 2%
(wt:wt) of total carbohydrates present in the diet. In total, 4 diets were prepared: control,
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Chapter 6
probiotic, prebiotic, and synbiotic (containing 2% Immunofortis® and B. breve M-16V). The
diets were stored at -20oC prior to use.
Three-week-old specific pathogen-free female C3H/HeOuJ mice were purchased from
Charles River Laboratories. Animal care and use were performed in accordance with the
guidelines of the Dutch Committee of Animal Experiments. Sham- and whey-sensitized mice
were fed a control, B. breve M-16V, GOS/FOS, or synbiotic diet (n = 6/group). Two weeks
before the first sensitization, the dietary intervention was started and continued during the
whole study protocol. Mice were sensitized i.g. with 0.5 mL homogenized whey (40 g/L
PBS) with CT (20 mg/L PBS) as an adjuvant using a blunt needle. Control mice received CT
alone. Mice were boosted weekly for a period of 5 wk as described previously (27). One
week after the last sensitization, feces were collected and mice were challenged with whey
intradermally (i.d.) in the ear to determine the acute allergic skin response and anaphylaxis
scores. After 24 h, mice were challenged i.g. with 1.0 mL homogenized whey (100 g/L PBS)
and 30 min later, blood samples were collected in Minicollect Z Serum Sep vials (Greiner bioone). Collected blood was centrifuged at 16,000 X g; 15 min and sera were stored at –70oC.
The weight of the mice was monitored weekly.
Acute allergen-specific skin response
The acute allergen-specific skin response was measured after i.d. injection with 20 mL
homogenized whey protein (0.5 g/L in PBS) in the ear pinnae. Ear thickness was measured
in duplicate using a digital micrometer (Mitutoyo) at t = 0 and 1 h after challenge. Delta
ear swelling (μm) was calculated by subtracting the specific whey-induced ear swelling
in sensitized mice after 1 h from the basal (t = 0 h) ear thickness. Additionally, the mean
Δ ear swelling from the group of sham-sensitized mice fed the corresponding diet was
subtracted.
Systemic anaphylaxis
Anaphylaxis was scored after i.d. challenge in the ear. Symptoms of systemic anaphylaxis
were scored (adapted from Li et al. [30]): 0 = no symptoms; 1 = pilar erecti, scratching
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6
and rubbing around the nose and head; 2 = pilar erecti, reduced activity; 3 = activity after
prodding and lowered body temperature; 4 = no activity after prodding, labored respiration,
and lowered body temperature; and 5 = death.
Measurement of specific serum Ig and mouse mast cell protease-1
6
Concentrations of whey-specific IgE, IgG1 and IgG2a, were determined in serum using ELISA
as described previously [27]; specific IgA was measured in feces. In short, microlon plates
(Greiner) were coated with 100 μL whey (20 mg/L), washed, and blocked with 5% bovine
serum albumin. Samples were applied in several dilutions; for determinations in feces, 1.0
g feces was dissolved in 10 mL PBS and centrifuged at 16,000 X g; 5 min. The supernatant
was collected for IgA measurements. Samples were incubated for 2 h at room temperature.
Plates were washed and incubated with biotin-labeled rat anti-mouse IgE, IgG1, IgG2a, or
IgA (1 mg/L), washed, and incubated with streptavidin-horseradish peroxidase, washed,
and developed with o-phenylendiamine. The reaction was stopped with 4 mol/L H2SO4 and
absorbance was measured at 490 nm on a Benchmark microplate reader (Bio-Rad). Results
were expressed as arbitrary units with pooled sera from whey- and alum-intraperitoneally–
immunized mice used as a positive reference serum to make the titration curve. Serum
concentrations of mouse mast cell protease-1 (mMCP-1) were determined according to the
manufacturer’s protocol using a commercially available ELISA kit (Moredun Scientific).
Preparation of fecal samples for PCR of B. breve M-16V
For PCR analysis, 0.1 g feces was placed in a Lysing Matrix E tube with 1.0 mL stool lysis
buffer (Qiagen). These tubes were placed in a mini-BeadBeater (BioSpec Products) for 3
min, heated for 5 min at 95oC, and centrifuged at 14,000 X g; 5 min. Then 200 μL Lysozym
(10 g/L MiliQ) was added to the supernatant, heated for 30 min at 37oC, and centrifuged
at 4000 X g; 10 min. DNA was extracted from the collected supernatant using a NucliSENS
easyMAG (BioMerieux). PCR was performed as described previously [21] using B. breve
M-16V-specific primers. Briefly, primers and probes of the B. breve M-16V were used in a
temperature profile consisting of 2 min at 50oC and 10 min at 95oC, followed by 45 cycles of
15 s at 95oC and 60oC for 1 min.
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Chapter 6
Statistics
All data were analyzed using 1-way ANOVA and post hoc Bonferroni’s multiple comparison
test except for the anaphylaxis data, these were analyzed using the Kruskal-Wallis test.
Statistical analyses were conducted using GraphPad Prism software (version 4.03). P-values
<0.05 were considered significant. Data are represented as means ± SEM.
Results
Acute allergic skin response
One hour after dermal challenge, the Δ ear swelling in the whey-sensitized mice was 117.6
± 8.6 μm (remaining ear swelling upon subtraction of ear swelling in sham-sensitized mice
fed control diet, see Methods section). The ear swelling of the whey-sensitized mice fed the
pro- and prebiotic diet was lower than in those fed the control diet (p<0.05). In mice fed the
synbiotic diet, this effect was even more pronounced (p<0.001; Figure 1). The ear swelling
of mice fed the synbiotic diet was reduced (p<0.01) compared with mice fed the pro- and
prebiotic diets. The diets themselves did not affect ear swelling in sham-sensitized control
mice (data not shown).
Figure 1. Acute allergic skin response of whey-sensitized mice fed a control, B. breve, GOS/
FOS, or synbiotic diet. The acute ear swelling response was measured 1 h after i.d. challenge
with whey. Values are means ± SEM, n=6. Means without a common letter differ, p<0.05.
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6
Figure 2. Anaphylactic symptom scores of sham-sensitized mice fed a control diet and wheysensitized mice fed a control, B. breve, GOS/FOS, or synbiotic diet. Values are medians, n=6.
Medians without a common letter differ, p<0.01.
6
Figure 3. Serum mMCP-1 concentrations of sham-sensitized mice fed a control diet and
whey-sensitized mice fed a control, B. breve, GOS/FOS, or synbiotic diet. Values are means ±
SEM, n=6. Means without a common letter differ, p<0.01.
Systemic anaphylaxis after i.d. challenge
The mice were i.d. challenged in the ear with whey 1 wk after the last sensitization. In the
whey-sensitized group fed the control diet, the anaphylaxis score was enhanced compared
with control sham-sensitized mice (p<0.01; Figure 2). In the synbiotic group, anaphylaxis
scores were lower than whey-sensitized mice fed the control diet (p<0.01). Neither the prenor the probiotic diet alone affected shock scores.
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Chapter 6
Decreased mMCP-1 levels in serum
To assess the effect of dietary intervention on mucosal mast cell degranulation, mMCP-1
concentrations were determined in the serum. Serum mMCP-1 was lower in mice fed the
synbiotic diet than in those fed control diet (p<0.01; Figure 3) but was unaffected in mice fed
the prebiotic or probiotic diet.
Whey-specific Ig levels in serum or feces
Thirty minutes after oral challenge, serum was obtained from all mice. Whey-specific serum
IgE, IgG1, IgG2a, and fecal IgA were analyzed using ELISA. In whey-sensitized mice, specific
IgE and IgG1 increased compared with sham-sensitized mice (p<0.01; Figure 4a, b). Overall,
dietary intervention did not affect serum IgE and IgG1 levels. In contrast, the probiotic,
prebiotic, and synbiotic diets enhanced whey-specific IgG2a levels in whey-sensitized mice
(p<0.01) compared with sham-sensitized mice (Figure 4c). The IgG2a levels in whey-sensitized
mice fed the control diet were not significantly enhanced compared with sham-sensitized
mice. Specific IgA levels were increased in feces of whey-sensitized mice fed the control
or B. breve M-16V diet (p<0.01; Figure 4d) and in mice fed the GOS/FOS or synbiotic diet
(p<0.001) compared with sham-sensitized mice fed the control diet.
Figure 4. Whey-specific Ig of sham-sensitized mice fed a control diet and whey-sensitized
mice fed a control, B. breve, GOS/FOS, or synbiotic diet. Whey-specific serum IgE a, serum
IgG1 b, serum IgG2a c, and fecal IgA D levels were measured. Values are means ± SEM, n=4–6.
Means without a common letter differ, p<0.01.
111
6
PCR analysis
PCR analysis on the fecal samples was performed to verify whether the B. breve M-16V was
present in the feces of the control or pro-, pre-, and synbiotic treated mice. Indeed, the
presence of B. breve M16-V was confirmed only in the mice fed the pro- and synbiotic diets,
whereas feces of control mice and mice fed Immunofortis®did not contain B. breve M-16V
(data not shown).
Discussion
6
Both pro- and prebiotics modulate immune function and have been shown to reduce the
risk of developing allergic disease in human studies [17, 31, 32]. Host factors, bacterial
strain differences, dosage, and timing of supplementation may underlie the differences
in effectiveness. There is general agreement that, although there are promising effects,
more data are needed [23, 33]. In the present study, dietary intervention with synbiotics
combining probiotic strain B. breve M-16V with prebiotic GOS/FOS mixture Immunofortis®
was shown to protect against the development of allergic symptoms in mice orally sensitized
with whey. Synbiotics effectively reduced the acute allergic skin response, the anaphylaxis
scores, and mast cell-derived mMCP-1 concentrations. Pro- and prebiotics alone were less
effective. The skin response is a robust parameter and was associated with enhanced serum
IgE and mast cell-derived mMCP-1 in previous studies [27]. Mice fed pro- and prebiotic
diets had a significantly reduced acute allergic skin response upon whey challenge in the
ear compared with whey-sensitized mice fed the control diet. The synbiotic diet was even
more effective and almost completely prevented occurrence of the acute skin response
as well as anaphylactic shock reactions. The clinical effectiveness of the prebiotic mixture
used in this study has previously been shown to reduce the incidence of AD in infants at
risk. The newborn infants were randomly assigned to standard formula milk or formula milk
containing Immunofortis® until 6 mo of age, reducing the incidence of AD by >50% [17].
Furthermore, infants at high risk for allergy supplemented with the prebiotic mixture had a
slight reduction in total IgE and IgG1–3 and specific IgG1 [34]. IgG4 tended to increase, which
might be suggestive for tolerance induction/outgrowing food allergy. Also probiotics have
been shown to reduce AD development in infants. When pregnant mothers with a family
history of allergic disease and their babies were supplemented with Lactobacillus GG, the
cumulative incidence of atopic eczema was still reduced after 7 y by 30% compared with
placebo controls [35]. Although the incidence of eczema was reduced, IgE levels were not
affected. In a similar study, Lactobacillus reuteri reduced the incidence of IgE-associated
eczema and the occurrence of a positive skin prick test for almost 50%, whereas IgE was not
affected [32]. As in the human studies, the allergic effector response in the current study
112
Chapter 6
using mice orally sensitized with whey was reduced upon dietary intervention with pro-,
pre-, and synbiotics, whereas IgE levels remained enhanced. In young infants, development
of AD can objectively be scored. Approximately one-third of these children are affected with
food allergy. Nevertheless, the occurrence of eczema in AD patients and the acute allergic
skin response as measured in mice form reliable measures for skin responses to allergens
and, in this respect, this parameter in mice might reflect, at least in part, some aspects of
AD. Indeed, a synbiotic diet has been shown to reduce AD-like skin lesions in mice [24].
Dietary intervention with pro-, pre-, and, in particular, synbiotics reduced the acute allergic
skin response and the anaphylaxis scores very effectively, which could not be explained by
a reduction in whey-specific TH2 type IgE or IgG1 levels. However, increased levels of wheyspecific IgG2a and enhanced fecal type IgA levels may reflect improved TH1 or regulatory T-cell
responses in the mice fed a pro-, pre-, or synbiotic diet [16, 36, 37]. In a preclinical murine
model, heat-treated L. rhamnosus GG reduced the development of AD probably through
the induction of interleukin (IL)-10 [38]. These studies and our study are in accordance
with at least 2 clinical studies in which probiotic treatment enhanced (fecal) IgA and IL-10
and alleviated allergic symptoms [23, 39]. Hence, dietary intervention with pre-, pro-, and
synbiotics in mice orally sensitized with whey may have supported TH1 responses and/or
regulatory T-cell function.
Besides the acute allergic skin response, anaphylactic scores upon i.d. challenge were
dramatically reduced in whey-sensitized mice fed the synbiotic diet. Although in humans the
shock usually appears after an oral challenge, at least for food allergens, anaphylaxis in our
mouse study occurred after i.d. challenge with whey proteins. Eigenmann et al. [40] studied
allergic shock in a murine model of orally induced β-lactoglobulin sensitization. In this study,
the anaphylactic shock reaction occurred after oral challenge. Oral inoculation with an
avirulent Salmonella typhimurium strain prior to allergic sensitization partially prevented
anaphylaxis. This is in accordance with the effects of the B. breve M-16V on anaphylaxis
as observed in the current study. So far, to our knowledge, there is no literature available
about the preventive effects of pre- and synbiotics on allergen induced anaphylaxis. The
dietary effects on the acute allergic skin response and the anaphylactic shock coincided.
Mast cell degranulation most probably underlies both symptom scores. Indeed, mMCP-1
serum concentrations reflecting mucosal mast cell degranulation were reduced, particularly
in mice fed the synbiotic diet. Hence, the diets may have altered mast cell function. In a
murine model of ovalbumin-induced allergy, oral administration of different bacterial
strains prevented mast cell degranulation [41]. IgE receptor expression on mast cells can
be diminished by CD4+CD25+ regulatory T-cells upon direct contact or via the secretion of
IL-10 and transforming growth factor-β [42]. Because whey-specific fecal IgA was enhanced
upon dietary intervention, this may be an indication of enhanced regulatory T-cell function.
In a clinical study with healthy infants receiving a GOS/FOS diet, fecal sIgA increased and
113
6
it was suggested to have a regulatory function and a positive effect on mucosal immunity
[16]. Alternatively, IgG-blocking antibodies may have inhibited IgE mediated anaphylaxis in
mice, as was shown by a study from Strait et al. [43]. IgG2a that was enhanced upon dietary
intervention may have a function as blocking antibody.
6
The current study shows very promising effects of the synbiotic concept in prevention of
the acute allergic skin response, reducing the incidence and severity of anaphylaxis and
reducing mMCP-1 a marker for mast cell degranulation. Little is known about the use of
synbiotics to prevent or cure allergic disease. A synbiotic mixture of Lactobacillus casei and
dextran reduced pollen allergy in humans [24]. Kukkonen et al. [25] showed that a synbiotic
mixture containing L. rhamnosus GG and LC705, B. breve Bb99, and Propionibacterium
freudenreichii ssp shermanii combined with GOS supplied to newborn infants was safe,
reduced the cumulative incidence of IgE-associated (atopic) diseases, and decreased the
occurrence of respiratory infections. In the present study, a preventive dietary intervention
study was performed comparing the effects of pro-, pre-, and synbiotics in mice orally
sensitized to whey. In particular, the synbiotic combination of Immunofortis® and the
probiotic strain B. breve M-16V, and to a lesser extent the pro- and prebiotic diets, reduced
the acute allergic skin response and anaphylactic scores, which are clinical relevant allergic
parameters. Although the exact mechanism of action is not yet known, dietary intervention
enhanced whey-specific TH1 type serum IgG2a and specific IgA in feces while reducing mast
cell degranulation. Synbiotics comprise a promising concept that may be more effective in
reducing allergic symptoms than single preparations of pre- or probiotics.
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Scholtens, P.A., et al., Fecal secretory immunoglobulin A is increased in healthy infants
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Lenoir-Wijnkoop, I., et al., Probiotic and prebiotic influence beyond the intestinal tract. Nutr
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Chapter 6
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6
117
7
Oligosaccharide induced whey-specific CD25+
regulatory T-cells are crucial in the suppression of
cow’s milk allergy in mice
Bastiaan Schouten1
Gerard A. Hofman1
Louis Boon3
Johan Garssen1,2
1
2
3
Bioceros BV, Utrecht, The Netherlands
Oligosaccharide induced whey-specific CD25+ regulatory T-cells are crucial
in the suppression of cow’s milk allergy in mice
Abstract
Background: Dietary intervention with a unique prebiotic non-digestible carbohydrate
mixture has been shown to reduce the development of allergic disease in infants at risk. In
this study the involvement of CD25+ regulatory T-cells in the carbohydrate induced effects
was investigated in mice orally sensitized with whey using adoptive transfer experiments.
Methods: Donor mice were sensitized with whey and fed a diet containing short-chain
galacto-, long-chain fructo- and acidic-oligosaccharides or control diet starting two weeks
before sensitization. The acute allergic skin reaction upon intra dermal whey challenge was
determined and whey-specific immunoglobulins were measured. Splenocytes of the donor
mice were transferred to naïve recipient mice after ex vivo depletion of CD25+ splenocytes.
Results: The prebiotic diet clearly diminished the acute allergic skin reaction. Wheysensitized recipient mice receiving splenocytes from whey-sensitized prebiotic fed donor
mice displayed almost complete prevention of the acute allergic skin reaction compared to
mice receiving cells from sham-sensitized prebiotic fed donor mice. Depletion of CD25+ cells
inhibited these effects, although IgE sensitization was not prevented.
Conclusion: This study points to a crucial role for whey-specific CD25+ regulatory T-cells
in suppression of the allergic effector response induced by dietary intervention with
prebiotics.
7
120
Chapter 7
Introduction
The gastro-intestinal tract is exposed to a wide variety of dietary proteins. In general,
these proteins are tolerized by the gastro-intestinal immune system resulting in local and
systemic non-responsiveness. This orally induced tolerance may be a result of anergy or
active suppression, which is determined by both antigen dosage [1] and age [2]. In food
allergic patients the oral tolerance to specific dietary proteins is broken or not established
[3]. Currently, there is no specific therapy for the treatment of food allergy. The only option
to avoid symptoms is to circumvent exposure to the responsible food. The most common
food allergy in childhood is cow’s milk allergy (CMA) which affects about 2-3% of all infants
[4]. It is caused by an allergic inflammatory response to milk proteins [5]. Cow’s milk
proteins (3.5% w/v in milk) consist of casein and whey proteins. Whey proteins comprise
β-lactoglobulin (the main allergen), α-lactalbumin, albumin, lactoferrin, immunoglobulins
and proteosepeptone [6]. In Europe most of the infant formula feedings are whey based and
are supplemented with prebiotics.
In food allergy the delegate balance between oral tolerance and hypersensitivity is regulated
by cells of the immune system. Cells with regulatory properties include TGF-β-producing TH3
cells, IL-10-producing Tr1 cells and NKT cells [7-9]. In addition, the TGF-β and IL-10 producing
naturally occurring CD4+CD25+Foxp3+ regulatory T-cells (Treg) were described to be important
for maintaining peripheral tolerance and for suppression of proliferation of CD4+ and CD8+
T-cells [10, 11]. Treg represent 5-10% of the CD4+ T lymphocyte population and express the
transcription factor forkhead box p3 (Foxp3), which is confirmed as a key gene for generation
and maintenance of Treg. Adaptive regulatory T-cells have similar surface markers, however
their inhibitory capacities act via cell-cell contact instead of cytokines. Currently, the
involvement of Treg in tolerance induction by dietary components is unknown. Human milk
consists of numerous components, including but not limited to oligosaccharides, which may
have many immunological properties and might facilitate tolerance [12, 13]. Prebiotic fibers
manufactured to resemble some of these oligosaccharides are defined as “non-digestible
food ingredients that beneficially affect the host by selectively stimulating the growth and/
or activity of one or a limited number of bacterial species already resident in the colon, and
thus attempt to improve host health” [14]. Short-chain galacto-oligosaccharides (scGOS)
and long-chain fructo-oligosaccarides (lcFOS) are dominating the prebiotic literature.
Beneficial immunological properties of these scGOS and lcFOS are confirmed by an improved
vaccination response in a murine model [15] and lowered incidence of atopic dermatitis in
children [16]. In addition, a similar prebiotic mixture reduced the acute allergic skin response
in mice orally sensitized with whey [17]. Besides scGOS and lcFOS, the pectin derived acidicoligosaccharides (pAOS) were identified as a potential prebiotic fiber [18].
121
7
In the present study a mouse model for orally induced sensitization to whey proteins was
used to study the immunological mechanisms that might underlie the induction of tolerance
against whey using prebiotics [19]. The role of Treg in the preventive capacities of dietary
intervention with a prebiotic mixture (2 w/w% (9:1:1) scGOS, lcFOS and pAOS) was assessed
using adoptive transfer experiments. It was demonstrated that CD25+ regulatory T-cells play
a pivotal role in the prebiotic dietary induced inhibition of cow’s milk allergy in mice.
Materials and methods
Chemicals
Whey proteins were obtained from DMV International (Veghel, The Netherlands). Cholera
toxin was purchased from Quadratech Diagnostics (Epsom, UK). PBS from Cambrex Bio
Science (Verviers, Belgium). Biotin-labeled rat anti-mouse IgE, IgG1 and IgG2a from BD
Biosciences (Alphen aan den Rijn, The Netherlands). PC61 (anti-CD25), a monoclonal
antibody from a rat PC61 hybridoma cell line, was a kindly gift of Bioceros BV (Utrecht, The
Netherlands). All other chemicals were obtained from Sigma-Aldrich-Chemie (Zwijndrecht,
The Netherlands).
