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The Development of ImmuneResponses and Allergy in Children
from Farming and Non-farmingEnvironments
Laura Orivuori
Immune Response UnitDepartment of Vaccination and Immune Protection
National Institute for Health and WelfareHelsinki, Finland
and
Children’s HospitalUniversity of Helsinki and Helsinki University Central Hospital
Helsinki, Finland
A C A D E M I C D I S S E R T A T I O N
To be presented, with the permission of the Faculty of Medicine of theUniversity of Helsinki, for public examination in Lecture Hall 2,
Biomedicum Helsinki, Haartmaninkatu 8, on 18th September 2015, at 12noon.
Helsinki 2015
SupervisorProfessor Outi Vaarala, MD, PhDClinicum, Children’s HospitalUniversity of HelsinkiHelsinki, Finland
ReviewersDocent Petri Kulmala, MD, PhDDepartment of PediatricsUniversity of OuluOulu, Finland
Marita Paassilta, MD, PhDDepartment of PediatricsUniversity of TampereTampere, Finland
OpponentProfessor Maria Jenmalm, PhDDepartment of Clinical and Experimental MedicineUniversity of LinköpingLinköping, Sweden
ISBN 978-951-51-1400-6 (printed)ISBN 978-951-51-1401-3 (PDF: http://ethesis.helsinki.fi)
Juvenes Print- Finnish University Print LtdHelsinki, Finland 2015
To Anton and Hannele
1
ABSTRACT
Allergies have become a major public health problem in recent decades.
Approximately 60 million people in Europe, and one billion worldwide,
are affected by allergies. In Europe, approximately 30% of the population
suffers from allergic rhinoconjunctivitis, approximately 20% from
asthma, and 15% from allergic skin conditions. The farming environment
has been shown to protect against allergies. In the present thesis, we
studied the development of Immunoglobulin E (IgE) sensitization,
allergic diseases, gut permeability and inflammation in children from
farming and non-farming environments who participated in the
PASTURE project (Protection against allergy: study in rural
environments).
Our results showed that early-age inflammation, measured with serum
high-sensitivity C- reactive protein (hs-CRP) at the age of 1 year, was
associated with decreased risk of IgE-sensitization at the age of 4.5 years.
Our findings suggest that poor inflammatory response at infancy may
predispose to IgE-sensitization.
The allergy-protective effect of breastfeeding is debated. Our findings
illustrated that increased breast milk soluble immunoglobulin A (sIgA)
levels were associated with decreased risk of atopic dermatitis (AD) up to
the age of 2 years. When the ingested dose of sIgA during the first year of
life was considered, it was associated with decreased risk of AD up to
ages of 2 and 4 years. Elevated sIgA levels in breast milk were associated
2
with environmental factors indicating increased environmental microbial
load.
Serum immunoglobulin A (IgA) and immunoglobulin G (IgG) against
cow’s milk β- lactoglobulin (BLG) and wheat gliadin are induced by the
antigen exposure, but the antibody levels are also affected by the gut
permeability and inflammation. Elevated serum IgA or IgG antibody
levels against BLG at the age of 1 year increased the risk of sensitization
to at least one of the measured allergens or food allergens at the age of 6
years. Elevated levels of IgG against gliadin at the age of 1 year increased
the risk of sensitization to any, at least one inhalant, or at least one food
allergen at the age of 6 years. Our findings suggest that an enhanced
antibody response to food antigens reflects alterations in mucosal
tolerance, such as altered increased gut permeability and/or microbiota,
which is later seen as sensitization to allergens.
Fecal calprotectin is a marker of intestinal inflammation. We studied the
association of early-infancy fecal calprotectin levels to the later
development of allergic diseases in children from farming and non-
farming environments, and further evaluated the effect of gut microbes on
the levels of fecal calprotectin. Infants from a farming environment had
higher levels of fecal calprotectin at the age of 2 months when compared
to the infants from a non-farming environment. However, farming did not
explain the very high levels of calprotectin, i.e. the levels above the 90th
percentile. The infants with remarkably high fecal calprotectin levels at
the age of 2 months, i.e. levels above the 90th percentile, had an increased
risk of developing AD and asthma/asthmatic bronchitis by the age of 6
3
years. Infants who had high fecal calprotectin concentrations had less
Escherichia in their feces. Our findings suggest that strong intestinal
inflammation at early infancy represents a risk of allergic diseases, and
the colonization with Escherichia coli is an important regulator of the
inflammation implicating long-term health effects mediated by early
intestinal colonization.
In conclusion, the findings in this thesis show that the environmental
microbial load plays an important role in the development of the immune
system in infants and ultimately may affect the risk of developing allergic
diseases. In addition to the direct effects on the infant, the effects of the
environmental microbial load are also mediated indirectly through
alterations in maternal milk composition. Interestingly, we found that an
early intestinal inflammation is associated with the later risk of allergic
diseases and gut colonization indicating a crucial role of gut colonization
in the development of allergy.