7
Diets
Semi-purified cow’s milk protein free AIN-93G-based diets were composed and mixed
with prebiotics by Research Diet Services (Wijk bij Duurstede, The Netherlands). The
prebiotic diet contained 2 w/w% (9:1:1) short-chain GOS (scGOS; enzymatic elongation
of lactose with galactose by β-galactosidase), long-chain FOS (lcFOS; from chicory inulin)
and pAOS (produced from pectin). scGOS (Vivinal GOS, FrieslandCampina Domo, Zwolle,
The Netherlands) contains oligosaccharides with a degree of polymerisation (dp) of 3–8.
lcFOS (Raftiline HP, Orafti, Wijchen, The Netherlands) contains oligosaccharides with an
average degree of polymerisation of >23. The scGOS/lcFOS spray-dried powder consists of
approximately 50% scGOS and lcFOS, 19% maltodextrin (glucidex 2, Roquette, France), 16%
lactose, 14% glucose and 1% galactose [15]. The pAOS powder (Südzucker AG, Mannheim,
Germany) have a degree of polymerisation of 1-20, consisting of approximately 75%
galacturonic acid oligomers, 10% monomers and 15% of moisture and ash [18]. All prebiotics
were exchanged for the same amount of total carbohydrates, this resulted in a comparable
overall carbohydrate composition in the diets.
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Chapter 7
Three-week-old specific pathogen free female C3H/HeOuJ mice were purchased from Charles
River Laboratories (Maastricht, The Netherlands) and housed in the animal facility. Animal
care and use were performed in accordance with the guidelines of the Dutch Committee of
Animal Experiments. Two weeks before the first sensitization animals received the different
diets; mice remained on these diets during the whole study protocol. Mice were sensitized
intra gastric (i.g.) with 0.5 mL homogenized whey (40 mg/mL PBS) with cholera toxin (CT,
20 μg/mL PBS) as an adjuvant, using a blunt needle. Control mice received CT alone. Mice
were boosted weekly for a period of 5 weeks as described previously [19]. One week after
the last sensitization feces were collected and mice were challenged intra dermally (i.d) in
the ear to determine the acute allergic skin response. After 24 hours blood samples were
collected in Minicollect® Z Serum Sep vials (Greiner bio-one) and mice were sacrificed by
cervical dislocation. Collected blood was centrifuged 15 min at 13500 rpm and sera were
stored at –70oC.
Acute allergen specific skin reaction
The acute allergen specific skin response was measured after i.d. injection with 20 μL
homogenized whey protein (0.5 mg/mL in PBS) in the ear pinnae. Ear thickness was
measured in duplicate using a digital micrometer (Mitutoyo, Veenendaal, The Netherlands),
at t=0 and 1 hour after challenge. The ear swelling (μm) was calculated by subtracting the
basal (t=0 hour) from the specific whey induced ear swelling after 1 hour ear thickness and
additionally (Δ ear swelling) subtracting the mean ear swelling from the group of shamsensitized mice fed the corresponding diet.
Adoptive transfer experiments
To test whether splenocytes are able to transfer prebiotic induced tolerance adoptive
transfer experiments were carried out. After the prebiotic supplemented (donor) mice were
sacrificed, the spleens were obtained and pooled per group (n=12). Single cell suspensions
of the four groups (sham, sham prebiotic, whey and whey prebiotic) were i.v. injected (100
μL, 1x107 cells/mL) in naïve mice, in total eight recipient groups of n=6/group were formed
(Table 1 experiment A). The recipient mice were fed the control diet and sensitized (sham or
whey) as described above. The acute allergic skin reaction was measured and whey-specific
serum immunoglobulins were measured to determine allergic sensitization.
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7
In another set of experiments single spleen cell suspensions of whey-sensitized donor mice
fed the control or prebiotic diet were pooled per group (n=16) and half of the cells were
treated with 50 μg per spleen rat anti-CD25 (PC61). Two mL Biomag Goat anti-rat IgG (Bangs
Laboratories Inc., Fishers, USA) was added to the treated cells and placed on a rotator for at
45 min, the bead-cell mixture was placed in a magnetic separator for 5 min and supernatant
was transferred and again placed on the magnetic separator for 5 min. Depletion of CD25
positive cells was analyzed using flow cytometric analysis. The splenocytes of the four
groups were i.v. injected (100 μL, 1x107 cells/mL) in recipient mice, in total eight groups
were formed (n=6/group) (Table 1 experiment B). The recipient mice were sham- or wheysensitized as described above. The acute allergic skin reaction was measured and wheyspecific serum immunoglobulins were measured to determine allergic sensitization.
Flow cytometry
7
Single cell suspensions were prepared from mouse spleens. Cells were counted using a Coulter
Z1 particle counter (Beckman Coulter) and diluted to a concentration of 2x106 cells/mL. For
identification of CD4+CD25+Foxp3+ cells, anti-CD4-FITC (1:100) and anti-CD25-PE (1:100) were
used (BD, Alphen aan den Rijn, The Netherlands). Foxp3 staining was performed following the
manufacturer’s protocol (eBioscience, Breda, The Netherlands). Flow cytometry analysis was
performed using FACSCalibur (BD, Alphen aan den Rijn, The Netherlands), CellQuest Pro Analysis
software (BD, Alphen aan de Rijn, The Netherlands) and isotype controls were subtracted.
Concentrations of whey-specific IgE, IgG1, IgG2a were determined in serum by means of
ELISA as described before [19]. In short, plates were coated with 100 µL whey (20 mg/L.
Plates were incubated with biotin-labeled rat anti-mouse IgE, IgG1 and IgG2a (1 mg/L),
and incubated with streptavidin-horseradish peroxidase (HRP). Results were expressed as
arbitrary units (AU), calculations were based on titration curves of pooled sera from whey
and alum-i.p. immunized mice as a positive reference serum.
Statistics
All data were analyzed using the one way ANOVA and post hoc Bonferroni’s Comparison
124
Chapter 7
Table 1. Overview of experimental set-up.
Experiment Sensitization Diet
of donors
A
sham
whey
B
whey
Treatment of Sensitization Diet
splenocytes
of recipients
control
-
sham
control
control
-
whey
control
prebiotic
-
sham
control
prebiotic
-
whey
control
control
-
sham
control
control
-
whey
control
prebiotic
-
sham
control
prebiotic
-
whey
control
control
-
sham
control
control
anti-CD25
sham
control
control
-
whey
control
control
anti-CD25
whey
control
prebiotic
-
sham
control
prebiotic
anti-CD25
sham
control
prebiotic
-
whey
control
prebiotic
anti-CD25
whey
control
Test or Mann-Whitney U test. For the immunoglobulin levels an ANOVA was performed on
log transformed data. Statistical analyses were conducted using GraphPad Prism software
(version 4.03). P-values of * p<0.05, ** p<0.01 and *** p<0.001 were indicated when
analyses reached statistically significance. Data are represented as means ± SEM.
Results
Acute allergic skin reaction of donor mice
To investigate the preventive effects of the prebiotic diet on sensitization, whey-sensitized
mice and sham-sensitized mice were challenged i.d. in the ear pinnae with whey, one week
after the last sensitization. The delta ear swelling in the whey-sensitized mice was 157 ±
11 μm, the prebiotic diet largely prevented the acute allergic skin reaction of the wheysensitized mice (67 ± 5.8 μm, p<0.001, Figure 1). The diet did not affect ear swelling of shamsensitized mice (data not shown).
125
7
Figure 1. The prebiotic (scGOS/lcFOS/pAOS) diet effectively reduces the acute allergic skin
reaction. Whey induced acute ear swelling in whey-sensitized mice fed the control or prebiotic
diet was measured 1 hour after i.d. challenge in the ear pinnae. This graph is a representative
of four identical experiments. Means ± SEM, *** p<0.001 and n=12 per group.
7
Figure 2. Reduced whey-IgE and whey-IgG1 in
whey-sensitized mice fed the prebiotic (scGOS/
lcFOS/pAOS) diet. Whey-specific a IgE, b IgG1,
and c IgG2a levels in serum of whey-sensitized
mice fed the control or prebiotic diet. Means ±
SEM, * p<0.05 and n=16 per group.
126
Chapter 7
Whey-specific immunoglobulin levels of donors
Whey-specific serum IgE, IgG1 and IgG2a levels were increased in whey-sensitized mice
compared to sham-sensitized mice (data not shown). Whey-specific IgE was reduced in
whey sensitized mice fed the prebiotic diet compared to mice fed the control diet (116 ±
27 vs 477 ± 167 AU, p<0.05, Figure 2). Also whey-specific IgG1 was reduced by the prebiotic
diet (68120 ± 10650 vs 42560 ± 6045 AU, p<0.05). Dietary intervention did not affect wheyspecific IgG2a levels in whey-sensitized mice fed control diet compared to prebiotic diet (665
± 167 vs 425 ± 89 AU).
Acute allergic skin reactions in recipient mice adoptively transferred with splenocytes
Sham- as well as whey-sensitized recipient mice receiving splenocytes (i.v.) of whey-sensitized
7
Figure 3. Adoptive transfer of splenocytes of whey-sensitized mice fed the prebiotic (scGOS/
lcFOS/pAOS) diet protects recipient mice from developing an acute allergic skin response.
Recipient mice receiving splenocytes from whey-sensitized mice fed the control diet
developed an acute skin response if sham- or whey-sensitized. Transfer of splenocytes of
whey-sensitized mice fed prebiotic diet prevented these responses. However the acute skin
response still developed in whey-sensitized recipients transferred with prebiotic fed donor
mice that were sham-sensitized. The delta ear swelling was calculated by subtracting ear
swelling of sham-sensitized recipient mice injected with cells of sham-sensitized donor mice
fed corresponding control or prebiotic diet. Means ± SEM, * p<0.05, *** p<0.001 and n=6
per group.
127
donor mice fed the control diet had severe allergic skin reactions in the ears 1 h after whey
injection (Figure 3). In contrast, the transfer of donor splenocytes of whey-sensitized mice
fed the prebiotic diet protected against development of an acute allergic skin reaction in
sham-sensitized as well as whey-sensitized recipient mice (12 ± 5 and 22 ± 8 µm, p<0.001
compared to recipients transferred with control diet fed donor splenocytes). This protection
was whey-specific since whey-sensitized recipient mice injected with splenocytes of shamsensitized donors receiving the prebiotic diet still developed a severe ear swelling response
(125 ± 12 µm).
Whey-specific immunoglobulin levels of recipients
7
Serum samples were collected of the recipient mice and analyzed for whey-specific
immunoglobulins. Whey-specific IgE levels of recipient mice receiving splenocytes of
sham-sensitized donor mice fed the control diet were enhanced upon whey-sensitization
compared to sham-sensitization (352 ± 243 vs 6.0 ± 3.0 AU, p<0.01, Figure 4a). Similar results
were obtained when recipients were transferred with splenocytes from sham-sensitized
donor mice fed the prebiotic diet (256 ± 112 vs 5.2 ± 2.4 AU, p<0.01). Whey-specific IgE
levels tended to increase in sham-sensitized recipient mice adoptively transferred with
splenocytes of whey-sensitized mice. In contrast, transfer of splenocytes of whey-sensitized
mice lowered the capacity to develop whey-specific IgE in recipients upon sensitization with
whey. However, no difference in IgE levels were observed between recipients transferred
with donor cells of mice fed the control or prebiotic diet.
Similar results were found for IgG1 and for IgG2a (Figure 4b and 4c). Whey-sensitized
recipient mice transferred with cells of sham-sensitized donor mice, regardless of the donor
diet had enhanced IgG1 and IgG2a levels compared to sham-sensitized recipients transferred
with splenocytes of sham-sensitized donors. Sham-sensitized recipients transferred with
splenocytes of whey-sensitized mice fed either the control or prebiotic diet, had the
lowest IgG1 and IgG2a levels. The IgG1 and IgG2a levels of recipient whey-sensitized mice
transferred with splenocytes of whey-sensitized mice, regardless of the received diet, were
of intermediate levels.
Overall, no differences in sensitization of the recipient mice transferred with cells of donor
mice receiving control or prebiotic diet were observed regarding the antibody levels.
128
Chapter 7
7
Figure 4. Whey-specific immunoglobulin levels in recipient mice of which the donor mice
were fed control or prebiotic (scGOS/lcFOS/pAOS) diet. No differences were observed
between diet groups. a Whey-specific IgE b whey-specific IgG1 and c whey-specific IgG2a.
Means ± SEM, * p<0.05, ** p<0.01, *** p<0.001 and n=5-6 per group.
129
b
rat IgG1
anti‐CD25
3.6%
7.9%
3.9%
0.0%
prebiotic
7
CD25
control
6.5%
isotype
Foxp3
Figure 5. Ex vivo depletion of CD25+ Treg in spleen cell suspension of donor mice using PC61
antibody and magnetic beads. a Percentage of CD4+CD25+Foxp3+ Tregs. b Representative dot
plots of CD25+Foxp3+ cells in the CD4+ population. These spleen cell fractions were used for
adoptive transfer experiments. Means ± SEM, ** p<0.01, n=3-6.
130
Chapter 7
Acute allergic skin reaction of recipients transferred with Treg depleted
donor cells
Splenocytes from whey-sensitized mice fed the control or prebiotic diet were transferred
with or without prior ex vivo CD25+ cell depletion using the PC61 antibody. There was no
difference in number of CD4+CD25+Foxp3+ Treg in the spleen of whey-sensitized mice fed the
prebiotic diet (6.1 ± 0.3%, Figure 5) compared to the control diet (6.4 ± 0.3%). In both groups
the ex vivo Treg depletion was similar, resulting in a 67% reduction in the prebiotic and
61% reduction in the control diet donor spleen suspension compared to the non-depleted
controls (p<0.01).
Acute allergic skin reaction of recipients using Treg depleted donor cells
To investigate the role of Treg in the transfer of dietary prebiotics induced tolerance,
splenocytes of whey-sensitized mice fed the control or prebiotic diet were ex vivo
depleted for Treg cells and transferred. The acute allergic skin reaction in recipient mice
was determined. Upon whey-sensitization the ear swelling response of recipient mice
transferred with complete or Treg depleted spleen cell suspensions from whey-sensitized
7
Figure 6. Depletion of Treg in transferred spleen cell suspensions of whey-sensitized donors
fed the prebiotic (scGOS/lcFOS/pAOS) diet abolished the protection against the development
of an acute allergic skin reaction. Donor cells of whey-sensitized mice fed the control diet did
not protect against the development of the acute allergic skin response in recipient mice and
Treg depletion did not alter this response. Means ± SEM, ns = not significant, * p<0.05, **
p<0.01, *** p<0.001 and n=5-6 per group.
131
7
Figure 7. Whey-specific immunoglobulin levels in serum of recipient mice after adoptive
transfer with sham or Treg depleted splenocytes. There are no differences between control
and prebiotic (scGOS/lcFOS/pAOS) fed groups. a whey-specific IgE b whey-specific IgG1 and
c whey-specific IgG2a. Means ± SEM, ns = not significant, * p<0.05, ** p<0.01 and n=6 per
group.
132
Chapter 7
donor mice fed the control diet significantly enhanced compared to the sham-sensitized
recipient mice (149 ± 8.1 or 179 ± 22 µm vs 78 ± 10 µm, p<0.01, Figure 6). Donor cells from
whey-sensitized mice fed the prebiotic diet effectively prevented the occurrence of an acute
skin response in recipients upon whey-sensitization compared to sham-sensitization and the
ear swelling response was significantly lower compared to recipients transferred with cells
of mice fed control diet (149 ± 8.1 vs 90 ± 15 µm; p<0.05). Treg depletion prior to transfer
completely abolished the preventive effect of adoptively transferred donor cells of mice fed
the prebiotic diet resulting in severe ear swelling in recipient mice upon whey-sensitization
(224 ± 11 µm, p<0.001).
Whey-specific immunoglobulin levels of recipients of Treg depleted donor
cells
Sham- or whey-sensitized recipient mice receiving spleen cell suspensions of control or
prebiotic diet fed whey-sensitized donor mice showed similar whey-specific IgE levels.
Recipient mice receiving Treg depleted spleen cell suspensions of whey-sensitized donor
mice fed the control diet had higher whey-specific IgE levels in comparison with recipient
mice receiving non-Treg depleted spleen cell suspensions (12.5 ± 3.6 vs 169 ± 71 AU,
p<0.05, Figure 7a). A similar tendency was found in recipient mice receiving Treg depleted
splenocytes of donor mice fed the prebiotic diet (32.5 ± 9.8 vs 170 ± 79 AU).
Also whey-specific IgG1 or IgG2a levels enhanced when recipient mice were transferred
with Treg depleted donor spleen cell suspensions, however no differences were observed
between recipient mice transferred with donor cells of mice fed the control or prebiotic diet
either in the presence or absence of Treg (Figure 7b and 7c).
Discussion
Genetic predisposition, environmental factors, timing, duration and extent of exposure
are important determinants of allergic sensitization [20]. So far, no effective treatment for
allergy is available, although murine and human studies have investigated the effectiveness
of prebiotics to prevent the development of allergic disease. Dietary supplementation
of oligosaccharides in babies and young infants has been found to reduce the incidence
of atopic dermatitis [16] and infections in infants at risk [21]. Recently, we have shown a
preventive effect of dietary prebiotics on the allergic effector response in an oral mouse
model for whey allergy [22]. In the current study this model was used to investigate the
133
7
contribution of CD25+ regulatory T-cells (Treg) in the development of tolerance to whey
induced by the prebiotic diet using adoptive transfer experiments and ex vivo depletion of
CD25+ Treg.
The prebiotic mixture of scGOS/lcFOS/pAOS used in this murine model for cow’s milk allergy
ameliorated the severity of allergic symptoms, since the acute allergic skin response in mice
fed the prebiotic diet during whey-sensitization was strongly reduced. A slight reduction
in TH2 immunoglobulin levels (whey-specific IgE and IgG1) was observed, whereas TH1 type
immunoglobulin IgG2a levels remained unaffected high. Previously, these prebiotics have
been shown to improve the vaccination response in mice, which is mainly TH1 driven [18].
The results suggest an improved TH1/TH2 balance upon dietary intervention with prebiotics
which could partly explain their effect, but not conclusively.
7
In mice orally sensitized with whey the acute allergic skin response determines the level
of systemic response as a result of a break in oral tolerance [19]. This allergen-specific ear
swelling response has similarities with the human skin prick test. Since the prebiotic diet
was found to reduce the severity of the acute allergic skin response, adoptive transfer
studies were performed to determine whether this tolerance could be transferred using
splenocytes. Adoptive transfer of splenocytes from whey-sensitized donor mice fed the
control diet transferred the allergy to recipient mice that were sham-sensitized, since they
developed a whey-specific acute skin response. Guttormsen et al. [23] also found adoptively
transferred whole spleen single cells to transfer immunologic memory for a vaccine. In
contrast to the whey-sensitized recipient mice receiving splenocytes of whey-sensitized mice
fed the control diet, the splenocytes of prebiotic fed whey-sensitized mice protected against
the development of allergic symptoms. The whey-specific acute allergic skin reaction was
prevented, hence tolerance for whey induced in the donor mice by the prebiotic diet could
be transferred to the recipient mice.
We hypothesized the CD4+CD25+ Treg, which occurs naturally or can be induced, to be a
potential cellular candidate mediating the prebiotic induced oral tolerance to whey. Two
phases of oral tolerance can be distinguished, clonal deletion or active suppression of the
immune response by Treg cells [24]. The prebiotic diet did not enhance the number of
Treg cells in Peyer’s patches, mesenteric lymph nodes and spleen when mice were fed the
prebiotic diet (data not shown). However, in vivo anti-CD25 depletion revealed that Treg
cells play an important role in the oral sensitization with whey in mice (Van Esch et al.,
submitted). Furthermore Gri et al.[25] showed that Treg cells are able to inhibit mast cell
degranulation and acute allergic responses which may relate to the reduced acute allergic
skin reaction observed in the present study in mice fed the prebiotic diet.
The involvement of Treg in the induction of tolerance by prebiotics was studied using
134
Chapter 7
adoptive transfer of splenocytes of whey-sensitized mice fed the prebiotic diet that were ex
vivo depleted for CD25+ Treg cells using an anti-CD25 antibody (PC61). Indeed, Treg depletion
prior to transfer of these donor cells abrogated the transfer of tolerance which protected the
recipient mice from developing a severe allergic skin reaction after whey-sensitization. It has
been shown that the anti-CD25 mAb selectively depletes the CD25high cells which are CD4+
[26]. Indeed, in this study we confirmed the ex vivo depletion of CD4+CD25+Foxp3+ cells to be
more than 60%. Also in other studies it was shown that complete depletion is not necessary
to be effective and can be used to investigate Treg function [27-30]. Our data implicate the
CD25+ Treg cells to be involved in the adoptive transfer of the tolerizing effect of dietary
prebiotics. The transfer of tolerance observed in the recipient mice was whey-specific since
transfer of cells of non-sensitized donor mice receiving the prebiotic diet did not protect
recipient mice. This suggests involvement of an antigen-specific Treg cell population and
there is growing evidence of a subset of Treg cells that have antigen-specific suppression
characteristics [31, 32].
To determine allergic sensitization, usually plasma levels of allergen-specific IgE are
measured. However, we and others found that this parameter is not conclusive for the
generation of allergic symptoms [33]. In contrast to the acute allergic skin reaction in wheysensitized recipient mice no differences in immunoglobulin levels were observed after
adoptive transfer with cells of control or prebiotic fed mice. In a mouse ovalbumin model,
transfer of Treg cells reduced the allergic response and reduced numbers of eosinophils in
the lungs. Furthermore, airway remodeling was prevented. However, also in this study IgE
levels remained unaffected high [34]. Given the extensive long half-life of the measured
antibodies, the actual experimental period might be too short to identify potential difference
on the long term in these levels caused by the intervention. However, the acute allergic
skin response reflects clinical symptoms which occur upon degranulation of allergic effector
cells. It can be concluded that the reduced skin response cannot be explained by differences
in antibody titers between recipients transferred with cells of prebiotic or control diet fed
mice.
In the present study it is shown that whey-sensitization is transferable using adoptive
transfer of single spleen cell suspensions while also the protective effect of a prebiotic
mixture on allergic symptoms could be transferred. The prebiotic diet provided only a minor
reduction in whey-specific Igs and the reduced acute allergic skin response of the recipient
mice transferred with donor cells of mice fed the prebiotic diet could not be explained by
a reduction in immunoglobulin levels. Ex vivo Treg cell depletion abrogated the protective
effects of the adoptive transfer with donor cells of whey-sensitized mice fed the prebiotic
diet. The results suggest the involvement of whey-specific Treg cells in the transfer of
tolerance.
135
7
References
1.
Friedman, A. and H.L. Weiner, Induction of anergy or active suppression following oral
tolerance is determined by antigen dosage. Proc Natl Acad Sci U S A, 1994. 91(14): p. 668892.
2.
Lundin, B.S., et al., Oral tolerization leads to active suppression and bystander tolerance in
adult rats while anergy dominates in young rats. Scand J Immunol, 1996. 43(1): p. 56-63.
3.
Burks, A.W., S. Laubach, and S.M. Jones, Oral tolerance, food allergy, and immunotherapy:
implications for future treatment. J Allergy Clin Immunol, 2008. 121(6): p. 1344-50.
4.
Hol, J., et al., The acquisition of tolerance toward cow’s milk through probiotic
supplementation: a randomized, controlled trial. J Allergy Clin Immunol, 2008. 121(6): p.
1448-54.
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2005. 24(6 Suppl): p. 582S-91S.
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p. 1277-86.
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20.
Kimber, I. and R.J. Dearman, Factors affecting the development of food allergy. Proc Nutr
Soc, 2002. 61(4): p. 435-9.
21.
life. J Nutr, 2008. 138(6): p. 1091-5.
22.
Schouten, B., et al., Cow Milk Allergy Symptoms Are Reduced in Mice Fed Dietary Synbiotics
during Oral Sensitization with Whey. J Nutr, 2009.
23.
Guttormsen, H.K., et al., Immunologic memory induced by a glycoconjugate vaccine in a
murine adoptive lymphocyte transfer model. Infect Immun, 1998. 66(5): p. 2026-32.
24.
25.
26.
Amu, S., et al., B-cell CD25 expression in murine primary and secondary lymphoid tissue.
Scand J Immunol, 2006. 64(5): p. 482-92.
27.
Shreffler, W.G., et al., Association of allergen-specific regulatory T cells with the onset of
clinical tolerance to milk protein. J Allergy Clin Immunol, 2009. 123(1): p. 43-52 e7.
28.
Kohm, A.P., et al., Cutting Edge: Anti-CD25 monoclonal antibody injection results in the
functional inactivation, not depletion, of CD4+CD25+ T regulatory cells. J Immunol, 2006.
176(6): p. 3301-5.
29.
Lewkowich, I.P., et al., CD4+CD25+ T cells protect against experimentally induced asthma
and alter pulmonary dendritic cell phenotype and function. J Exp Med, 2005. 202(11): p.
1549-61.
30.
Matsushita, N., et al., Comparative methodologies of regulatory T cell depletion in a murine
melanoma model. J Immunol Methods, 2008. 333(1-2): p. 167-79.
31.
Zhang, Z.X., et al., Identification of a previously unknown antigen-specific regulatory T cell
and its mechanism of suppression. Nat Med, 2000. 6(7): p. 782-9.
32.
Koenen, H.J. and I. Joosten, Antigen-specific regulatory T-cell subsets in transplantation
tolerance regulatory T-cell subset quality reduces the need for quantity. Hum Immunol,
2006. 67(9): p. 665-75.
33.
van der Gugten, A.C., et al., Usefulness of specific IgE levels in predicting cow’s milk allergy.
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Kearley, J., D.S. Robinson, and C.M. Lloyd, CD4+CD25+ regulatory T cells reverse established
allergic airway inflammation and prevent airway remodeling. J Allergy Clin Immunol, 2008.
122(3): p. 617-24 e6.
7
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7
139
8
Dietary non-digestible carbohydrates inhibit the
allergic effector response via induction of
casein-specific CD25+ regulatory T-cells in orally
sensitized mice
Bastiaan Schouten1
Gerard A. Hofman1
Sander de Kivit1
Louis Boon3
Johan Garssen1,2
1
2
3
Bioceros BV, Utrecht, The Netherlands
Dietary non-digestible carbohydrates inhibit the allergic effector response via
induction of casein-specific CD25+ regulatory T-cells in orally sensitized mice
Abstract
8
Background: Dietary prebiotics have been shown to prevent the development of orally
induced cow’s milk allergy in mice. In this study the contribution of CD25+ regulatory T-cells
in casein allergy prevention by prebiotics was investigated using in vivo CD25+ depletion and
adoptive transfer studies.
Methods: Mice were sensitized with casein and fed a prebiotic diet containing 2% shortchain galacto-, long-chain fructo-, and acidic-oligosaccharides or a control diet. Anti-CD25
(PC61) or isotype control antibody was injected intraperitoneally two times during the
dietary intervention. In adoptive transfer experiments, splenocytes of donor mice sensitized
with casein and fed the prebiotic or control diet were transferred to naïve recipients. CD25+
cells were ex vivo depleted using PC61. Recipient mice were casein- or sham-sensitized and
received the control diet. The acute allergic skin reaction upon intra dermal casein challenge
was determined and casein-specific serum immunoglobulins were measured. Furthermore,
TH1 and TH2 cell numbers were analyzed in the mesenteric lymph nodes (MLN) using flow
cytometry.
Results: The prebiotic diet strongly reduced development of the casein-specific acute
allergic skin reaction (p<0.001) and in vivo anti-CD25 treatment abrogated this. The diet
was found to enhance the percentage of TH1 and tended to reduce the percentage of TH2
cells in MLN of casein-sensitized mice. In casein-sensitized recipient mice, transferred with
splenocytes from casein-sensitized prebiotic fed donor mice, development of the acute
allergic skin reaction was prevented (p<0.001), while cells from sham-sensitized prebiotic
fed donor mice were not able to down regulate the allergic response in recipients. This
highlights the antigen specificity of the anti-allergic effects of the prebiotic diet. Ex vivo
depletion of CD25+ regulatory T-cells prevented this transfer of tolerance without affecting
casein specific immunoglobulin levels.
Conclusion: CD25+ regulatory T-cells play a pivotal role in the anti-allergic effects induced by
a unique mixture of non-digestible dietary carbohydrates.
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Chapter 8
Introduction
Immune modulation by dietary intervention is a growing field in immunological sciences. In
particular the effect of non-digestible carbohydrates is currently under investigation by many
research groups. Non-digestible carbohydrates such as short-chain galacto oligosaccharides
(scGOS) and long-chain fructo oligosaccharides (lcFOS) are added to clinical nutrition and
infant formulas since they may have health and immune promoting properties [1, 2].
Acidic oligosaccharides derived from pectin (pAOS) are recently developed non-digestible
carbohydrates that mimic properties of human acidic oligosaccharides such as present in
breast milk [3]. Recent research is focusing on the influence of prebiotic carbohydrates
on the development of allergic disease, dietary intervention in most studies started
immediately after birth. In a mouse model for orally induced cow’s milk allergy (CMA), a
specific mixture of scGOS and lcFOS (Immunofortis®) has been shown to reduce the allergic
effector response [4]. Furthermore, in a clinical study with infants at high risk for allergy
the same carbohydrate mixture reduced the incidence of atopic dermatitis significantly
[5]. Oligosaccharides may exert their immunomodulatory effects via direct interaction
with the mucosal immune system and/or via adaptation of the intestinal microbiota, since
oligosaccharides such as scGOS and lcFOS are selectively fermented by lactic acid producing
Bifidobacteria and Lactobacilli in the gut [6-8].
One of the first manifestations of atopic disease is the development of CMA in young
infants. There are two major allergenic fractions in cow’s milk: casein and whey. Casein, and
in particular αS1-casein, is the main allergenic protein [9, 10]. Normally the immune system
is generating local and systemic tolerance to harmless cow’s milk proteins, however in some
infants who develop cow’s milk allergy this oral tolerance is not induced, resulting in an
allergic response to these proteins. Oral tolerance induction is in majority generated within
the gut associated lymphoid tissue in particular the Peyer’s patches (PP) and the mesenteric
lymph nodes (MLN). From the MLN tolerance can be transferred to the systemic immune
system. Especially regulatory T-cells play a crucial role in this process [11, 12]. Cells with
regulatory properties include TGF-β-producing TH3 cells, IL-10-producing Tr1 cells and iNKT
cells [13-16]. However, CD4+CD25+Foxp3+ naturally occurring de novo generated regulatory
T-cells (Treg) were described to be important for maintenance of peripheral tolerance or
acquired tolerance for e.g food antigens, respectively [17-19]. Treg cells represent 5-10%
of the CD4+ T lymphocyte population and express the transcription factor forkhead box
p3 (Foxp3), which is confirmed as a key gene for generation and maintenance of Treg cell
functionality. The contribution of CD25+ Treg cells in the induction of oral tolerance has been
shown in children who have outgrown cow’s milk allergy, however also other mechanisms
may be involved as well [20-22].
143
8
The underlying mechanism of action of allergy prevention by non-digestible carbohydrates
is largely unknown. In the present study, mice were fed a 2% scGOS/lcFOS/pAOS prebiotic
mixture during oral sensitization with casein which was found to reduce the allergic effector
response. In this model the contribution of CD25+ Treg cells to this preventive effect of the
diet was studied using in vivo anti-CD25 depletion and adoptive transfer of splenocytes ex
vivo depleted for CD25+ Treg cells.
Materials and Methods
Chemicals
Casein proteins were obtained from DMV International (Veghel, The Netherlands). Cholera
toxin was purchased from Quadratech Diagnostics (Epsom, UK). PBS from Cambrex Bio Science
(Verviers, Belgium). Biotin-labeled rat anti-mouse IgE, IgG1 and IgG2a from BD Biosciences
(Alphen aan den Rijn, The Netherlands). PC61 (anti-CD25), a rat monoclonal antibody PC61
hybridoma cell line, was a kindly gift of Bioceros BV (Utrecht, The Netherlands). All other
chemicals were obtained from Sigma-Aldrich-Chemie (Zwijndrecht, The Netherlands).
Diets
8
Semi-purified cow’s milk protein free AIN-93G-based diets were composed and mixed with
the oligosaccharides by Research Diet Services (Wijk bij Duurstede, The Netherlands). The
scGOS/lcFOS/pAOS diet contained 2 w/w% (9:1:1) short-chain GOS (enzymatic elongation
of lactose with galactose by β-galactosidase), long-chain FOS (from chicory inulin) and
pAOS (produced from pectin). GOS (Vivinal GOS, FrieslandCampina Domo, Zwolle, The
Netherlands) contains oligosaccharides with a degree of polymerisation (dp) of 3–8. FOS
(Raftiline HP, Orafti, Wijchen, The Netherlands) contains oligosaccharides with an average
dp of >23. The GOS/FOS spray-dried powder consists of approximately 50% GOS and FOS,
19% maltodextrin (glucidex 2, Roquette, France), 16% lactose, 14% glucose and 1% galactose
[23]. The pAOS powder (Südzucker AG, Mannheim, Germany) have a dp of 1-20, consisting
of approximately 75% galacturonic acid oligomers, 10% monomers and 15% of moisture and
ash [24]. All oligosaccharides were exchanged for the same amount of total carbohydrates,
which resulted in a comparable overall carbohydrate composition in the diets.
144
Chapter 8
Three-week-old specific pathogen free female C3H/HeOuJ mice were purchased from Charles
River Laboratories (Maastricht, The Netherlands) and housed in the animal facility. Animal
care and use were performed in accordance with the guidelines of the Dutch Committee
of Animal Experiments. Mice were fed the control or scGOS/lcFOS/pAOS diet during the
whole study period, the diet was started two weeks before the first sensitization. Mice
were sensitized intra gastric (i.g.) with 0.5 mL homogenized casein (40 mg/mL PBS) with
cholera toxin (CT, 20 μg/mL PBS) as an adjuvant, using a blunt needle. Control mice received
CT alone. Mice were boosted weekly for a period of 5 weeks as described previously [25].
One week after the last sensitization feces were collected and mice were challenged intra
dermally (i.d) in the ear pinnae to determine the acute allergic skin response. After 24 hours
blood samples were collected in Minicollect® Z Serum Sep vials (Greiner bio-one) and mice
were sacrificed by cervical dislocation. Collected blood was centrifuged 15 min at 13,500
rpm and sera were stored at –70oC.
The acute allergen specific skin response was measured after i.d. injection with 20 μL
homogenized casein protein (0.5 mg/mL in PBS) in the ear pinnae. Ear thickness was
basal (t=0 hour) from the specific casein induced ear swelling after 1 hour ear thickness and
additionally (Δ ear swelling) subtracting the mean ear swelling from the group of shamsensitized mice fed the corresponding diet.
Anti-CD25 depletion studies
In the first set of experiments in vivo depletion of CD25+ regulatory T-cells was performed
(Table 1). One day before the first sensitization, sham- and casein-sensitized mice fed the
control or scGOS/lcFOS/pAOS diet were i.p. injected with 100 µL (2 mg/mL) rat anti-mouse
CD25 mAb or rat IgG1 isotype control antibody as a control. A second CD25+ depletion was
carried out one day before the third (day 14) sensitization.
145
8
Adoptive transfer experiments
To test whether splenocytes are able to transfer scGOS/lcFOS/pAOS induced prevention
of the allergic effector response, the contribution of CD25+ regulatory T-cells was studied
in adoptive transfer experiments. After the scGOS/lcFOS/pAOS supplemented (donor)
mice were sacrificed, the spleens were obtained and pooled per group (n=18). Single cell
suspensions of the four groups (sham, sham scGOS/lcFOS/pAOS, casein and casein scGOS/
lcFOS/pAOS) were pooled. Per group 2/3 part of the spleen cell suspension was treated with
600 μg anti-CD25 (PC61) mAb in 60 mL RPMI with 5% FCS for half an hour. After washing
with RPMI with 5% FCS two mL Biomag Goat anti-rat IgG magnetic beads (Bangs Laboratories
Inc., Fishers, USA) were added to the treated cells and placed on a rotator for at 45 min. The
bead-cell mixture was placed in a magnetic separator (Bangs Laboratories Inc., Fishers, USA)
for 5 min and supernatant was transferred and again placed on the magnetic separator for 5
min. Depletion of CD25 positive cells was analyzed using flow cytometric analysis. Cells were
counted using the Coulter Z1 particle counter (Beckman Coulter) and the splenocytes of the
donor mice ex vivo depleted for CD25+ cells or complete 1/3 part non-depleted suspensions
were i.v. injected (100 μL, 1x107 cells/mL) in sixteen different groups of recipient mice, n=6/
group (Table 1). The recipient mice were sham- or casein-sensitized as described above. The
Table 1. Overview of experimental set-up of adoptive transfer experiments.
Sensitization
of donors
sham
8
casein
146
Diet
Treatment of
splenocytes
control
Sensitization Diet
of recipients
-
sham
control
control
-
casein
control
control
anti-CD25
sham
control
control
anti-CD25
casein
control
scGOS/lcFOS/pAOS
-
sham
control
scGOS/lcFOS/pAOS
-
casein
control
scGOS/lcFOS/pAOS
anti-CD25
sham
control
scGOS/lcFOS/pAOS
anti-CD25
casein
control
control
-
sham
control
control
-
casein
control
control
anti-CD25
sham
control
control
anti-CD25
casein
control
scGOS/lcFOS/pAOS
-
sham
control
scGOS/lcFOS/pAOS
-
casein
control
scGOS/lcFOS/pAOS
anti-CD25
sham
control
scGOS/lcFOS/pAOS
anti-CD25
casein
control
Chapter 8
acute allergic skin reaction was measured and casein-specific serum immunoglobulins were
measured to determine allergic sensitization.
Flow cytometry
Single cell suspensions were prepared from mouse spleens and blocked for 30 min in PBS
containing 1% BSA and 5% FCS. In addition, for the isolation of MLN lymphocytes, MLN
were treated with RPMI containing collagenase D and DNAse (Roche Diagnostics, Almere,
The Netherlands) and incubated for 30 min at 37oC twice. Collagenase D and DNAse were
inactivated using RPMI supplemented with 5% FCS. Cells were counted using a Coulter Z1
particle counter (Beckman Coulter) and diluted to a concentration of 2x106 cells/mL. For
identification of different subsets of cells, cells were incubated for 30 min at 4oC with different
Abs (all from BD, Alphen aan den Rijn, The Netherlands, unless otherwise stated). AntiCD4-FITC (1:100), anti-CD4-PerCP-CY5.5 (1:50), anti-CD25-PE (1:100), anti-CD69-FITC (1:50),
anti-CD69-PE (1:50), anti-CXCR3-PE (1:50) and anti-T1/ST2-FITC (1:50, MD Biosciences, St.
Paul, Minnesota, USA) and isotype controls. Foxp3 staining was performed following the
manufacturer’s protocol (eBioscience, Breda, The Netherlands). Flow cytometry analysis
was performed using FACSCalibur (BD, Alphen aan den Rijn, The Netherlands) and CellQuest
Pro Analysis software (BD, Alphen aan de Rijn, The Netherlands) and isotype controls were
subtracted.
Concentrations of casein-specific IgE, IgG1 and IgG2a were determined in serum by means of
ELISA as described before [25]. In short, plates were coated with 100 µL casein (20 mg/L),
washed and several serum dilutions were applied for 1 h. Plates were washed and incubated
with biotin-labeled rat anti-mouse IgE, IgG1 and IgG2a (1 mg/L), washed and incubated
with streptavidin-horseradish peroxidase (HRP). For detection A490 nm absorption was
determined using spectrophotometer (Bio-Rad). Results were expressed as arbitrary units
(AU), calculations were based on titration curves of pooled sera from casein and alum-i.p.
immunized mice as a positive reference serum.
147
8
Statistics
All data were analyzed using the one way ANOVA and post hoc Bonferroni’s Comparison
Test or Mann-Whitney U test. For the immunoglobulin levels the ANOVA was performed on
log transformed data. Statistical analyses were conducted using GraphPad Prism software
(version 4.03). P-values of *<0.05, ** p<0.01 and *** p<0.001 were indicated when analyses
reached statistically significance. Data are represented as means ± SEM.
Results
Adoptive transfer experiments; donor mice
8
Two weeks prior to the first sensitization mice were fed control or scGOS/lcFOS/pAOS diet.
One day before the first sensitization mice were i.v. injected with anti-CD25 or isotype
control. To study CD25+ Treg cells involvement in the preventive effects of the scGOS/lcFOS/
pAOS diet adoptive transfer experiments were performed. Initially, four groups of donor
mice were casein- or sham-sensitized and fed the control diet or scGOS/lcFOS/pAOS (Table I)
and the acute allergic skin reaction was determined (Figure 1). The scGOS/lcFOS/pAOS diet
effectively reduced the acute allergic skin reaction of casein-sensitized donor mice when
compared to the control diet (109 ± 4.3 vs 61 ± 5.0 µm, p<0.001). To explore underlying
mechanistic pathways of scGOS/lcFOS/pAOS the TH1 and TH2 cells in the MLN were analyzed
using flow cytometry (Figure 2). The percentage of activated TH1 cells (CD4+CD69+CXCR3+)
was enhanced in the scGOS/lcFOS/pAOS fed casein-sensitized mice when compared to
control fed casein-sensitized mice (p<0.05, Figure 2a). The percentage of activated TH2 cells
tended to be reduced (Figure 2b).
To study the contribution of CD25+ regulatory T-cells to the preventive effects of the scGOS/
lcFOS/pAOS diet on the acute allergic skin reaction, sham- and casein-sensitized mice were
injected (i.p.) with anti-CD25 antibody or isotype control. The delta ear swelling in the
isotype control casein-sensitized mice was 71 ± 5.5 μm and the scGOS/lcFOS/pAOS diet
largely prevented this (28 ± 6.0 μm, p<0.001, Figure 3). The diet did not affect ear swelling of
sham-sensitized mice. The protective effect of the scGOS/lcFOS/pAOS diet was completely
abrogated by anti-CD25 mAb treatment (76 ± 5.4 vs 78 ± 4.9 μm).
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Chapter 8
Figure 1. Acute allergic skin reaction of casein-sensitized donor mice fed control or scGOS/
lcFOS/pAOS diet. The ear swelling response of sham-sensitized mice was subtracted in order
to calculate the delta ear swelling. Splenocytes of these mice were used for adoptive transfer
experiments. Means ± SEM, *** p<0.001 and n=17-18 per group.
c
control diet
sham
scGOS/lcFOS/pAOS diet
sham
casein
casein
7.0%
8.9%
7.2%
isotype
21.3%
0.4%
CD69
8
CXCR3
3.3%
3.2%
2.2%
0.3%
CD69
4.3%
T1/ST2
Figure 2. Flow cytometric analysis of activated TH1 and TH2 cells in MLN cells of sham- and
casein-sensitized mice fed control or scGOS/lcFOS/pAOS diet. a CD4+CD69+CXCR3+ (TH1) cells
and b CD4+CD69+T1/ST2+ (TH2) cells. c Representative dot plots of CXCR3+CD69+ and T1/
ST2+CD69+ in the CD4+ population. Means ± SEM, * p<0.05 and n=6-8 per group.
149
Figure 3. The scGOS/lcFOS/pAOS diet effectively reduced the acute allergic skin reaction which
was abrogated by anti-CD25 mAb treatment. Casein induced acute ear swelling in caseinsensitized mice fed the control of scGOS/lcFOS/pAOS diet was measured 1 hour after i.d.
challenge. Anti-CD25 or isotype control Abs were i.p. injected. This graph is a representative
of multiple identical experiments. Means ± SEM, *** p<0.001 and n=6 per group.
8
b
rat IgG1
2.9%
isotype
0.0%
CD25
5.4%
anti‐CD25
Foxp3
Figure 4. a Confirmation of in vivo depletion of CD4+CD25+Foxp3+ regulatory T-cells in the
spleen after the second anti-CD25 treatment. b Representative dot plots of CD25+Foxp3+
cells in the CD4+ population following isotype or anti-CD25 treatment. The percentage of
CD4+CD25+Foxp3- cells was unaffected. Means ± SEM, ** p<0.01 and n=5-6 per group.
150
Chapter 8
Confirmation depletion of CD25+ cells using flow cytometry
Two groups of casein-sensitized mice were sacrificed at day 15, two days after the second
anti-CD25 or isotype control treatment and one day after the third sensitization. The
splenocytes were analyzed to confirm CD25+ Treg cell depletion. CD4+CD25+Foxp3+ regulatory
T-cell percentages were declined by more than 65% (Figure 4, p<0.01). A difference was
observed in the percentage of the CD4+ cells between isotype rat IgG1 and anti-CD25 treated
groups (12.9 ± 1.0 vs 10.1 ± 0.3% of total lymphocytes, p<0.05, data not shown), anti-CD25
treatment did not reduce CD4+CD25+Foxp3- cells which includes the effector T-cell population
(5.1 ± 0.4 vs 5.5 ± 0.5% of CD4+ cells; data not shown).
Casein-specific immunoglobulin levels of in vivo Treg cells depleted mice
Serum samples were collected and analyzed for casein-specific immunoglobulin levels. As
described before [25] casein-specific IgE levels were not enhanced in comparison with shamsensitized mice. However, after Treg cell depletion casein-specific IgE levels were enhanced
(p<0.05, Figure 5a) in comparison with sham-sensitized mice. No differences were observed
between the control diet and scGOS/lcFOS/pAOS diet with respect to casein-specific IgE
levels in isotype or anti-CD25 treated mice.
Casein-specific IgG1 levels of mice fed the control diet were not enhanced due to two nonresponders. However, after Treg cell depletion casein-specific IgG1 levels were enhanced
compared to sham-sensitized mice (p<0.01). Casein-sensitized mice fed scGOS/lcFOS/
pAOS diet had low casein-specific IgG1 levels. However, this was not significant lowered as
compared to mice fed the control diet. After Treg cell depletion casein-specific IgG1 levels
were significantly enhanced as compared to both sham- as casein-sensitized mice fed the
scGOS/lcFOS/pAOS diet (p<0.05, Figure 5b).
No differences in casein-specific IgG2a levels of mice fed the control diet were found. Caseinspecific IgG2a levels of casein-sensitized mice fed the scGOS/lcFOS/pAOS diet were enhanced
as compared to casein-sensitized control fed mice upon anti-CD25 treatment (p<0.05, Figure
5c). However, also sham-sensitized mice fed the scGOS/lcFOS/pAOS tended to be enhanced
(p<0.05, Figure 5c). After Treg cell depletion, in both diet groups, casein-specific IgG2a levels
were enhanced, although only the scGOS/lcFOS/pAOS group reached significance compared
to sham sensitized controls (p<0.01).
151
8
8
Figure 5. Casein-specific immunoglobulin levels in sham- or casein-sensitized mice. Mice
were i.p. injected with anti-CD25 or isotype control antibodies and fed control or scGOS/
lcFOS/pAOS diet. a Casein-specific IgE, a casein-specific IgG1 and c casein-specific IgG2a.
Means ± SEM, * p<0.05, ** p<0.01 and n=6 per group.
152
Chapter 8
Figure 6. Adoptive transfer of splenocytes in absence or presence of ex vivo depleted CD25+
cells. Casein-sensitized recipient mice adoptively transferred with splenocytes from caseinsensitized scGOS/lcFOS/pAOS fed donor mice were protected against the development of
an acute allergic skin reaction whereas sham-sensitized donor cells did not transfer this
tolerance. Ex vivo depletion of CD25+ Treg cells from splenocytes of scGOS/lcFOS/pAOS fed
donor mice abolished this casein-specific protection. Donor cells of casein-sensitized mice fed
the control diet did not protect against the development of the acute allergic skin response
in recipient mice and Treg cell depletion did not alter this response. Delta ear swelling was
calculated, subtracting the mean ear swelling of corresponding sham-sensitized recipient
mice. Means ± SEM, ** p<0.01, *** p<0.001 and n=4-6 per group.
Acute
allergic skin response of recipient mice in adoptive transfer
experiments
Splenocytes of sham- or casein-sensitized mice fed control or scGOS/lcFOS/pAOS diet were
adoptively transferred to recipient mice (Table I). Recipient mice, fed the control diet, were
sensitized and the acute allergic skin response was measured after i.d. challenge with casein.
All recipient mice developed an acute allergic skin reaction, which was not prevented using
splenocytes of casein-sensitized mice fed the control diet (Figure 6). In contrast, adoptive
transfer of splenocytes of casein-sensitized mice fed the scGOS/lcFOS/pAOS diet almost
completely prevented development of the acute allergic skin reaction. This effect was casein
specific since splenocytes of sham-sensitized mice fed the control diet did not transfer this
tolerance. Ex vivo CD25+ Treg cell depletion completely abrogated the protective effect
(p<0.001).
153
8
Casein-specific
immunoglobulins of recipient mice in adoptive transfer
experiments
Casein-specific IgE levels of sham-sensitized recipient mice receiving splenocytes from
sham-sensitized donor mice was low (control diet 4.0 ± 0.22, scGOS/lcFOS/pAOS 4.8 ± 0.71
AU; data not shown) and did not enhance upon sensitization (Figure 7a). However, when the
casein-sensitized recipient mice were transferred with splenocytes from casein-sensitized
donors (control as well as scGOS/lcFOS/pAOS fed) the casein-specific IgE levels tended to
enhance. Ex vivo CD25+ Treg cell depletion did not affect this. A similar tendency was found
for casein-specific IgG1 and IgG2a levels (Figure 7b and 7c). Casein-sensitization of recipient
mice transferred with splenocytes from sham-sensitized donor mice resulted in enhanced
levels of casein-specific IgG1 when compared to sham-sensitization (control fed 105 ± 4.4 and
scGOS/lcFOS/pAOS fed 103 ± 20 AU; data not shown; Figure 7b). Transfer with splenocytes
of casein-sensitized donors tended to enhance this response. Ex vivo CD25+ Treg cell
depletion did not affect this and no differences were found between recipients transferred
with splenocytes of mice fed the control or scGOS/lcFOS/pAOS diet. Casein-specific IgG2a
levels were not enhanced upon sensitization (data not shown). However, casein-specific
IgG2a levels of casein-sensitized recipient mice receiving splenocytes from casein-sensitized
control diet fed donor mice were enhanced as compared to recipients receiving splenocytes
of sham-sensitized control diet fed donor mice (p<0.001; Figure 7c). The scGOS/lcFOS/pAOS
diet showed the same tendency and ex vivo CD25+ Treg cell depletion did not alter this
response.
8
In general, no differences in casein-specific immunoglobulin levels were found between
recipient mice receiving splenocytes of control or scGOS/lcFOS/pAOS fed mice and ex vivo
CD25+ Treg cell depletion did not affect immunoglobulin levels.
Discussion
In this study, pivotal involvement of CD25+ regulatory T-cells (Treg) is demonstrated in the
protective effects of a specific oligosaccharide mixture on the induction of cow’s milk allergy
in mice orally sensitized with casein. Dietary intervention with scGOS/lcFOS/pAOS was
started two weeks prior to the first sensitization and continued during the sensitization phase
and was found to reduce the acute allergic skin response. These findings are in accordance
with a study in infants at high risk for allergies; when these children were fed hypo-allergic
milk formulas supplemented with scGOS and lcFOS during the first two years of life, the
incidence of atopic dermatitis decreased and reduced allergic manifestations and infections
were observed compared to control formula fed infants [5, 26]. We hypothesized that the
154
Chapter 8
8
Figure 7. Casein-specific immunoglobulin levels in serum of recipient mice after adoptive
transfer with whole spleen suspension or ex vivo CD25+ Treg cells depleted splenocytes. No
differences in immunoglobulin levels were observed in mice transferred with splenocytes
of control and scGOS/lcFOS/pAOS fed mice and ex vivo Treg cell depletion did not affect
immunoglobulin levels. a Casein-specific IgE b casein-specific IgG1 c casein-specific IgG2a.
Means ± SEM, * p<0.05, ** p<0.01 and n=6 per group.
155
diet affects the mucosal immune system and actively interferes in oral tolerance induction.
Allergies develop as a consequence of a disparity in the TH1/TH2 balance towards TH2 [27].
Different Treg cell subsets actively regulate this balance locally in the gastro-intestinal
tract. Antigens, or dendritic cells loaded with antigen, travel from the Peyer’s patches to
the mesenteric lymph nodes (MLN) and are presented to T-cells. Antigen-responsive T-cells
(CD4+) travel subsequently through the bloodstream into the mucosa or to the peripheral
immune system (e.g. spleen) [11, 12]. Antigen specific T-cells from the MLN in this respect
can relocate systemic tolerance. In the present study the scGOS/lcFOS/pAOS mixture was
found to affect the TH1/TH2 balance in the MLN of casein-sensitized mice. The percentage
of activated TH1 cells was enhanced 24 h after oral challenge with casein while the number
of activated TH2 cells tended to reduce. This effect was only observed in the scGOS/lcFOS/
pAOS fed casein-sensitized mice and associated with a reduced systemic acute allergic skin
response. The percentage of TH1 in casein-sensitized mice fed control diet was unaltered
compared to sham-sensitized mice. This may indicate active regulation of the mucosal
immune response as consequence of dietary intervention with scGOS/lcFOS/pAOS occurring
in the presence of casein. These effects in the MLN may relate to the peripheral reduction of
acute allergic effects. Local induced tolerance can be transferred to the periphery resulting
in peripheral tolerance, in particular Treg cells have been indicated to contribute to this
phenomenon [12]. Sun et al. [28] showed that the oral administration of Ag induced oral
tolerance both locally (mucosal lymphoid organs) and systemically (spleen) was induced by
CD4+CD25+Foxp3+ cells.
8
To study the contribution of CD25+ Treg cells in the prevention of the acute allergic skin
reaction by the diet in vivo depletion studies were performed. Monoclonal anti-CD25
antibody (mAb) treatment resulted in a decrease of regulatory CD4+CD25+Foxp3+ cells in the
spleen, whereas CD4+CD25+ effector cells were unaffected. It has been shown that the PC61
anti-CD25 mAb selectively depletes the CD25high cells which are CD4+ [29]. Indeed, in the
current study the in vivo depletion of CD4+CD25+Foxp3+ cells was confirmed to be more than
65%. Also in other studies it was shown that complete in vivo depletion is not necessary in
order to investigate Treg cell function [30-33]. In the present study in vivo depletion of CD25+
Treg cells was found to abrogate the protective effects of the scGOS/lcFOS/pAOS diet, when
treated with anti-CD25 scGOS/lcFOS/pAOS fed mice did develop a severe acute allergic skin
response. Indicating a role for CD25+ Treg cells in the preventive effects caused by the diet.
However, the depletion of CD25+ Treg cells also resulted in enhanced casein-specific
immunoglobulin levels. This was in particular found in mice fed the scGOS/lcFOS/pAOS
diet, since the diet tended to suppress casein specific TH2 type IgE and IgG1 levels upon
sensitization. In vivo Treg cell depletion enhanced casein specific IgG1 and tended to enhance
IgE in these mice compared to treatment with isotype control antibodies, indicating an
exacerbated TH2 response upon in vivo anti-CD25 treatment. The imbalance in favor of TH2
156
Chapter 8
in allergy is regulated by Treg cells resulting in a suppression of the allergic TH2 response,
depletion of Treg cells can therefore result in exacerbation of allergic sensitization. Also
Sumi et al. [34] showed that in a murine model for allergic conjunctivitis treatment with
anti-CD25 mAb resulted in increased levels of specific IgE as well as IgG1. Therefore, in vivo
anti-CD25 mAb treatment may have enhanced the capacity of B-cells to produce antibodies
since regulation by Treg cells was disrupted. The increased antibody levels upon in vivo antiCD25 mAb treatment could have underlain abrogation of the protective effect of the scGOS/
lcFOS/pAOS diet.
To discriminate between these interfering effects adoptive transfer experiments with
splenocytes were conducted to confirm the contribution of Treg cells in the protective
effects of the diet. Splenocytes, untreated or ex vivo depleted for CD25+ cells, from shamand casein-sensitized donor mice fed control and scGOS/lcFOS/pAOS diet were transferred
to naïve recipient mice that were sham- or casein-sensitized and fed control diet. Caseinsensitized recipients receiving splenocytes from scGOS/lcFOS/pAOS fed casein-sensitized
donor mice were protected from the development of an acute allergic casein-specific skin
response, which was abrogated after ex vivo anti-CD25 mAb treatment of the splenocytes.
These findings were in line with our previous finding using in vivo CD25+ Treg cell depletion.
However ex vivo depletion did not alter the levels of casein-specific immunoglobulins when
compared to recipient mice transferred with whole spleen cell suspensions of caseinsensitized donor mice. Hence, the adoptive transfer studies confirmed Treg cells to be
involved in the protective effects provided by the scGOS/lcFOS/pAOS diet.
In casein-sensitized recipients transferred with splenocytes of casein-sensitized donor mice,
IgG2a levels were increased and IgE and IgG1 showed similar tendency, when compared to
recipients transferred with sham-sensitized donor splenocytes. Guttormsen et al. [35] already
found that whole spleen single cell suspensions were able to transfer immunologic memory
for a vaccine in adoptive transfer experiments. This is comparable to the observations in the
casein allergic mice.
The donor diets did not differentially alter the immunoglobulin levels of the recipient mice
and Treg cell depletion also was found to leave antibody levels unaffected high. Typically,
the acute allergic skin response was completely prevented in recipient mice transferred
with cells of casein-sensitized donor mice fed the scGOS/lcFOS/pAOS diet. This suggests a
Treg cell mediated suppression of the effector immune response independent of antibody
production. Gri et al.[36] showed that Treg cells are able to inhibit acute allergic responses
which may relate to the reduced allergic skin reaction observed in the present study in mice
fed the scGOS/lcFOS/pAOS diet. Mast cells are most probably involved in this acute reaction
and Treg cells are believed to alter mast cell degranulation by IL-10 production or cell-cell
contact [36, 37]. In addition, Treg cells have been shown to suppress Fc-receptor expression
157
8
on mast cells which would reduce the capacity of these cells to degranulate and induce an
acute skin response [38].
Both the in vivo and ex vivo CD25+ Treg cell depletion studies suggest a central participation
of CD25+ Treg cells in the preventive effects of scGOS/lcFOS/pAOS diet on casein induced
acute allergic responses. Additionally, these data suggest the existence of antigen-specific
CD25+ Treg cells, since only splenocytes from casein-sensitized and not splenocytes from
sham-sensitized donor mice fed the scGOS/lcFOS/pAOS diet could prevent the acute allergic
response in recipient mice. In a review by Koenen and Joosten [39], protocols to identify and
proliferate antigen-specific Treg cells are provided and the use of these cells in preventing
solid organ transplant rejection is hypothesized. Multiple studies have speculated on an
antigen-specific Treg cell population and their application in different diseases [39-41].
In the present study it is shown that dietary intervention with scGOS/lcFOS/pAOS reduces
the development of an acute allergic response upon antigen challenge, although specific
immunoglobulins levels remain high. Both in vivo as well as ex vivo depletion of CD25+ Treg
cell abrogated the diminished acute allergic response and imply crucial involvement of
antigen specific CD25+ Treg cells in the suppression of the allergic effector response.
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161
9
iNKT cells contribute to the allergic response in
cow’s milk protein sensitized mice
Bastiaan Schouten1
Stephanie M. Lim1
Gerard A. Hofman1
Johan Garssen1,2
Arjan P. Vos2
1
2
To be submitted for publication
Abstract
9
Background: Little is known about the contribution of the invariant natural killer T (iNKT)cells in the onset of food allergy. Using a mouse model for cow’s milk allergy the function of
iNKT cells was investigated. α-Galactosylceramide (αGalCer), a specific iNKT cell agonist, was
used to stimulate iNKT cells.
Methods: Female C3H/HeOuJ mice were sensitized five times orally with casein or whey
proteins. One hour before the sensitizations mice were treated intraperitoneally with
αGalCer (2 µg/mice) or control. One week after the last sensitization mice were challenged
intradermally in the ear and acute allergic skin reactions were measured. Sera were analyzed
for specific immunoglobulins. Furthermore, in liver, spleen and mesenteric lymph nodes
(MLN) percentages of iNKT cells were analyzed and liver lymphocyte restimulation assays
were performed.
Results: Mice treated with αGalCer showed enhanced acute allergic skin reactions
in comparison with sham treated casein- or whey-sensitized mice. This finding was
associated with increased levels of whey-specific immunoglobulins, while casein-specific
immunoglobulins were not affected. The percentage of iNKT cells in the liver of casein- and
whey-sensitized mice was reduced when compared to sham-sensitized mice. However, in
whey-sensitized mice IL-4, IFN-γ and IL-10 secretion upon ex vivo αGalCer restimulation was
unaffected. The iNKT cell frequency was enhanced in the spleen and MLN of whey- and not
in casein-sensitized mice. Strikingly, αGalCer treatment was found to deplete iNKT cells in
the liver of sensitized as well as sham-sensitized mice, and the hepatocytes did not respond
to ex vivo restimulation with αGalCer.
Conclusion: iNKT cell frequencies were reduced in the liver of casein- and whey-sensitized
mice and enhanced in the spleen and MLN of whey allergic mice. Due to repeated application
of αGalCer, hepatocytes were functionally depleted for iNKT cells. This resulted in an
increased allergic effector response which was most pronounced in whey-sensitized mice
and associated with enhanced whey-specific immunoglobulin levels. In conclusion, iNKT
cells may suppress cow’s milk allergic symptoms in mice and may differentially regulate oral
sensitization for casein and whey.
164
Chapter 9
Introduction
Cow’s milk allergy (CMA) affects 3-5% of the infants and is outgrown in most of the cases
within 3-5 years [1, 2]. IgE-mediated CMA predisposes infants to other food allergies and
even asthma later in life [3, 4]. In most individuals the condition comprises skin, respiratory
tract and gastrointestinal tract symptoms and in some cases, although rare, even systemic
anaphylaxis [5, 6]. Oral tolerance is believed to be induced in the mesenteric lymph nodes
(MLN) and Peyer’s patches (PP) by regulatory T-(Treg)cells [7, 8]. However, invariant Natural
Killer T- (iNKT) cells play a pivotal role as well in oral tolerance by inducing Treg cells and in
addition, by clonally deleting antigen-specific CD4+ T-cells [9]. Natural Killer T- (NKT) cells
are a subset of cells that express a T cell receptor (TCR), which makes them T-lymphocytes.
There are several subsets of NKT cells, however the most studied NKT cell is the invariant
NKT (iNKT), or type I NKT cell, which express an invariant Vα14Jα18 TCR chain in combination
with a Vβ2, Vβ7 and Vβ8 in mice [10]. iNKT-cells, T-cells and NK-cells share cell surface
markers like CD161 and NKR-P1 [11]. Whereas T-cells interact with peptides presented
on MHC-class I or II molecules by antigen presenting cells, iNKT cells do not interact with
MHC-class I or II molecules. However, iNKT cells recognize glycolipids presented by CD1d,
a non-classical antigen presenting molecule [11, 12]. Therefore, iNKT cells can generate an
adaptive immune response towards a class of antigens which is not covered by conventional
T-cells. These antigens include mycobacterial cell wall antigens, gangliosides, tumor-derived
phospholipids and nonlipidic molecules [13, 14]. The ability of iNKT cells to show flexibility in
their cytokine response have led to the proposition that iNKT cells have regulatory properties
and might be involved in homeostasis of the mucosal immune system [12, 15, 16]. In mice,
these cells are present at low frequencies in the thymus, bone marrow, spleen, MLN and PP
(<2.0%) [17] (Schouten et al., unpublished data). In contrast, hepatic lymphocytes consist of
approximately 20% iNKT cells. The best characterized and potent specific iNKT cell agonist
is a glycolipid isolated from the marine sponge Agelas mauritianus, α-galactosylceramide
(αGalCer) [18-20]. The complex of CD1d plus αGalCer binds to the iNKT cell TCR and activates
the cell. After iNKT stimulation interleukin (IL)-4 and interferon (IFN)-γ are rapidly produced
and secreted. More recently, it has been shown that iNKT cells produce also TH17 cytokines,
both IL-17 and IL-22 [21, 22].
The function of iNKT cells has been studied in several diseases including diabetes [23],
multiple sclerosis [23], colitis [24] and cancer [25]. However, the contribution of iNKT cells,
using αGalCer, in the onset of allergy is, to our knowledge, only studied in allergic asthma
models [26-28]. With regard to food allergy and in particular cow’s milk allergy so far, no
data are available on the role of iNKT cells in induction or maintenance of allergy to food
ingredients and/or tolerance. Therapeutic approaches include the elimination of cow’s
milk proteins from the diet and several forms of immunotherapy [29-31]. To assess the
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9
mechanisms underlying CMA, the present study focused on the contribution of iNKT cells in
casein and whey protein induced hypersensitivity. The effects of αGalCer application, as an
iNKT-specific stimulus, were studied during oral sensitization in a murine CMA model.
Methods
Chemicals
Casein and whey proteins were obtained from DMV International (Veghel, The Netherlands).
Cholera toxin was purchased from Quadratech Diagnostics (Epsom, UK). PBS from Cambrex
Bio Science (Verviers, Belgium). Biotin-labeled rat anti-mouse IgE, IgG1, IgG2a and IgA from BD
Biosciences (Alphen aan den Rijn, The Netherlands). αGalCer was a kindly gift from Kyowa
Hakko Kirin Co., Ltd (Japan). All other chemicals were obtained from Sigma-Aldrich-Chemie
(Zwijndrecht, The Netherlands).
Oral sensitization, treatment and challenge of mice
9
Three-week-old specific pathogen free female C3H/HeOuJ mice were purchased from
Charles River Laboratories (Maastricht, The Netherlands), bred and raised on a cow’s milk
protein free diet and housed in the animal facility. Animal care and use were performed
in accordance with the guidelines of the Dutch Committee of Animal Experiments. Mice
were sensitized intragastrically (i.g.) with 0.5 mL homogenized casein or whey (40 mg/mL
PBS) with cholera toxin (CT, 20 μg/mL PBS) as an adjuvant, using a blunt needle. Control
mice received CT alone. Mice were boosted weekly for a period of 5 weeks as described
previously [32]. One hour before every sensitization mice were intraperitoneally (i.p.)
treated with 100 μL αGalCer (20 µg/mL PBST) or control (PBS and 0.5% Tween). One day
after the last sensitization feces were collected and one week after the last sensitization
mice were challenged intradermally (i.d) in the ear pinnae to determine the acute allergic
skin reaction and challenged i.g. with 0.5 mL homogenized casein or whey (100 mg/mL PBS).
After 16 hours blood samples were collected in Minicollect® Z Serum Sep vials (Greiner bioone) and mice were sacrificed by cervical dislocation. Collected blood was centrifuged 15
min at 16000 x g and sera were stored at –70oC.
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The acute allergen specific skin reaction was measured after i.d. injection with 20 μL
homogenized casein or whey protein (0.5 mg/mL in PBS) in the ear pinnae. Ear thickness was
basal (t=0 hour) from the specific casein or whey induced ear swelling after 1 hour.
Concentrations of casein- and whey-specific IgE, IgG1 and IgG2a were determined in serum
whereas specific IgA was measured in feces by means of ELISA as described before [32]. In
short, plates were coated with 100 µL casein or whey (20 mg/L) and after washing several
serum or fecal water dilutions were added. Bound whey or casein specific antibodies were
detected using biotin-labeled rat anti-mouse IgE, IgG1, IgG2a or IgA (1 mg/L) and streptavidinhorseradish peroxidase (HRP). Results were expressed as arbitrary units (AU), calculations
were based on titration curves with pooled reference sera from casein or whey alum-i.p.
immunized mice.
Flow cytometry
Single cell suspensions were prepared from mouse livers, liver lymphocytes were isolated
using 40% Percoll gradient, spleens and MLN and blocked for 30 min in PBS containing 1%
BSA and 5% FCS. Cells were counted using a Coulter Z1 particle counter (Beckman Coulter)
and diluted to a concentration of 2x106 cells/mL. For identification of iNKT cells, cells were
incubated for 30 min at 4oC with anti-CD3-PerCP-CY5.5 (1:100), anti-CD4-FITC (1:100), antiCD19-APC and CD1d-tetramer loaded with αGalCer (1:100). Antibodies were purchased
from BD (Alphen aan den Rijn, The Netherlands), except for CD1d-tetramer preloaded with
αGalCer, which were purchased from ProImmune Ltd. (Oxford, UK). iNKT cells were gated
using CD3+CD19-CD4+CD1d/αGalCer tetramer+. Flow cytometric analysis was performed
using FACSCalibur (BD, Alphen aan den Rijn, The Netherlands) and CellQuest Pro Analysis
software (BD, Alphen aan de Rijn, The Netherlands).
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9
Restimulation assay
In hepatocyte culture supernatants of single cell suspensions (1x106/mL) restimulated using
αGalCer (100ng/ml) or control were harvested at 48 h and concentrations of granulocyte
macrophage colony-stimulating factor (GM-CSF), IFN-γ, tumor growth factor (TNF)-α, IL2, IL-4, IL-5, IL-10 and IL-12p70 were determined by multiplex cytokine bead assay (BioRad, Veenendaal, The Netherlands) using a Luminex 200 Analyzer (Nuclilab, Ede, The
Netherlands). Data were analyzed using xPonent software 3.0.380.0 (Luminex Corporation,
Austin, USA).
Statistics
All data were analyzed using the one way ANOVA and post hoc Bonferroni’s Comparison Test
or Mann-Whitney U test. Analyses of the immunoglobulin levels and cytokine concentrations
were performed on log transformed data. Statistical analyses were conducted using
GraphPad Prism software (version 4.03). P-values of * p<0.05, ** p<0.01 and *** p<0.001
were indicated when analyses reached statistically significance. Data are represented as
means ± SEM.
9
Figure 1. The acute allergic skin reaction of shamcasein- or whey-challenged mice, control
or αGalCer treated. Mice were sensitized i.g. with cholera toxin (CT) and whey or casein
for 5 times during weekly intervals, CT alone was used for sham-sensitization. Before each
sensitization mice were i.p injected using αGalCer or control. Means ± SEM, * p<0.05 and
*** p<0.001 and n=5-6 per group.
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Chapter 9
Results
One week after the last sensitization mice were challenged i.d. in the ear with casein or
whey. After one hour the acute allergic skin reaction was determined. αGalCer treatment
did not affect the ear swelling reaction of sham-sensitized mice (60.7 ± 3.3 vs 76.0 ± 10.1
µm for casein challenge and 53.7 ± 4.6 vs 54.5 ± 8.0 µm for whey challenge). However, both
casein- and whey-sensitized mice developed an increased acute allergic skin reaction after
αGalCer treatment (137.2 ± 4.5 vs 162.5 ± 7.5 µm for casein-sensitized mice, p<0.05 and
136.4 ± 12.7 vs 209.9 ± 10.2 µm for whey-sensitized mice; p<0.001; Figure 1).
Allergen-specific immunoglobulin levels
Sixteen hours after oral challenge, serum was obtained from all mice. Caseinspecific IgE, IgG1 and IgA levels were enhanced in casein- and whey-sensitized mice
when compared to sham-sensitization (Figure 2a, b, d, e, f and h). Specific IgG2a was
only enhanced in whey-sensitized mice and tended to enhance in casein-sensitized
mice (Figure 2c, g). αGalCer treatment did not affect immunoglobulin levels in
casein-sensitized mice but further enhanced whey-specific IgE, IgG1, IgG2a, but not IgA in
whey-sensitized mice (Figure 2e, f, g and h).
Flow cytometry
The effect of sensitization and αGalCer treatment on iNKT cell frequency was determined
using flow cytometry. Casein- (14.6 ± 2.5%) and whey-sensitization (14.0 ± 2.8%) was found
to reduce the percentage of hepatic iNKT cells in comparison with sham-sensitized mice (22.9
± 0.6%; p<0.05; Figure 3a). In addition, αGalCer treatment was found to deplete hepatic iNKT
cells in both sham- and casein- or whey-sensitized mice in comparison with control treated
mice (p<0.001; Figure 3a). Low percentages of iNKT cells were found in the spleen and the
MLN possibly as a consequence of migration. Whey-sensitization was found to enhance
iNKT cell percentages in the spleen of whey-sensitized mice (2.4 ± 0.2%) as compared to
sham- (1.4 ± 0.2) and casein-sensitized (1.2 ± 0.1%) mice and αGalCer treatment was found
to abrogate this (1.5 ± 0.1%; p<0.001; Figure 3b). In addition, whey-sensitization was found
to enhance iNKT cell percentages in the MLN of whey-sensitized mice (0.92 ± 0.04%) as
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9
9
Figure 2. Serum specific immunoglobulins and fecal specific IgA in control or αGalCer treated
casein- or whey-sensitized mice. a-d Casein-specific immunoglobulins and e-h whey-specific
immunoglobulins. Means ± SEM, * p<0.05, ** p<0.01 and *** p<0.001 and n=5-6 per
group.
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Chapter 9
sham‐sensitized
d
sham
casein‐sensitized
αGalCer
whey‐sensitized
sham
αGalCer
sham
αGalCer
1.2%
11.7%
0.8%
13.2%
0.9%
1.9%
0.9%
1.2%
1.4%
2.4%
1.4%
0.4%
0.3%
0.45%
0.6%
0.85%
0.75%
CD4
MLN
spleen
liver
21.7%
CD1d loaded with αGalCer
Figure 3. Flow cytometric analysis of iNKT cells in control or αGalCer treated sham- caseinor whey-sensitized mice. Cells were isolated and stained with CD3, CD4 and αGalCer-loaded
CD1d tetramers. Percentages are calculated within the CD3+ population and representative
dot plots are shown. a iNKT in the liver b in the spleen and c in the mesenteric lymph nodes.
d Representative dot plots of the CD4+CD1d tetramers loaded with αGalCer+ in the CD3+
population. Means ± SEM, * p<0.05 and *** p<0.001 and n=5-6 per group.
compared to sham- (0.4 ± 0.0%) and casein-sensitized mice (0.47 ± 0.02% (p<0.05, p<0.01;
Figure 3c). The frequency of MLN iNKT in whey-sensitized mice was higher than caseinsensitized mice (p<0.05). αGalCer treatment was not found to affect the percentage of NKT
cells in whey-sensitized mice but did increase MLN iNKT cells of casein-sensitized mice vs
control treated casein-sensitized mice (0.7 ± 0.09 vs 0.5 ± 0.02%; p<0.05; Figure 3c).
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9
Figure 4. Analysis of αGalCer induced cytokine
responses in control and αGalCer treated shamcasein- and whey-sensitized hepatocytes. a IL4, b IFN-γ and c IL-10 secretion was measured
48 hours after stimulation. Unstimulated cells
did not secrete any cytokines. Means ± SEM,
*** p<0.001 and n=5-6 per group.
Restimulation assay
9
Single cell suspensions that were used for flow cytometric analysis were restimulated using
αGalCer and analyzed. αGalCer was used to specificity target the iNKT cells and to investigate
their cytokine response. Restimulation of splenocytes using casein or whey did not result in
detectable levels of cytokines. Cytokine production of control-stimulated hepatocytes was
below detection limit (data not shown). Ex vivo restimulation of hepatocytes from sham, casein- or whey-sensitized mice with αGalCer resulted secretion of all eight measured
cytokines and cytokines show similar patterns. iNKT produced IL-4, IL-10 and IFN-γ are
known to influence TH1/TH2 balance and allergy development, therefore these are shown
(Figure 4). No differences were detected between the groups, however no correction for
the percentage of iNKT cells in the liver was performed. These were significantly lower in
casein- and whey-sensitized mice in comparison with sham-sensitized mice. In vivo αGalCer
treatment rendered the hepatocytes unresponsiveness to ex vivo αGalCer restimulation, no
cytokine production was detected.
Discussion
Recent evidence implies iNKT cells to be involved in oral tolerance induction [33]. iNKT cells
can be found in large numbers in the liver, but can also be found within the intestine since
2% of the mouse intestinal lamina propria lymphocyte population consists of iNKT [34]. The
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contribution of iNKT cells in the pathophysiology of cow’s milk allergy (CMA) is unknown,
therefore the effect of oral sensitization on iNKT cell populations in the liver, spleen and
MLN was investigated. Furthermore, αGalCer, a specific iNKT agonist, was used to intervene
with the development of allergy during oral sensitization with casein or whey in mice.
Both casein- and whey-sensitization resulted in decreased iNKT cell percentages in the liver
of cow’s milk allergic mice. Typically, in whey-sensitized mice iNKT cells percentages were
increased in the spleen and MLN, this was not observed in casein-sensitized mice. These data
show that the iNKT cell population is affected during sensitization and may imply that these
cells contribute to the development of allergic disease or are involved in the suppression of
(food) allergic disorders. iNKT cells have been shown to induce or break oral tolerance [27,
33] and may suppress or enhance allergic disease in mice [18, 35]. These dual mechanisms
may partly be explained by the existence of different subsets of iNKT. In humans it was
shown that part of the population of αGalCer reactive iNKT cells produced high amounts of
IL-4, IL-10 and IFN-γ and induced IL-12 producing DC in vitro, resulting in TH1 skewing of the
effector response while another subset produced relatively low amounts of IL-4 and IL-10
and induced DC cytolysis and a TH2 polarized immune response [36]. Typically, in particular
iNKT derived IL-4 and IL-10 were found to contribute to oral tolerance induction [33].
To further explore the involvement of iNKT in allergic sensitization for cow’s milk proteins,
agonist studies using αGalCer were performed. αGalCer is the best characterized and potent
specific iNKT cell agonist [18-20]. iNKT treatment was intended to restore the frequency
and/or activity of hepatic iNKT cells in casein- or whey-sensitized mice in order to inhibit
the acute allergic skin reaction. However, multiple i.p. injections of αGalCer resulted in an
almost complete depletion of iNKT cells in the liver, both in sensitized as well as in control
mice, and exacerbation of the allergic response in sensitized mice. A strong decrease in
hepatic iNKT cells could be explained by migration of these cells towards other lymphoid
organs or the periphery. However, FACS analysis did not show enhanced iNKT numbers in
spleen or MLN, suggesting that the iNKT cells were depleted instead of stimulated. Parekh
et al. [37, 38] demonstrated that a single high dose administration of αGalCer (5 µg/mouse)
causes anergy of iNKT cells for at least one month. Matsuda et al. [20] as well as Hachem et
al. [18] showed that a single lower dose of αGalCer (2 µg/mouse) resulted in an inhibition
of allergic airway inflammation in an ovalbumin model. More in line with our results is the
study by Meyer et al. [39] who showed an increased airway hyper reactivity after two doses
of αGalCer (2 times 2 µg/mouse), although the number of iNKT cells were not analyzed
in this study and the study by Kim et al. [28] who showed that intranasally application of
αGalCer (3 times 1 µg/mouse) worsened allergic asthma in mice.
Although oral sensitization resulted in reduced numbers of iNKT cells in the liver, these
cells functionally reacted upon restimulation with αGalCer, since several cytokines were
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9
produced, such as IL-4, IFN-γ and IL-10 which are known to contribute to either TH1 skewing
or tolerance induction [33, 36]. This increase in cytokine production upon ex vivo αGalCer
restimulation is in agreement with multiple other studies [26, 35, 40]. The produced cytokine
concentrations in restimulated hepatocyte iNKT cells can be corrected for the percentage
of iNKT cells in the liver. Lower frequencies of hepatic iNKT cells in control treated wheysensitized mice produced the same amounts of cytokines as sham-sensitized mice, whereas
equal numbers would have produced higher levels of cytokines. Therefore, whey-sensitized
mice produced relatively more IL-4, IL-10 and IFN-γ, whereas the cytokine production per
iNKT cell was probably not altered in casein-sensitized mice. The observation that the
iNKT cells were depleted by the in vivo αGalCer treatment was in correspondence with
the cytokine production of hepatocytes restimulated with αGalCer. An almost complete
reduction of all cytokines was found in comparison with control treated mice, indicating
iNKT unresponsiveness or absence in association with an increased allergic reaction.
αGalCer treatment in particular was found to enhance the acute allergic skin response in
whey-sensitized mice. In these mice reduced iNKT numbers in the liver were compensated
by enhanced cytokine production per cell. Furthermore, in the spleen and MLN of wheysensitized mice the iNKT cell numbers were enhanced which was abrogated in the spleen by
in vivo αGalCer treatment. This may imply a contribution of iNKT in the suppression of CMA
in particular in whey-sensitized mice.
9
Differences between casein- and whey-sensitized mice in cytokine production by liver iNKT
cells and spleen and MLN iNKT frequencies indicate a different role for iNKT in casein and
whey allergy in mice. The semi-invariant T-cell receptor of NKT cells in particular recognizes
CD1d presented glycolipid antigens presented by antigen presenting cells or intestinal
epithelial cells [34]. Casein and whey proteins differ with regard to physical, physiological
and dietary properties [41-44]. The three dimensional shape of the proteins differ. Individual
caseins (11.6 - 25.2 kDa) are crosslinked and form aggregates, these nanoclusters form large
structures called micelles, whereas whey (14.2 - 76.2 kDa) forms linear structures of mostly
monomers and dimers. Furthermore, the glycolipid composition of whey and casein may
differ. Antigen presentation therefore might be different for either casein or whey affecting
iNKT-cell properties.
The dramatic augmentation of the acute allergic skin response in αGalCer treated wheysensitized mice was found to be associated with significantly enhanced whey-specific IgE
and IgG1 when compared to control treated whey-sensitized mice. In casein-sensitized mice
the enhancement of the acute skin response was much less severe and casein-specific
immunoglobulins were not further enhanced upon αGalCer treatment. αGalCer treatment
also significantly increased TH1 or regulatory T-cell-related immunoglobulin IgG2a levels
in whey-sensitized mice and not in casein-sensitized mice. Specific IgA levels remained
increased in both casein-sensitized mice as well as whey-sensitized mice in comparison to
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Chapter 9
their sham-sensitized control mice, with no further enhancement after αGalCer treatment.
These immunoglobulin data suggest an immunoglobulin dependent mechanism for wheysensitized mice, whereas for casein-sensitized mice another mechanism may underlie the
increase in acute allergic skin response due to in vivo αGalCer treatment. Other studies
showed that the acute allergic skin response in casein-sensitized mice shows immunoglobulin
free light chain dependency (Schouten et al., submitted). Immunoglobulin free light chains
are believed to form an alternative pathway to mast cell degranulation [45-48].
It can be concluded that iNKT cells might play a regulatory role in the onset of CMA in mice.
Multiple i.p. injections of αGalCer with weekly intervals depleted hepatic iNKT cells and to
a lesser extent spleen and MLN iNKT cells. Depletion of iNKT cells exacerbated the allergic
response in mice orally sensitized with cow’s milk proteins, this was most pronounced for
whey.
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9
179
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General discussion
General discussion
Status quo
The incidence of allergic diseases is growing and therefore the need for accurate diagnostics
as well as therapeutics increases. Food allergies are diagnosed by e.g. titers of specific IgE
using the radioallergosorbent test and/or a positive skin prick test. In addition, the golden
standard, a double-blind placebo-controlled oral food challenge can be performed [1-3].
Until now there is no satisfying treatment available and the treatment so far comprises
strict allergen avoidance, several forms of immunotherapy and treatment of symptoms by
pharmaceuticals.
Allergen avoidance, for cow’s milk allergic patients, can be realized using partial or extensive
hydrolysates, or in the case of persistent and severe clinical reactions, an amino acid based
formula. Time consuming immunotherapies involve subcutaneous injections of allergen
extracts, incrementally increased swallowed allergen (oral immunotherapy) or incrementally
increased allergen doses held under the tongue and then swallowed (sublingual
immunotherapy) [4-6]. For non-invasive preventive purposes several immune modulatory
dietary components are added to infant milk formulas which have been shown to have high
potential. One clinical study has shown to reduce the incidence of atopic diseases by using
non-digestible oligosaccharides [7]. Clear underlying immunological mechanisms are still
unknown [8].
10
In healthy individuals, harmless food proteins induce oral tolerance. In contrast, in allergic
patients a serious effector response is being generated resulting in allergic inflammation
upon allergen challenge. The current insights in the mechanisms underlying allergic diseases
point to an imbalance of TH1 and TH2 effector responses, polarizing towards TH2 and too
little suppressive capacities of regulatory T-cells (Figure 1). However, it is largely unknown
why this imbalance exists and what is needed for the induction of oral tolerance. It has
been hypothesized that a lower exposure to microbes might cause hypersensitivity of the
immune system to normally harmless agents such as food ingredients. Another risk factor
for developing an allergic disease is the genetic predisposition [9]. A child with either a
mother or father (or both) with a history of allergic rhinitis, asthma or atopic eczema has a
higher risk of developing an allergy than a child without any parental history of allergy.
This thesis provides preclinical data concerning the effectiveness of several dietary immune
modulatory components (e.g. prebiotics, probiotics and synbiotics) and, in part, describes
their underlying mechanisms of action.
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Chapter 10
Figure 1. Schematic representation of the organization of a humoral immune response by cells
of the murine adaptive immune system resulting in mast cell sensitization and degranulation
responsible for the allergic symptoms. TH: T helper cell; iTreg: inducible regulatory T-cell;
nTreg natural regulatory T-cell; NKT; natural killer T-cell; Tr1: T regulatory type 1 cell.
183
10
General discussion
A mouse model for cow’s milk allergy
To provide in the requirements for preclinical efficacy testing of components that may
prevent or treat cow’s milk allergy and to study underlying mechanisms, a reliable model
is essential. Therefore we adapted an existing mouse model for orally induced cow’s milk
allergy (CMA) and extended the available parameters to measure the allergic outcome [1012]. In Europe, infant formulas are whey protein based, whereas in the USA these formulas
are based on caseins. Oral sensitization of mice using whey or casein in the absence of
cholera toxin results in oral tolerance for the given proteins and therefore a mucosal
adjuvant is needed to break oral tolerance. Cholera toxin is an enterotoxin produced by
Vibrio cholera with strong adjuvant function at the mucosal surface [13]. The TH2 polarizing
effect of cholera toxin is diverse. The most important are the enhanced antigen presentation
by various antigen presenting cells and increased permeability of the mucosal epithelium
resulting in the production of TH2 related immunoglobulins (IgE and IgG1) and TH2 produced
cytokines, i.e. IL-4, IL-5, IL-6 and IL-10 [14].
10
The whey and casein allergic models were optimized and validated and the acute allergic
skin response was used as a sensitive and stable major parameter throughout all the
experiments. The mice were sensitized (leading to casein- and whey-specific IgE and IgG1
serum levels) and developed a systemic allergic reaction. To determine the challenge
reaction, the acute allergic skin response (ear swelling upon i.d. allergen challenge) was
measured as an equivalent of the skin prick test in humans. The acute allergic skin response
was significantly enhanced in both caseinas well as whey-sensitized mice if challenged
with the specific allergen. However, an important difference between the casein and the
whey model was that only in whey-sensitized mice increased levels of specific IgE were
demonstrated, whereas both models exhibit increased levels of specific IgG1. Upon allergen
challenge, serum concentrations of mouse mast cell protease-1 were enhanced. In addition,
the numbers of mast cells were reduced in the colon of both caseinas well as wheysensitized mice as demonstrated by histological procedures. These data indicate that the
mast cell is a key player in this acute food allergy model. However also other cell types
(especially basophils, although also monocytes, eosinophils, Langerhans cells and dendritic
cells [15]) display FcεRI and may have a contribution to in the acute allergic effector response
as well. However, the precise role of these cells in this model for cow’s milk allergy remains
to be analyzed in more detail.
Gastro-intestinal discomfort is one of the most prominent symptoms of CMA, which can lead
to abdominal pain, diarrhea or sometimes constipation [16, 17]. In our models we used the
labor intensive isometric organ bath technique to investigate contraction of colon segments.
Casein-sensitized mice had significantly reduced motility when compared to sham- or whey184
Chapter 10
sensitized mice. Both caseinas well as whey-sensitized mice had slightly increased water
levels in their feces, reflecting diarrhea. Differences between casein- and whey-induced
allergy in mice was not only observed in the humoral immune response also functional
intestinal differences were found. Casein and whey proteins differ with regard to physical,
physiological and dietary properties [18-21]. The three dimensional shape of the proteins
differ significantly. Individual caseins (11.6 - 25.2 kDa) are cross-linked and form aggregates.
These nanoclusters form large structures called micelles. In contrast, whey (14.2 - 76.2 kDa)
form linear structures of mostly monomers and dimers. Antigen presentation by dendritic
cells and B-cells might be different for either protein, as whey proteins are smaller when
compared to casein micelles affecting the establishment of the adaptive immune response.
As a consequence, this may have led to different local intestinal allergic responsiveness
resulting in altered contractility in casein-sensitized mice in combination with the specific
physicochemical characteristics of the casein or whey proteins.
Focus on discrepancy
The surprising difference in antibody response between the whey and casein model led us to
investigate this phenomenon in more detail. Coincidently, part of our group was interested
in an alternative pathway of mast cell degranulation via immunoglobulin free light chains
(Ig-fLC) [22, 23]. Therefore Ig-fLC levels were determined and blocking experiments were
performed using F991, a specific 9 mer peptide Ig-fLC antagonist. In casein-sensitized mice
total levels of Ig-fLC were enhanced. Whereas only in the casein-sensitized mice the acute
allergic skin response was blocked using F991 (i.v.). Furthermore, to provide evidence that
this mechanism was humoral and not cellular, concentrated spleen supernatants were
transferred i.v. into naïve recipient mice. Ear swelling, as well as blocking of the ear swelling
by F991, was confirmed. The relevance for Ig-fLC in diseases is under investigation. For
example, in patients suffering from multiple sclerosis free kappa light chains correlate with
disability prognosis [24] and in rheumatoid arthritis, there is a significant correlation between
kappa and lambda fLC and the disease activity score [25]. Furthermore, in non-IgE mediated
rhinitis patients both lambda and kappa Ig-fLC levels are increased in nasal secretions (Powe
et al., JACI in press). Notably, children with severe AD have higher levels of lambda and
kappa light chain in serum [26]. Plasma samples from infants at high risk for allergy who
participated in a prebiotic intervention study were measured for both kappa and lambda
Ig-fLC levels. The infants supplemented with the specific short-chain galacto-oligosaccharide
(scGOS) and long-chain fructo-oligosaccharide (lcFOS) prebiotic mixture (Immunofortis®)
had a reduced incidence of atopic dermatitis and infections when compared to the control
group [7, 27, 28]. In line with our preclinical findings both kappa and lambda concentrations
were diminished in infants with AD in comparison with infants without AD (Schouten et
185
10
General discussion
al., submitted). The groups were heterogeneous, as the inclusion criteria were a parental
history of atopic eczema, allergic rhinitis or asthma. Therefore more clinical study data with
cow’s milk allergic infants or other forms of allergies are necessary to further confirm these
findings and are currently under analysis.
Biotics
10
After establishing the murine model of CMA, dietary intervention studies were conducted to
determine the preventive effects of pre-, pro-, and synbiotics on the allergic outcome. Two
weeks prior the first sensitization mice were fed the specific prebiotic diet (Immunofortis®),
the probiotic diet (Bifidobacterium breve M-16V) or the synbiotic diet (the combination of
both) (Figure 2). A preventive instead of a treatment set up was chosen to obtain maximum
efficacy. As hypothesized, all three diets were capable, in some extent, to prevent the
allergic outcome. However, the synbiotic diet showed the best results. The acute allergic
skin response and anaphylaxis were almost completely prevented and mouse mast cell
protease levels were lower as compared to control mice. Nevertheless, sensitization could
not be prevented as both specific IgE and specific IgG1 serum levels remained elevated,
although both specific-fecal IgA and specific-serum IgG2a levels, indicative of a regulatory
and/or TH1 type immune responses, were enhanced, which might indicate an improved TH1/
TH2 balance and/or Treg function. In addition, it was shown that supplementation of UVkilled bacteria of the same strain had similar effects (Schouten et al., unpublished data).
These findings are in accordance with the outcome of clinical studies using pre- and/or
probiotics for the prevention of atopic disease [29-32]. When pregnant mothers, with a
family history of allergic disease, and their babies were supplemented with Lactobacillus
GG the cumulative incidence of atopic eczema was still reduced after 7 years by 30%
when compared to placebo controls [29]. Although the incidence of eczema was reduced,
no effects were found on IgE levels. In a similar study Lactobacillus reuteri reduced the
incidence of IgE-associated eczema and the occurrence of a positive skin prick test for almost
50%, whereas no effect on IgE was observed [30]. On the other hand, several studies show
little effect of dietary intervention with pre-, pro- or synbiotics [32, 33]. Generally, various
factors influence the outcome of these studies, especially dietary composition, duration of
treatment, differences in probiotic (e.g. single strain or mixtures) that have been used and
doses play an important role. This is one of the first studies that show additional effects of
the combination of prebiotics (scGOS/lcFOS) and probiotics (B. breve M-16V). Ogawa et al.
[34] showed additional effects of Lactobacillus casei together with dextran in the clinical skin
severity scores in a mouse model. In contrast, Passeron et al. [35] did not find an additional
effect of synbiotics over prebiotics in children with AD. Taken together, the pre-, pro- and
in particular the synbiotic diets effectively reduced the allergic outcome of mice sensitized
186
Chapter 10
with whey proteins. However, underlying mechanisms (e.g. production of specific IgA and
IgG2a) are still largely unknown and the remaining studies in this thesis focused on the role
of the CD25+ regulatory T (Treg)-cell.
Three of a kind
Besides scGOS and lcFOS another potent prebiotic, a pectin-derived acidic oligosaccharide
(pAOS), was manufactured in order to represent the acidic oligosaccharide fraction
which is present in human breast milk. Conjugation of acidic oligosaccharides with
bovine β-lactoglobulin showed less allergenicity in both in vitro experiments and after
i.p. immunization in mice [36, 37]. Furthermore, in a vaccination model and in a model
for allergic asthma the combination of scGOS, lcFOS and pAOS suppressed allergy in mice
[38, 39]. In addition, the combination of the three non-digestible carbohydrates were well
tolerated and did not result in differences of growth, crying, vomiting and regurgitation
pattern when added to milk formula as compared to control formula nourished infants [40,
41].
In both casein- and whey-sensitized mice the scGOS/lcFOS/pAOS mixture effectively
prevented the acute allergic skin response. However, sensitization was not prevented and both
specific-IgE and IgG1 levels remained enhanced, although whey-specific immunoglobulins
were slightly lower when compared to control fed mice. The discrepancy between the
observed clinical symptoms (acute allergic skin response) and antigen-specific serum levels is
difficult to explain. In chapter 3, a correlation between IgE and/or IgG1 and the acute allergic
skin response was shown in whey- sensitized mice fed control diet. However, after dietary
intervention this correlation was lost and the symptoms were reduced whereas the specific
serum
Control or intervention diet
‐14
‐7
0
7
14
21
28 33 34 days
Sensitization by gavage with whey + CT or CT alone (control)
10
ear swelling
anaphylaxis
FACS
Figure 2. A schematic overview of the dietary intervention, sensitization and challenge
protocol and the main parameters that are analyzed.
187
General discussion
immunoglobulin levels remain elevated. In human studies, without any dietary intervention,
the correlation between high specific IgE levels and clinical reactivity is complex as well and
depends e.g. on the age of the patient [42-44]. This makes reliable diagnosis very difficult.
Furthermore, desensitization is associated with elevated levels of specific IgG4. It is known
that inhibition of mast cell activation occurs after coengagement of IgG and IgE receptors
and therefore, at least, partly prevents mast cell degranulation [45, 46].
Regulatory affairs
The preventive capacities of scGOS/lcFOS/pAOS on the clinical outcome of disease was
demonstrated at least in several mouse models for allergy. However, the mechanism of
action was still unidentified. In 2007 Van Wijk et al. [47] showed that CD4+CD25+ Treg cells
regulate the intensity of a hypersensitivity response in a murine model of peanut allergy.
To investigate this Van Wijk et al. used a rat anti-CD25 monoclonal antibody (mAb; PC61)
for in vivo depletion of the Treg, which resulted in an exacerbation of the allergic response
to peanut. Although, several other studies showed that the use of PC61 is effective enough
and can be used to investigate Treg cell function [48-51]. Recent studies showed that the
combination of both PC61 and another anti-CD25 mAb 7D4 was more effective in depletion
or inactivation of Treg cells [52]. Furthermore, debate is ongoing whether using either of the
mAbs depletes or inactivates only the Treg cells.
Also in CMA clinically reactive patients, functional CD4+CD25+ Treg cells were found to
regulate the allergen specific response since in vitro Treg depletion was shown to increase
allergen specific T-cell proliferation [53]. In addition, children who outgrew their allergy
had higher frequencies of Treg cells in circulation, whereas (again) depletion of Treg cells
resulted in a fivefold induced proliferation response ex vivo [54]. Therefore we hypothesized
that the preventive effect of the scGOS/lcFOS/pAOS diet on the acute allergic skin response
to cow’s milk proteins could be due to improved Treg cell function (Figure 1).
10
To test this hypothesis, mice were injected i.p. with αCD25 mAb 1 day before the first oral
sham- or casein-sensitization (which was 13 days after starting the scGOS/lcFOS/pAOS diet)
and the Treg cell depletion was repeated 1 day before the 3th oral sensitization. One week
after the 5th and last oral sensitization the acute allergic skin response was measured. In
mice fed the scGOS/lcFOS/pAOS diet the acute allergic skin response was reduced, whereas
in vivo Treg depletion abolished this protective effect, although immunoglobulin levels
were increased as well in the Treg depleted mice. In vivo anti-CD25 mAb treatment may
have enhanced the capacity of B-cells to produce antibodies since regulation by Treg cells
was disrupted. The increased antibody levels upon in vivo anti-CD25 mAb treatment could
188
Chapter 10
have resulted in an enhanced acute skin response or the depletion of Treg as such indeed
effectively abrogated the protective effect of the diet. To pin down the Treg cells as crucial
cell type by which the scGOS/lcFOS/pAOS diet mechanistically acts, adoptive transfer studies
were performed. After transfer of splenocytes of sensitized mice fed the scGOS/lcFOS/
pAOS diet into naïve recipient mice fed the control diet and thereafter sensitized to casein
or whey, the acute allergic skin response was still effectively suppressed upon casein or
whey challenge. In addition, the allergic memory of sensitized mice fed the control diet was
transferred as well. These experiments showed that a cellular mechanism was involved and
that using splenocytes for adoptive transfer is adequate. Splenocytes were already shown
to be effective in transferring immunological memory for a vaccine [55]. Furthermore,
our experiments revealed that this prevention was antigen specific, since splenocytes of
sham-sensitized mice fed the scGOS/lcFOS/pAOS diet did not prevent the acute allergic skin
response in the recipient mice. There is growing evidence, in particular in transplantation
biology, that although few in numbers, antigen-specific Treg cells can suppress both TH1 and
TH2 [56, 57].
The following step was to repeat the experiments; although before the transfer, splenocytes
were depleted for Treg cells, using PC61. Recipient mice receiving splenocytes from scGOS/
lcFOS/pAOS fed donor mice depleted for Treg cells were no longer able to protect against
the induction of the acute allergic skin response after intra dermal allergen challenge. Both
casein- and whey-sensitized mice showed comparable results with regard to adoptive transfer
experiments and involvement of Treg cells, although some slight differences were observed.
In whey-sensitized recipients, transferred with ex vivo Treg depleted splenocytes of wheysensitized mice, an increased TH1 and TH2 immunoglobulins were observed, whereas caseinsensitized mice did not show this increase. This effect is indifferent of the diet the donors
received, once more suggesting differences in the adaptive immune response developed as
a consequence of sensitization with either casein or whey. Another mechanism of, probably,
B-cell activation or switch takes place, explaining the differences observed in sensitization,
although the net overall effect, the acute allergic skin response, was similar. In this set of
experiments transfer of the peripheral immune cells (e.g. splenocytes) showed that the
effect of the scGOS/lcFOS/pAOS diet acts systemically. However, currently it is unknown
whether local immune cells (e.g. MLN or Peyer’s patches) have the same capabilities. In
these organs oral tolerance is induced and can be transferred to the systemic compartment
via the MLN involving Treg cells [58]. In a murine ovalbumin model MLN cells were able to
transfer allergy to naïve mice and diarrhea developed upon oral ovalbumin challenge [59].
In addition, transfer of MLN or Peyer’s patches cell suspensions of mice orally sensitized
to β-lactoglobulin into naïve mice resulted in an increased production of specific IgG1,
indicative for transfer of sensitization [60]. Furthermore, MLN cells of ovalbumin tolerized
mice could transfer oral tolerance in naïve mice [61]. These experiments suggest a crucial
contribution for local lymphoid organs in the onset and regulation of allergy and tolerance.
189
10
General discussion
Additional experiments are needed to pinpoint the specific T cell subset in the regulation of
oral tolerance in cow’s milk allergy.
Lethal regulation
Invariant Natural Killer T (iNKT) cells form another subset of cells with regulatory properties
and might be involved in homeostasis of the mucosal immune system. In mice these cells are
present at low frequencies in the spleen, MLN and Peyer’s patches (<1.0%) [62] (Schouten
et al., submitted). However, hepatic lymphocytes consist of approximately 20% iNKT cells
and therefore the liver was chosen to study iNKT cells. The best characterized and potent
specific iNKT cell agonist is a glycolipid isolated from the marine sponge Agelas mauritianus
and is called α-galactosylceramide (αGalCer) [63-65]. We found that after oral sensitization
with either whey or casein the percentage of hepatic iNKT cells, using flow cytometry, was
decreased. Therefore it was hypothesized that after stimulation of these cells the severity of
the acute allergic skin reaction might be reduced. However, stimulation of the iNKT cells with
αGalCer (2 µg/mouse) 1 h before oral sensitization resulted, unexpectedly, in an increased
acute allergic skin response and a much further decrease in hepatic iNKT counts, in control
mice as well. We speculated on a migration of these cells towards the other lymphoid organs,
although this was not confirmed by FACS analyses, determining iNKT cell numbers in Peyer’s
patches and MLN, and gave rise to in the interpretation that the iNKT cells were depleted
instead of stimulated. This interpretation was supported after analyzing cytokine production
of hepatocytes restimulated with αGalCer. Hepatocytes from mice treated with αGalCer
showed almost a complete reduction of cytokine profiles in comparison with control treated
mice. Parekh et al. [66] demonstrated that a single high dose administration of αGalCer (5
µg/mouse) causes anergy of iNKT cells for at least one month. In contrast, Matsuda et al.
[65] as well as Hachem et al. [63] showed that a single lower dose of αGalCer (2 µg/mouse)
resulted in an inhibition of allergic airway inflammation in an ovalbumin model. More in line
with our results is the study by Meyer et al. who showed that the airway hyper reactivity
increased after two doses of αGalCer (2 times 2 µg/mouse), although the number of iNKT
cells were not analyzed in this study.
10
iNKT cells might play a role in the onset of CMA in mice, although further studies are needed
to confirm these findings. A single low dose before the first sensitization might stimulate the
iNKT cells effectively and might partially prevent the development of an acute allergic skin
response. In addition, the contribution of iNKT cells in the immune modulatory effect of
dietary components like scGOS/lcFOS/pAOS requires further investigation.
It is difficult to combine our research on Treg cells and iNKT cells. However, Kim et al. [67]
190
Chapter 10
showed that iNKT cells play a pivotal role in oral tolerance by inducing Treg cells and in
addition by clonally deleting antigen-specific CD4+ T cells. Therefore it can be argued that, in
line with our results, both Treg cells and iNKT cells are involved in the regulation of CMA in
mice. However, studies which investigate both the role of Treg cells as well as iNKT cells in
CMA are needed to further assess this regulatory mechanism.
To conclude the discussion
The studies performed in this thesis have resulted in a better understanding of the
immunological mechanisms underlying the pathophysiology in mice orally sensitized with
caseins or whey proteins. It was found that sensitization with cow’s milk proteins can lead to
both IgE- and Ig-fLC-dependent allergic hypersensitivity responses. Immune modulation by
dietary intervention with probiotics, synbiotics, and in particular the prebiotic scGOS/lcFOS/
pAOS diet was investigated and small parts of their working mechanisms are unraveled. Treg
cells have been shown to contribute to the immune modulatory effects of prebiotics. These
results should be further confirmed in clinical studies which are currently being performed.
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10
195
Miscellaneous
Summary
Dankwoord
Curriculum Vitae
Abbreviations
Summary
Summary
The prevalence of allergic diseases is rising to almost 20%. This includes one percent of the
population affected with an adverse immune reaction to food components. In developed
countries 2-4% and in some countries even higher percentages of infants exhibit cow’s milk
allergy (CMA). Although most infants outgrow CMA before their fifth year, immunoglobulin
(Ig)E-mediated CMA predisposes the development of other (food) allergies and even
asthma later in life. Furthermore CMA is one of the leading causes of food allergy in adults.
Approximately 0.1-0.5% have clinical symptoms after ingestion of milk containing products.
Clinical symptoms may involve the skin, respiratory tract and gastrointestinal tract, and can
even lead to a systemic anaphylactic reaction. Cow’s milk contains two main protein classes,
which are the caseins (30 g/L) and whey proteins (5 g/L). The caseins consist mainly of αS1-,
αS2-, κ- and β-casein, whereas whey proteins comprise of β-lactoglobulin, α-lactalbumin,
bovine serum albumin, serum immunoglobulins and lactoferrin. Large population studies
with cow’s milk allergic infants have shown that the major allergens are β-lactoglobulin,
αS1-casein and α-lactalbumin. Animal models of CMA provide a tool to reveal mechanisms
involved in the pathophysiology of CMA and to explore new preventive and/or therapeutic
approaches. This thesis focuses on the use of dietary intervention with pre-, pro- or synbiotics
to prevent the development of cow’s milk allergy in mice. In most of the animal models
currently available to study food allergy, sensitization is not performed via the oral route but
via intra peritoneal injection of the allergen. In reality humans are mostly sensitized upon
ingestion of the inflicting food. In the current thesis a mouse model for orally induced CMA
was introduced using whey or casein proteins. Furthermore, new tools were developed to
address pathological changes in the casein and whey allergic mice. Measurement of the
acute allergic skin response, as an equivalent of the human skin prick test, was introduced
to reflect systemic allergy as one of the consequences of oral sensitization (chapter 3).
M
Striking differences were observed comparing the sensitizing capacities of whey and casein
(chapter 3). These proteins have different characteristics e.g. their three-dimensional
structures, whey proteins are linear and casein proteins are globular. Both whey and casein
allergic mice induced systemic acute allergic symptoms as reflected by an increase in ear
swelling upon intradermal challenge with the specific antigen. However in casein allergic
mice specific IgE titers were not detectable. In addition, only in casein sensitized mice
intestinal contractility changes occurred, reflecting local pathophysiological functional
changes. Since casein sensitized mice appeared to have allergic symptoms in the absence
of casein specific IgE it was speculated that another mechanisms might be involved in the
onset of the acute allergic response, e.g. immunoglobulin free light chain (Ig-fLC). Ig-fLC is
produced by B cells and it has been shown to induce an acute allergic response. Indeed,
using a specific Ig-fLC antagonist (F991) a complete reduction of the allergic skin response
198
Miscellaneous
was established in the casein sensitized mice only. This was confirmed in a passive transfer
study in which spleen supernatant was injected into naïve recipient mice. F991 protected
these mice from the development of an acute allergic skin response, when challenged in the
ear with casein (chapter 4).
To confirm the relevance for Ig-fLC in our preclinical model, sera from a cohort of term
born infants at risk for allergy were analyzed for Ig-fLC. A difference in Ig-fLC concentrations
was found between infants that developed atopic dermatitis (AD) after six months when
compared to infants that remained unaffected thus with no detectable AD (chapter 4). The
infants of this cohort received a control milk formula or Immunofortis® (for details see next
paragraph) supplemented milk formula. The addition of the Immunofortis® mixture was
found to lower the incidence of AD by 50%. In the sera of these Immunofortis® treated
infants Ig-fLC concentrations were found to be significantly reduced which may relate to the
lower incidence of AD (chapter 5).
The clinical study, as described partially in chapter 4 and 5, showed a clear preventive
effect of these specific prebiotics. In other human as well as animal studies pre-, pro- and
synbiotics have been shown to be effective in the reduction of allergic disorders as well. In
order to investigate the underlying mechanisms of action preventive intervention studies
were carried out using the established mouse model for CMA. In order to investigate
the preventive effects mice were fed a control or short-chain galacto-oligosaccharides
(scGOS) and long-chain fructo-oligosaccharides (lcFOS) containing diet during oral wheysensitization. This scGOS/lcFOS (ratio 9:1) mixture (Immunofortis®) contains non-digestible
carbohydrates that selectively stimulate the growth of bacteria that support the health of
the host. This prebiotic mixture appeared to inhibit the allergic symptoms highly significantly.
If probiotic bacteria were added allergic symptoms were downregulated as well. The best
preventive concept, however, was a mixture of the prebiotic oligosaccharides and a probiotic
Bifidobacterium breve M-16V strain. This latter mixture is called a synbiotic. In sum, dietary
intervention with the pre-, pro- and in particular the synbiotics was found to diminish the
acute allergic skin response and anaphylactic scores upon allergen challenge. However,
no reduction in specific IgE and IgG1 levels was observed and the number of regulatory
T-cells (CD4+CD25+FoxP3+) in the spleen, mesenteric lymph nodes and Peyer’s patches was
unaltered. IgG2a, indicative for a regulatory T-cell or TH1 response, was affected positively by
the different diets (chapter 6).
In order to examine the role of regulatory T-cells in the prebiotic effects the functional
contribution of regulatory T-cells using in vivo regulatory T-cell depletion and adoptive transfer
experiments were performed. For this purpose pectin-derived acidic-oligosaccharides
(pAOS), a third group of non-digestible carbohydrates simulating the acidic oligosaccharide
structures present in breast milk, were added to the scGOS/lcFOS mixture.
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Summary
Both whey- and casein-sensitization was performed using dietary intervention with a scGOS/
lcFOS/pAOS (ratio 9:1:1) mixture. The outcome was comparable to the effect of scGOS/
lcFOS. Dietary intervention was found to reduce the acute allergic skin response in whey- as
well as casein-sensitized mice, however there was still no significant effect on the serological
parameters and the number of regulatory T-cells. Nevertheless it was hypothesized that the
regulatory T-cells played an important functional role in the allergic outcome. Therefore
a different set of experiments was initiated using a monoclonal antibody to deplete the
regulatory T-cells in vivo (PC61, an anti-CD25 monoclonal antibody). From these studies
it was concluded that regulatory T-cells played a crucial role in the preventive effects of
scGOS/lcFOS/pAOS (chapter 7 and 8).
To add additional evidence regarding the involvement of the regulatory T-cells a set of
adoptive transfer experiments was initiated. Single cell suspensions of spleens from allergic
(either whey or casein) or non-allergic mice fed a control or prebiotic diet were transferred to
naïve recipient mice. In addition, splenocytes were transferred which were ex vivo depleted
for CD25+ regulatory T-cells using magnetic beads. Using these experiments it was shown
that the dietary induced inhibition of allergy could be transferred, while selective depletion
of CD4+CD25+Foxp3+ regulatory T-cells abrogated this protective effect. Furthermore these
results showed that the induction of tolerance by the prebiotic diet was antigen specific,
since single cell suspensions of control sensitized mice fed the prebiotic diet did not protect
recipient mice from developing allergic symptoms (chapter 7 and 8).
M
Another potentially important regulatory cell type in allergy is the invariant Natural Killer T
(iNKT)-cell. Initial experiments showed that the number of iNKT-cells in the liver of caseinas well as whey-sensitized mice was diminished. Therefore we hypothesized that after
stimulation with α-galactosylceramide (αGalCer), a specific iNKT-cell agonist, the number
of iNKT-cells would be restored or at least their activity would increase, protecting the mice
from developing CMA. However, the acute allergic skin response was worsened instead of
improved, which was most pronounced in whey sensitized mice. In addition, whey-specific
IgE, IgG1 and IgG2a were enhanced as well, while in casein-sensitized mice αGalCer treatment
did not alter casein-specific immunoglobulin levels. Flow cytometric analysis revealed an
almost complete decrease in number of iNKT-cells. In addition, hepatocyte suspensions
of αGalCer treated mice failed to produce cytokines upon ex vivo restimulation. Therefore
we hypothesized that over-stimulation of the iNKT-cell population resulted in a depletion
instead of activation of these cells. Hence data presented in this chapter suggest iNKT-cells
to have immunomodulatory capacities, in particular relevant in mice orally sensitized with
whey (chapter 9).
In this thesis a new murine model for whey as well as casein has been described and
differences in underlying mechanism of action between the two allergenic proteins were
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revealed. Different dietary interventions showed preventive effects in the onset of CMA.
The synbiotic diet was most effective. The induction of protein specific regulatory T-cells was
revealed to be part of the immunological mechanism involved in both whey- and caseinsensitized mice. Furthermore, the contribution of iNKT cells to the pathophysiology was
investigated and might be a new potential target for dietary intervention in particular in case
of whey-sensitization. Future studies using the newly developed murine models for whey
and casein allergy will focus on further unraveling the underlying mechanisms of CMA and
to study new dietary interventions aimed at the prevention and/or treatment of CMA.
M
201
De prevalentie van allergische aandoeningen is 20%, hiervan is ongeveer 1% gericht tegen
eiwitten uit voeding. Koemelkallergie (KMA) vormt een serieus probleem in de westerse
wereld. Twee tot drie procent van de baby’s krijgt last van KMA in de eerste levensjaren.
Gelukkig groeit 70 tot 80% van deze kinderen over de allergie heen voordat ze vijf jaar oud
zijn. Een bijkomend probleem is echter dat deze kinderen een verhoogd risico lopen op het
ontwikkelen van andere allergieën en astma in hun latere leven. De belangrijkste symptomen
van KMA zijn huidreacties (eczeem), luchtwegklachten, buikpijn en in sommige gevallen zelfs
een anafylactische shock. Allergie is een ziekte van het afweersysteem die zich kenmerkt
door de aanwezigheid van ongewenste specifieke immunoglobuline E (IgE) antilichamen in
het bloed. Kinderen, maar ook volwassenen, met koemelkallergie hebben veelal IgE dat zich
richt tegen koemelkallergenen. Om KMA te diagnosticeren wordt dit in het serum gemeten.
De twee belangrijke allergene eiwitgroepen in koemelk zijn caseïne- (30 g/L) en wei-eiwitten
(5 g/L). De caseïne-eiwitten worden onderverdeeld in αS1-, αS2-, κ- en β-caseïne, terwijl de
wei-eiwitten onderverdeeld worden in β-lactoglobuline, α-lactalbumine, serum albumine
en lactoferrine. Grote klinische studies hebben uitgewezen dat de belangrijkste allergenen
waarop de mens reageert β-lactoglobuline, αS1-caseïne en α-lactalbumine zijn.
Diermodellen voor KMA kunnen helpen bij het onderzoek naar de betrokken mechanismen
die leiden tot de ontwikkeling van KMA en bij het toetsen van nieuwe preventieve en
therapeutische behandelmogelijkheden. Een nadeel van de meeste bestaande diermodellen
is dat de dieren niet gesensibiliseerd (allergisch gemaakt) worden op de manier waarop
mensen allergisch raken. Vaak worden proefdieren namelijk op een kunstmatige wijze
gesensibiliseerd (bijvoorbeeld via een injectie in de buikholte) en niet via orale toediening
van het allergene eiwit. Om onderliggende mechanismen en nieuwe nutritionele concepten
voor preventie en/of behandeling van KMA te onderzoeken hebben we een diermodel
ontwikkeld waarin muizen via orale toediening van de koemelkeiwitten caseïne- of weiallergisch worden gemaakt. In deze muizen zijn de pathologische veranderingen bestudeerd
en middels dieetinterventies is onderzocht of de ontwikkeling van allergische symptomen
kon worden voorkomen. De allergische oortest (vergelijkbaar met de huidpriktest bij
mensen) is geïntroduceerd om sensibilisatie aan te tonen. Hierbij wordt caseïne of wei in de
huid van het oor gespoten. Als het dier allergisch is voor het eiwit zwelt het oor op en deze
verdikking van het oor wordt gemeten (hoofdstuk 3).
M
De muismodellen voor weien voor caseïne-geïnduceerde koemelkallergie ontwikkelden een
allergeen specifieke oorzwellingsrespons. Dit suggereert eenzelfde werkingsmechanisme
in beide modellen. Er werden echter opmerkelijke verschillen gevonden. Patiёnten met
koemelkallergie kunnen last hebben van diarree maar ook van constipatie. Als maat voor
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darmmotiliteit werd in orgaanbadstudies de contractiliteit van de darmspieren gemeten.
Met name in de dikke darm van muizen die allergisch waren gemaakt voor caseїne bleek
de mate van carbachol (een stof die het samentrekken van spieren opwekt) geïnduceerde
spiercontractie verhoogd ten opzichte van wei-allergische en controle dieren. Er is meer
onderzoek nodig om vast te kunnen stellen wat de reden van deze verschillen is.
Zoals eerder beschreven is allergie een ziekte van het afweersysteem die zich kenmerkt
door allergeen specifiek IgE in het bloed. In het wei-model bleek dit precies zo te zijn,
maar muizen die met caseïne gesensibiliseerd werden hadden een allergische respons
(oorzwelling) in afwezigheid van specifiek IgE. Onze hypothese was dat een ander
mechanisme eenzelfde reactie veroorzaakt. In een IgE gemedieerde allergische respons
bindt een eiwit, bijvoorbeeld wei, aan IgE, wat gebonden zit aan receptoren op mestcellen.
Door receptor crosslinking ontvangt deze allergische effectorcel een signaal waarna hij zijn
inhoud (o.a. histamine) uitscheidt in de omgeving. Hierdoor vindt er oedeemvorming en
huidirritatie plaats en worden andere cellen aangetrokken; de allergische reactie. Echter,
bij caseïne kan dit proces waarschijnlijk niet via IgE antilichamen lopen, omdat deze niet of
in erg lage hoeveelheden aanwezig zijn in deze muizen. Op onze afdeling wordt onderzoek
gedaan naar een alternatieve route voor het uitlokken van een allergische respons en het
betrokken molecuul wordt immunoglobuline vrije lichte keten genoemd (light chain). IgE
bestaat uit twee zware (grote) ketens en twee lichte (kleine) ketens die aan elkaar vastzitten.
De alternatieve route van allergeenbinding en mestcelldegranulatie zou mogelijk via deze
lichte ketens plaats kunnen vinden zonder dat ze aan de zware ketens vastzitten. In het
caseïne model is onderzocht of deze immunoglobuline vrije lichte ketens verhoogd aanwezig
waren in het bloed, dit bleek inderdaad het geval. Vervolgens injecteerden we F991 in de
muizen voordat de oorzwellingstest werd uitgevoerd. F991 is een zogenaamde antagonist
en vangt vrije lichte ketens weg. Deze zijn daardoor niet meer actief in de muizen. In F991
behandelde caseïne-allergische muizen vond hierdoor bijna geen oorzwelling meer plaats,
terwijl gebruik van F991 geen gevolg had voor de oorzwelling van wei-allergische muizen. Om
dit te bevestigen zijn miltcellen van een aantal caseïne-allergische muizen in kweek gebracht.
Deze cellen produceren antilichamen, waaronder vrije lichte ketens. Het supernatant van
deze miltcellen werd in een niet-allergische (naïeve) muis gespoten en daarna hebben we
bestudeerd of er een oorzwelling optrad na injectie van caseïne in het oor. De antilichamen
geproduceerd door miltcellen van caseine-allergische muizen bleken inderdaad naïeve
muizen allergisch te maken. De oorzwelingsrespons bleek immunoglobuline vrije lichte
keten afhankelijk, want F991 kon het optreden van de oorzwelling voorkomen (hoofdstuk
4).
Om te bestuderen of immunoglobuline vrije lichte ketens ook een rol kunnen spelen bij
allergische mensen zijn in een groep van zuigelingen die risico lopen om een allergie te
ontwikkelen, immunoglobuline vrije lichte ketens gemeten in het serum. In de groep
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van zuigelingen die atopisch eczeem ontwikkelden (vaak ontstaan door een allergische
reactie) lagen de waarden van de immunoglobuline vrije lichte ketens hoger dan in de
groep van zuigelingen die geen atopisch eczeem hadden. Verder bleken de concentraties
van immunoglobuline vrije lichte ketens lager in de groep zuigelingen die Immunofortis®
(betekenis wordt in detail in onderstaande alinea uitgelegd) in hun flesvoeding kregen. Juist
deze groep ontwikkelde minder vaak atopisch eczeem, in vergelijking met zuigelingen die
normale flesvoeding zonder toevoeging van Immunofortis® ontvingen (hoofdstuk 5).
Om de sensibilisatie te verminderen zijn muizenproeven uitgevoerd met verschillende
dieetinterventies. Als eerste zijn in het muis-allergiemodel voor wei-eiwitten de dieren
gevoerd met verschillende diëten. Een van de diëten bevatte niet-verteerbare koolhydraten
(een soort suiker), zogenaamde oligosacchariden. Deze oligosacchariden komen van nature
ook voor in moedermelk en in de beschreven studies is een mix gebruikt die erg lijkt op
bepaalde oligosacchariden in moedermelk. De gebruikte mix bevatte in totaal 2% korte keten
galacto-oligosacchariden (scGOS) en lange keten fructo-oligosacchariden (lcFOS; verhouding
9:1, bekend als Immunofortis®). Het is bekend dat deze mix bepaalde goede bacteriën in
de darm stimuleert en op deze manier de gezondheid van het individu ondersteunt. Dit
wordt een prebiotisch effect genoemd. De niet-verteerbare koolhydraten worden prebiotica
genoemd, de bacteriën zelf probiotica en de combinatie synbiotica. Als potentieel effectieve
probiotica is een bacteriestam geselecteerd, de Bifidobacterium breve M-16V. In onze
preventieve studie gebruikten we drie diëten: het prebiotische dieet, een probiotisch dieet
en de combinatie, een synbiotisch dieet. Alle drie de diëten bleken in staat om de allergische
reactie te onderdrukken, maar het synbiotische dieet deed dit het beste. De oorzwelling
was sterk verminderd en de anafylactische shock was terug op controleniveau. We vonden
geen verschillen in IgE (of IgG1) antilichaam-niveaus en het aantal regulatoire T-cellen.
Deze regulatoire T-cellen zijn te herkennen aan bepaalde receptoren op hun oppervlak
(celmembraan), in dit geval CD4, CD25 en FoxP3, en bewaken de immunologische balans
zodat deze niet doorslaat in de richting van allergieën of auto-immuunziekten. De niveaus
van IgG2a antilichamen waren verhoogd door de diëten. Dit duidt op een verbeterde balans
van de immuunrespons, mogelijk veroorzaakt door de activiteit van regulatoire T-cellen
(hoofdstuk 6).
M
Een derde groep van niet-verteerbare koolhydraten die erg lijkt op die uit moedermelk
werd toegevoegd aan de oligosaccharide mix; deze zure oligosaccharide is afgeleid van
pectine (pAOS). In een andere experimentenreeks hebben we deze in combinatie met de
vorige prebiotische mix getest (2% scGOS/lcFOS/pAOS in een verhouding van 9:1:1). Het
preventieve effect op de allergie van de muizen was wederom positief. De oorzwelling werd
bijna volledig voorkomen, maar er was nog steeds nauwelijks tot geen effect te zien op
de antilichaamniveaus en het aantal regulatoire T-cellen. Toch was onze hypothese dat
het dieet een effect had op de regulatoire T-cellen die de ontwikkeling van KMA mogelijk
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Miscellaneous
kunnen afremmen. Om dit te bevestigen zijn deze regulatoire T-cellen voor en tijdens de
sensibilisatie verwijderd uit de muizen met een monoklonaal antilichaam gericht tegen
de CD25-receptor die aanwezig is op deze cellen. Voor zowel wei- als caseïne-allergische
muizen deden we dezelfde ontdekking: als de muizen waarvan de regulatoire T-cellen
verwijderd waren gevoerd werden met het prebiotische dieet, kregen ze wel weer een sterke
oorzwelling. Hierdoor is de conclusie gerechtvaardigd dat regulatoire T-cellen betrokken zijn
bij de gunstige effecten van het prebiotische dieet (hoofdstuk 7 en 8).
Om meer zekerheid te verkrijgen omtrent deze bevinding, hebben we zogenaamde adoptieve
transfer-experimenten uitgevoerd. Cellen uit de milt van zowel wei- als caseïne-allergische
muizen die een sterk verminderde allergische reactie lieten zien doordat ze het prebiotische
dieet hadden ontvangen, zijn geïnjecteerd in naïeve muizen die nog niet allergisch waren
gemaakt. We ontdekten dat deze andere muizen niet allergisch gemaakt konden worden
en dat de cellen die verantwoordelijk zijn voor deze tolerantie, in de milt aanwezig zijn.
Vervolgens voerden we de experimenten een tweede keer uit, maar vóórdat we de miltcellen
injecteerden verwijderden we de regulatoire T-cellen uit de celsuspensie. Nu bleek dat de
ontvangende muizen niet meer beschermd waren tegen de sensibilisatie met wei of caseïne,
ze werden dus allergisch. Verder zijn er aanwijzingen dat bij deze doorbroken bescherming
wei- of caseïne-specifieke regulatoire T-cellen betrokken waren. Dit volgt uit het experiment
waarin we muizen wel voerden met het prebiotische dieet, deze muizen werden niet
gesensibiliseerd met caseine of wei. De miltcellen van deze niet-allergisch gemaakte muizen
werden geïnjecteerd in naïeve muizen. Deze muizen waren echter niet beschermd tegen
het ontwikkelen van allergie. Het oligosaccharide dieet ondersteunt dus de ontwikkeling
van wei- of caseïne-specifieke regulatoire T-cellen en juist deze antigeen-specifieke cellen
kunnen bescherming bieden tegen het ontwikkelen van allergie (hoofdstuk 7 en 8).
Naast de regulatoire T-cel waren we ook geïnteresseerd in de bijdrage van een ander
type regulatoire cel, namelijk de ‘Natural Killer T’ (NKT)-cel in de bescherming tegen
koemelkallergie. De eerste experimenten toonden aan dat zowel wei- als caseïneallergische muizen een verlaagd aantal van deze NKT cellen in de lever hadden. Uit dit
gegeven ontstond de hypothese dat wanneer we deze cellen zouden activeren of laten
vermenigvuldigen, de allergie zou verminderen. De activatie vond plaats door middel van de
stof α-galactosylceramide (kortweg αGalCer). αGalCer kan selectief de NKT-cel stimuleren. We
vonden echter geen verlaagde allergische respons, zoals verwacht, maar juist een verhoging
van de acute huidrespons (voornamelijk in de wei-allergische dieren). We onderzochten de
NKT-cellen in de lever en deze bleken zeer sterk in aantal verminderd door de toediening
van αGalCer. Ook migratie van deze cellen konden we vrijwel uitsluiten omdat we ook in
andere organen deze cellen hebben geteld (milt en mesenterische lymfeknopen). Uit
deze bevinding concludeerden we dat we, door de dieren een aantal maal met αGalCer te
stimuleren, de NKT-cellen niet geactiveerd, maar juist verwijderd hadden. De consequentie
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was dat de allergie toenam in plaats van te verminderen. De data die zijn beschreven in
dit hoofdstuk suggereren dat NKT-cellen immunomodulerende capaciteiten bevatten, met
name in muizen die wei-allergisch zijn (hoofdstuk 9).
In dit proefschrift zijn twee muismodellen voor koemelkallergie ontwikkeld. De muizen zijn
oraal gesensibiliseerd tegen wei- of caseïne-eiwitten en de verschillen in onderliggende
mechanismen tussen deze twee modellen zijn bestudeerd. Verschillende dieetinterventies
lieten een preventief beschermend effect zien op het ontstaan van KMA, vooral het
synbiotische dieet leek hierbij veelbelovend. De betrokkenheid van eiwit-specifieke
regulatoire T-cellen in het beschermende effect door de diëten is aangetoond in de weien caseïne-allergische muizen. Daarnaast is de bijdrage van NKT-cellen aan de allergische
respons tegen wei en caseïne onderzocht. NKT-cellen lijken een mogelijk target te zijn
voor dieetinterventie. De ontwikkelde muismodellen bieden de mogelijkheid om meer
mechanistisch onderzoek te verrichten naar het ontstaan van KMA. De hieruit opgedane
kennis kan in de toekomst mogelijk gebruikt worden om nieuwe therapeutische of
preventieve (voedings)interventies te ontwikkelen met als doel de klachten van KMA te
voorkomen of mogelijk zelfs te verhelpen.
M
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Dankwoord
Dankwoord
Zo, het zit erop! Na vier jaar van denken, uitvoeren, analyseren en schrijven, heel veel
schrijven, is het af. Wat kun je veel doen in vier jaar en toch vliegt de tijd voorbij. Nu alleen
het meest gelezen stuk van het proefschrift nog: het dankwoord. Het tot stand komen van dit
‘boekje’ is zeker geen soloactie en daarom neem ik de gelegenheid om iedereen te bedanken
voor zijn of haar bijdrage! Natuurlijk ga ik iemand vergeten, maar jij ook bedankt!
Zoals elke promotie begint, zo begon het ook bij mij: met een promotor. Johan, ik blijf me
verbazen over de hoeveelheid dingen die jij voor elkaar krijgt. En dan heb je zelfs nog tijd
om op het lab te helpen. Jij kunt als geen ander mensen motiveren voor onderzoek. Ik ben je
dankbaar dat je mij inwijdde in de wereld van de immunologie. Wat zal ik onze gesprekken
missen tijdens de ITH’s (meer in detail zal ik niet treden).
Linette, als co-promotor is het je taak om de dagelijkse begeleiding van je AIO’s te verzorgen.
Jij hebt dit heel letterlijk genomen en waar ik juist de grote lijnen zie en wel denk te weten
hoe het zit, ga jij een stap verder en wil je echt elk detail weten. Jij hebt het perfectionisme
tot een kunst verheven! Wat hebben wij een hoge mate van complementariteit. Zonder
jouw inzet en motivatie was de helft nog niet op papier gekomen.
Léon, als tweede co-promotor kwam je iets later bij de club, maar je was absoluut een aanwinst
voor ons. Je inzet en je kennis van allergiemodellen, samen met je relativeringsvermogen,
hebben zeer zeker bijgedragen aan dit proefschrift. Nu zijn we eindelijk formeel collega’s.
Bedankt voor je wijze input!
I own many thanks to the reading committee who had to judge about the thoroughness of
this manuscript. I was optimistic about your decision, though you were really enthusiastic!
Professor Joost Ruitenberg, Professor Georg Kraal, Professor Henk van Loveren, Professor
Günther Boehm en Dr. Maarten Hoekstra: thank you all for reading this manuscript!
Maandelijks hadden we overleg met het Utrecht Center for Food Allergy. Deze mensen kwam
ik elke keer ook weer tegen als ik ergens op congres was… Edward, Els, Carla, Maarten,
André, Suzanne, Henk, Raymond, Marissa, Marianne, Joost, Janine, Geert, Jolanda en alle
anderen, bedankt voor jullie waardevolle discussies!
Louis, jou wil ik ook bedanken voor je input, maar vooral voor je antilichamen. Zonder de
PC61 had ik de mooiste hoofdstukken niet gehad!
En dan natuurlijk het lab @ UIPS, daar is het echte werk uitgevoerd en daar heb ik het meest
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Dankwoord
gezeten. Allereerst Betty, wat had ik zonder jou gemoeten? Je hebt ervoor gezorgd dat we
een vliegende start konden maken en de data bleven maar binnenstromen. We hadden zelfs
zoveel dat er al snel besloten werd dat je voor jezelf zou gaan beginnen. Ik zie uit naar jouw
boekje! Het meest zal ik onze gezamenlijke congresbezoeken missen. Altijd gezellig, altijd
even de stad in om iets van de sfeer op te snuiven. Ik vind het super dat je mijn paranimf
wilt zijn!
Een kunstenaar in wat je doet ben jij, Gerard. Nooit te beroerd om iets uit te voeren en
bedankjes wilde je niet (‘doe maar in de emmer’). Hierbij dan toch: bedankt!
Dank ben ik ook verschuldigd aan mijn mede-AIO’s: Sven, Esmaeil, Anneke, Yousef en Hadi,
jullie gingen mij voor op het nobele pad der promovendi. Gelukkig gingen er niet alleen
mensen weg. Marco kwam de gelederen versterken (ook op foto-gebied!) en vervolgens
kwamen de Sassen (Saskia O. en Saskia B.) erbij (wanneer gaan we eigenlijk weer verder met
Janeway?). Bedankt voor de toffe tijd, ik mis jullie nu al.
Mijn UIPS-kamergenoten wil ik ook hartelijk danken! Anneke, je was mijn vraagbaak tijdens
de eerste twee jaar van mijn AIO-tijd en met jouw vertrek raakte ik mijn voorbeeld kwijt.
Gelukkig bleef ik niet alleen achter in mijn sobere multimediale verblijf. Daphne en Tom
(weer een AIO!), jullie kwamen mij vergezellen. Bedankt voor de goede gesprekken! Tom,
samen de wereld van Light chain verkennen was super! Veel dank aan the Light Chain team
onder aanvoering van Frank, dankzij jullie heb ik twee extra hoofdstukken in mijn boekje! En
Bart, het wordt toch lastig om mijn dankwoord langer te laten worden dan de rest, maar ik
doe mijn best! Het blotten voor mij is voorbij, denk ik…
En dan de rest van de FFF. Jullie hebben mij vier jaar lang als Fijne Farmacologie Familie
een echt tweede thuis gegeven! Frans, Gert, Aletta, Paul, Andries, Ferdi, Marjolein, Judith,
Maaike, Hans, Els, Dinij, Thea, Ingrid, Astrid, Naomi, Kim, Marije, Liz, Pim, Bin, Sel Gillina,
Abdi, Nahid, Simone, Tamara, Karin: bedankt!
M
Verder wil ik graag mijn studenten bedanken. Ook jullie hebben bijgedragen aan de kennis
die we nu hebben over koemelkallergie. Ik heb veel van jullie geleerd en ik hoop jullie ook van
mij. Ben, Richard, Darya, Karin, Louise, Simone, Nicole, Feike, Michael en Stephanie: super
bedankt. Mijn dank geldt ook de studenten van anderen die mij zijdelings hielpen: Sandra,
Diana, Tea, Roderick en anderen. Nu denken Lieke, Sander en Laura dat ik hen vergeten
ben. Nou, dat lukt me niet en ik zou het ook niet willen! Lieke; eerst onze bachelorstudent,
toen onze masterstudent en nu collega-AIO! Ik was blij dat je mijn in vitro taken kwam
overnemen, want ik voelde me steeds schizofrener worden. Hetzelfde geldt voor Sander, ik
was blij dat je onze koemelkgroep kwam versterken, ook eerst als student en nu als medeAIO. En Laura, na je stage bleef je maar komen en toen mocht je dan soort van blijven! Nog
208
Miscellaneous
een UIPS-AIO die werkt aan het interessante project voedselallergie.
Naast de FFF was er nog een afdeling waar ik gedurende het vorderen van mijn promotie
steeds meer aanwezig was: Danone. Mijn flex-plek werd langzaamaan omgezet in een vaste
plek, bij Joyce en Anita op de kamer. Ik wil jullie bedanken voor het kritisch meedenken,
zowel inhoudelijk als over de vorm. Gelukkig hebben jullie voorkomen dat er allemaal
pyjamaplaatjes in mijn boekje staan! En jij Joyce, super bedankt voor het proof readen
van mijn proefschrift, je zult je niet verveeld hebben tijdens de treinreis van 2x5 uur naar
Duitsland.
Veel dank ook aan de andere Danoontjes: Paul (straks nog een persoonlijk woord voor jou),
Sander, Niki, Prescilla, Nicole, Nienke, Jaqueline, Miranda, Jeroen, Bea, Alma, Karen, Hilde,
Suzan, en naast immunologen nog: Jan, Kaouther, Monique, Thijmen, Martijn, Eline V. Veel
PR voor mijn proefschrift is verzorgd door Mirjam; bedankt voor alle interviews. Misschien
gaan mensen mij op straat nog eens herkennen.
En dan de laatste groep van Danone, mijn nieuwe collega’s. Mieke en Patricia, jullie maakten
al eerder de switch van immunology naar klinische studies. Ik wil jullie bedanken voor de
emotionele hulp in de eindstrijd en het inwijden in de wonderlijk gestructureerde wereld
van de klinische studies. Verder de rest van het baby team: Jürgen, Paul, Tarik, Jenny, Dineke,
Martine, Xuchen, Stefanie, Sarah, Claudia, Carolien, Flemming, Liandré, Gerda. En natuurlijk
ook alle anderen van medical, ik voel mij al helemaal thuis!
Er is nog een groep mensen die mij al jaren door dik en dun steunt en mijn gedachten af en
toe wist los te rukken van mijn promotie: mijn vrienden. Wat moest ik zonder jullie, bedankt
voor de vriendschap!
Leendert, Nelleke, Jesse en Chaja, bedankt voor het gebruik van jullie tuin! We hopen er
binnenkort zelf een te hebben, maar we zullen blijven komen!
Gerrit en Dinella, de langste avond van het jaar houden jullie ons gezelschap, maar zelfs dan
komen we nauwelijks toe aan een potje Kolonisten. Nu jullie een eigen huisje hebben gaan
we zeker vaker bij jullie langskomen!
Christianne, jij bent een beetje de zus die ik nooit gehad heb en veel mensen zien ons ook
zo. Timon en Jolien, bedankt voor de gesprekken, ook die over slechte docenten, ik hoop
dat ik een betere was voor mijn studenten…Af en toe is het prettig om met een AIO uit een
ander vakgebied te praten over hoe het project loopt. Jan Harm en Myra, bedankt hiervoor
en jij JH, succes met de laatste loodjes!
Maarten en Suze, het was erg leuk en handig om een adresje in Utrecht te
hebben voor als ik moest ‘overblijven’. Dat is nu voorbij, maar we houden contact.
Jaap, Janneke, Lydia, Jort en Thirza; Paul Peter, Jolanda en Nathan: ik hoop dat onze kids nog
veel speelplezier samen zullen hebben.
Dan heb ik ook nog mijn volleybal vrienden: Christian, Tessa, Dirk, Annemien, Paul (alweer),
Bart en Erik, bij jullie kon ik wekelijks mijn frustratie eruit slaan.
209
M
Dankwoord
Het hele volleybalteam bestaat uit mensen uit de NGK en een aantal vervult een dubbelrol;
de kring gaf op de woensdagavonden een welkome ontspanning en in de muziek vond ik
ook afleiding om zaken te kunnen relativeren. Jan, Rense, Annemien, Christian, Christine,
Annelie, (kleine) Rense, Sandor, Lisette, Jaap, Petra, Jordy, Kimberly, Paul, Geja, Karlan,
Mincke, Johan, Ineke, Evert en Gerlies: bedankt voor de goede momenten, het is duidelijk
waar het in het leven om gaat.
Collega, volley-teamgenoot, kringleden, vrienden… jullie passen overal bij: Paul en Geja en
natuurlijk jullie kids, Noah en Florian. Jullie weten wat er voor nodig is om te promoveren
en Paul is in meerdere opzichten een voorganger en voorbeeld voor mij geweest. Tof dat we
zoveel met elkaar delen, laten we dat blijven doen! En Paul, bedankt dat je mij blijft steunen
tot en met de promotie zelf, super dat jij ook mijn paranimf wilt zijn.
Ben ik in het schrijven soms te kort en bondig, dit dankwoord is allerminst kort te noemen.
Toch sluit ik pas af nadat ik nog een aantal voor mij zeer belangrijke mensen bedankt heb.
Allereerst mijn schoonfamilie: Pa, Ma, Wiggert, Beate, Make en Opa Velema. Ik heb het
getroffen met jullie!
Opa en Oma Van Ham, al de keren dat ik vroeger bij jullie speelde hebben jullie mij laten zien
hoe je op een degelijke manier dingen maakt. Hiermee hebben jullie de basis gelegd voor
het doen van goed onderzoek, bedankt!
Oma Schouten, wat zou Opa dit graag meegemaakt hebben! Hij wist als geen ander
te analyseren en door te dringen tot de kern van de zaak. Precies een jaar geleden is hij
overleden. We denken nog vaak aan hem, en natuurlijk ook aan u!
Pa en Ma, bedankt voor de mogelijkheden die jullie mij gegeven hebben. Jullie stonden en
staan altijd voor ons klaar met tijd en ook met ruimte in meerdere opzichten!
Maarten en Susanne, broer en (schoon)zus, ik heb er echt een zus bij gekregen! Bedankt
voor jullie vriendschap!
En dan nu de belangrijkste mensen in mijn leven. Adriëlle, levensgezellin. Wat hebben wij
al veel beleefd. Telkens weer denken we dat de drukte wel voorbij is en dan verzinnen we
weer iets. Ik ben dankbaar voor je liefde en je hulp tijdens deze fase, zonder jou had ik het
niet gered! Met name voor het corrigeren van de Nederlandse teksten ben ik je veel dank
verschuldigd. Ik hou van jou!!!
Natuurlijk noem ik hier ook Joël en Micha en ook Kimberly. Jullie zijn samen het beste (en
vermoeiendste) dat mij is overkomen. Joël, je zei wel eens: ‘Papa, je oeft vandaa niete
werke…’. Dan was het niet makkelijk om de deur uit te stappen! Bedankt voor jullie liefde!
M
Als laatste geef ik alle eer aan mijn Hemelse Vader. Hij schiep alles zo fantastisch, dat we
zelfs na jaren van intensieve studie nog nauwelijks weten hoe het immuunsysteem in elkaar
zit.
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Miscellaneous
Zie ik de hemel, het werk van Uw vingers, de maan en de sterren door U daar bevestigd,
wat is de sterveling dat U aan hem denkt, het mensenkind dat U naar hem omziet?
Psalm 8:4 en 5
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211
Curriculum Vitae
Curriculum Vitae
Bastiaan Schouten was born in Utrecht on 27 February 1981 and graduated from the Menso
Alting College in Hoogeveen in 2000 and decided to study Biology at the Wageningen
University and Research Centre. During his first student internship he studied the estrogenic
potency of different food-packaging-associated plasticizers and antioxidants under
supervision of Dr. M.G.R. ter Veld, Prof. Dr. A.J. Murk and Prof. Dr. Ir. I.M.C.M. Rietjens at the
sub-department of Toxicology of the Wageningen University and Research Centre. During
his traineeship at Numico Research B.V. in Wageningen, he studied central fatigue during
chemotherapy in rats, under supervision of Dr. E.M. van der Beek and Dr. L.M. Broersen. His
second student internship concerned the biological, ethical, philosophical and theological
aspects of biometric identification under supervision of Prof. Dr. Ir. E. Schuurman of
Reformational Philosophy at the Applied Philosophy group of the Wageningen University
and Research Centre. During his study the author was chairman of the student corps Ichthus
Wageningen in 2004-2005.
After graduating, the author started as a PhD student at the division of Pharmacology and
Pathophysiology of the Utrecht Institute of Pharmaceutical Sciences (UIPS) of the Utrecht
University in September 2005. The project was entitled: “Role for pre- and probiotics in
immune-modulation, especially during allergic reactions to food components” under
supervision of Prof. Dr. J. Garssen, Dr. L.E.M. Willemsen (UIPS) and Dr. L.M.J. Knippels
(Danone Research) in collaboration with Danone Research – Centre for Specialised Nutrition
in Wageningen. During his PhD, the author was member of the PhD-council of the Utrecht
Institute of Pharmaceutical Sciences graduate school, member of the PhD-council of the
Eijkman graduate school, member of the Graduate School of Life Sciences and co-founder
and member of the PhD-council of the faculty of Science.
Currently, the author is employed as a Clinical Study Manager in the Clinical Studies Platform
of Danone Research – Centre for Specialised Nutrition, headed by Dr. Ir. J. van der Mooren.
He is married to Adriëlle Schouten-Loonstra and together they have two sons, Joël (2006)
and Micha (2008).
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212
Full papers
Cow’s milk allergy symptoms are reduced in mice fed dietary synbiotics during oral
sensitization with whey
Schouten B., Van Esch B.C.A.M., Hofman G.A., Van Doorn S.A.C.M., Nauta A.J., Garssen J.,
Willemsen L.E.M., Knippels L.M.J.
2009 The Journal of Nutrition139(7):1398-1403
Acute allergic skin response as a new tool to evaluate the allergenicity of whey hydrolisates
in a mouse model of orally induced cow’s milk allergy
Van Esch B.C.A.M., Schouten B., Hofman G.A., Van Baalen T., Nijkamp F.P., Knippels L.M.J.,
Willemsen L.E.M., Garssen J.
2009 Pediatric Allergy and Immunology, in press
Exposure of intestinal epithelial cells to UV killed Lactobacillus GG but not Bifidobacteria
breve enhances the effector immune response in vitro
Van Hoffen E., Korthagen N.M., De Kivit S., Schouten B., Bardoel B., Duivelshof A., Knol J.,
Garssen J., Willemsen L.E.M.
2009 International Archives of Allergy and Immunology, in press
Acute allergic skin reactions and intestinal contractility changes in mice orally sensitized
against casein or whey
Schouten B., Van Esch E.C.A.M., Hofman G.A., Van den Elsen L.W.J., Willemsen L.E.M.,
Garssen J.
2008 International Archives of Allergy and Immunology 147(2): p. 125-134
Schouten B., Van Esch B.C.A.M., Groot Kormelink T., Moro G.E., Boehm G., Arslanoglu A.,
Willemsen L.E.M., Knippels L.M.J., Garssen J.
Submitted
Contribution of IgE and Immunoglobulin free light chain in the allergic reaction to cow’s
milk proteins
Schouten B., Van Esch B.C.A.M., Blokhuis B., Groot Kormelink T., Hofman G.A., Moro G.E.,
Boehm G., Arslanoglu A., Willemsen L.E.M., Knippels L.M.J., Garssen J.
Submitted
213
M
Dietary non-digestible carbohydrates inhibit the allergic effector response via induction of
casein-specific CD25+ regulatory T-cells in orally sensitized mice
Schouten B., Van Esch B.C.A.M., Hofman G.A., De Kivit S., Boon L., Knippels L.M.J., Garssen
J., Willemsen L.E.M.
Submitted
Oligosaccharide induced whey-specific CD25+ regulatory T-cells are crucial in the
suppression of cow’s milk allergy in mice
Schouten B., Van Esch B.C.A.M., Hofman G.A., Boon L., Knippels L.M.J., Willemsen L.E.M.,
Garssen J.
Submitted
Schouten B., Van Esch B.C.A.M., Knippels L.M.J., Willemsen L.E.M., Garssen J.
Review in preparation
Schouten B., Van Esch B.C.A.M., Lim S., Hofman G.A., Knippels L.M.J., Willemsen L.E.M.,
Garssen J., Vos A.P.
In preparation
CD25+ regulatory T-cells play a prominent role in immune modulation by specific
oligosaccharides
Van ‘t Land B., Schijf M., Van Esch B.C.A.M., Van Bergenhenegouwen J., Schouten B.,
Bastiaans J., Boon L., Garssen J.
In preparation
Estrogenic potency of food-packaging-associated plasticizers and antioxidants as
detected in ERalpha and ERbeta reporter gene cell lines
Ter Veld M.G.R., Schouten B., Louisse J., Van Es D.S., Van der Saag P.T., Rietjens I.M.C.M.,
Murk A.J.
2006. J Agric Food Chem 54(12): p. 4407-16
Abstract publications
M
Immunoglobulin free light chains play a pivotal role in casein allergy in mice and might be
a biomarker for atopic dermatitis in infants
Schouten B., Van Esch B., Blokhuis B., Groot Kormelink T., Hofman G., Moro G., Boehm G.,
Arslanoglu S., Knippels L., Willemsen L., Redegeld F., Garssen J.
214
Miscellaneous
2009 Allergy, suppl 90, vol 64: p54
A probiotic mixture reduces the acute allergic skin response via regulatory T cells in mice
orally sensitised to whey
Schouten B., Van Esch B., Hofman G., Boon L., Knippels L., Willemsen L., Garssen J.
Induction of acute systemic anaphylaxis in a murine model of cow's milk allergy
Nauta A.J., Schouten B., Van Doorn S.A.C.M., Van Esch B.C.A.M., Willemsen L.E.M., Garssen
J., Knippels L.M.J.
Prebiotic oligosaccharides reduce casein sensitization in mice: a possible role for regulatory
T cells
Schouten B., Van Esch B.C.A.M., Hofman G.A., Boon L., Knippels L.M.J., Willemsen L.E.M.,
Garssen J.
Anti-CD25 treatment in a mouse model for cow's milk allergy modifies the acute skin
response from IgE- into Ig free light chain-dependent
Van Esch B.C.A.M., Schouten B., Blokhuis B.R.J., Hofman G.A., Boon L., Knippels L.M.J.,
Willemsen L.E.M., Garssen J., Redegeld F.A.
2008 Allergy, suppl 88, vol 63: p. 181
A specific mixture of short chain galacto-oligosaccharides and long chain fructooligosaccharides and a probiotic strain suppresses the allergic reaction in cow's milk
allergic mice
Schouten B., Van Esch B.C.A.M., Nauta A.J., Van Doorn S.A.C.M., Hofman G.A., Knol J.,
Garssen J., Willemsen L.E.M., Knippels L.M.J.
T-helper 1 skewing of the effector immune response through intestinal epithelial cells
exposed to Toll like receptor agonists
De Kivit S., Korthagen N., Schouten B., Van Hoffen E., Garssen J., Willemsen L.E.M.
Acute skin response in mice as a measure for cow's milk hydrolysate induced oral
tolerance to whey proteins
Van Esch B.C.A.M., Schouten B., Hofman G.A., Van Baalen T., Nauta A.J., Knippels L.M.J.,
Willemsen L.E.M., Garssen J.
215
M
A prebiotic oligosaccharide mixture inhibits pathophysiological changes in mice orally
Schouten B., Van Esch B.C.A.M., Hofman G.A., Willemsen L.E.M., Garssen J.
2008 Nederlands Tijdschrift voor Allergie, vol 8(1)
Intestinal epithelial cells exposed to probiotics or TLR/NOD2 agonists enhance Th1 type
cytokine secretion
Willemsen L.E.M., Korthagen N., De Kivit S., Schouten B., Duivelshof A., Knol J., Garssen J.
2008 Nederlands Tijdschrift voor Allergie, vol 8(1)
A new animal model for cow’s milk allergy: useful tool for epitope testing
Van Esch B.C.A.M., Schouten B., Hofman G.A., Willemsen L.E.M., Knol E., Van Hoffen E., Van
Baalen T., Knippels L.M.J., Garssen J.
2007 Allergy, suppl 83, vol 32, p57, 139
Differential pathophysiological changes in mice orally sensitized to casein or whey
proteins
Schouten B., Van Esch B.C.A.M., Hofman G.A., Van den Elsen L.W.J., Willemsen L.E.M.,
Garssen J.
2007 Gastroenterology, suppl 1, vol 132, nr 4, p460, M2156
Dietary intervention reduces inflammation and depression relevant to Alzheimer’s
Disease
De Wilde M.C., Schouten B., Balvers M., Kamphuis P.J., Van der Beek E.M., Broersen L.M.
2006 Alzheimer’s & Dementia vol 2(3): S237 p2-016
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216
Abbreviations
Abbreviations
AD:
CM:
CMA:
CT:
DBPCFC:
dp:
FA:
fLC:
GM-CSF:
I.d.:
IFN-γ:
Ig:
I.g.:
IL:
iNKT:
lcFOS:
MLN:
mMCP-1:
pAOS:
PP:
PUFA:
scGOS:
sIgA:
SPT :
TCR:
TH:
TNF-α:
Treg:
atopic dermatitis
cow’s milk
cow’s milk allergy
cholera toxin
double-blind placebo controlled food challenge
degree of polymerization
food allergy
free light chains
granulocyte macrophage colony-stimulating factor
intra dermal
interferon-γ
immunoglobulin
intra gastric
interleukin
invariant natural killer T (cells)
long-chain fructo oligosaccharides
mesenteric lymph node
mouse mast cell protease-1
acidic oligosaccharides derived from pectin
Peyer’s patch
polyunsaturated fatty acids
short-chain galacto oligosaccharides
secretory immunoglobulin A
skin prick test
T cell receptor
T helper cell
Tumor necrosis factor-α
regulatory T (cells)
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Cow`s milk allergy

Transcription

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