Keywords: Allergy, antibody, asthma, atopic dermatitis, farming environment, fecalcalprotectin, IgE-sensitization, inflammation.
4
TIIVISTELMÄ
Viimeisten vuosikymmenten aikana allergioista on muodostunut
huomattava terveysongelma. Arviolta 60 miljoonaa ihmistä Euroopassa ja
miljardi ihmistä koko maailmassa kärsii allergioista. Jopa 30%
eurooppalaisista kärsii allergisesta nuhasta ja siihen liittyvästä silmän
sidekalvotulehduksesta, 20% astmasta ja 15% allergisista iho-oireista.
Maatilaympäristöllä on havaittu olevan suojaava vaikutus allergioiden
kehittymisessä. Tässä väitöskirjassa tutkittiin IgE- herkistymisen,
allergisten sairauksien, suolen läpäisevyyden ja tulehduksen kehittymistä
maatilaympäristöstä kotoisin olevilla lapsilla ja lapsilla, jotka eivät
asuneet maatilaympäristössä. Lapset osallistuivat PASTURE- projektiin
(Allergiasuoja: tutkimus maatilaympäristöissä).
Tuloksemme osoittivat, että varhaisen iän tulehdusvaste, joka määritettiin
seerumista herkkä C-reaktiivisen proteiinin (hs-CRP) - mittauksella yhden
vuoden iässä, liittyi lapsen alentuneeseen riskiin olla IgE- herkistynyt 4.5
vuoden iässä. Tuloksemme viittaavat siihen, että heikko tulehdusvaste
imeväisiässä voi altistaa IgE- herkistymiselle.
Rintaruokinnan allergioilta suojaava vaikutus on herättänyt paljon
keskustelua. Tuloksemme osoittavat äidinmaidon kohonneiden liukoisen
immunoglobuliini A (sIgA)- pitoisuuksien laskevan atooppisen ihottuman
riskiä 2 vuoden ikään mennessä, kun taas lapsen ensimmäisenä
elinvuotena rintamaidon sIgA:n kohonnut kokonaismäärä vähensi
atooppisen ihottuman riskiä sekä 2 ja 4 vuoden ikään mennessä.
5
Kohonneet sIgA- pitoisuudet äidinmaidossa liittyivät ympäristötekijöihin,
jotka viittaavat korkeisiin mikrobimääriin.
Seerumin immunoglobuliini A (IgA) ja immunoglobuliini G (IgG)
lehmänmaidon β- laktoglobuliinia (BLG) ja vehnän gliadiinia vastaan
muodostuvat lapsen altistuessa näille ruoka-aineille. Vasta-aineiden
pitoisuuteen vaikuttavaa myös suolen läpäisevyys ja tulehdus. Lapsilla,
joilla seerumin IgA- tai IgG- vasta-ainepitoisuudet BLG:tä vastaan olivat
kohonneet 1 vuoden iässä, oli kohonnut riski olla herkistynyt vähintään
yhdelle mitatulle allergeenille tai ruoka-allergeenille kuuden vuoden
iässä. Kohonneet IgG- pitoisuudet gliadiinia vastaan 1 vuoden iässä
lisäsivät herkistymisriskiä vähintään yhdelle allergeenille, vähintään
yhdelle hengitysvälitteiselle ja vähintään yhdelle ruoka-allergeenille 6
vuoden iässä. Löydöksemme viittaavat siihen, että voimistunut vasta-
aineiden tuotto ruoan antigeenejä vastaan liittyy limakalvon sietokyvyn
muutoksiin, kuten suolen läpäisevyyden muutoksiin ja/tai muutoksiin
mikrobipopulaatiossa, mikä havaitaan myöhempänä herkistymisenä
allergeeneille.
Ulosteesta mitattu kalprotektiini on suolitulehduksen merkkiaine.
Tutkimme varhaisen iän ulosteen kalprotektiinin yhteyttä myöhempään
allergioiden kehittymiseen lapsilla, jotka asuivat maatilaympäristöissä ja
lapsilla, jotka eivät asuneet maatilaympäristössä. Tutkimme myös
suoliston mikrobien vaikutusta ulosteen kalprotektiinipitoisuuksiin.
Maatilaympäristössä asuvilla lapsilla oli korkeammat
kalprotektiiniipitoisuudet verrattuna lapsiin, jotka eivät asuneet
maatilaympäristössä. Maatilaympäristö ei kuitenkaan selittänyt erittäin
6
korkeita kalprotektiinipitoisuuksia, eli 90 prosenttipisteen ylittäviä tasoja.
Niillä lapsilla, joilla oli korkeat ulosteen kalprotektiinipitoisuudet 2kk
iässä, eli pitoisuus yli 90 prosenttipisteen, oli kohonnut riski sairastua
atooppiseen ihottumaan ja astmaan/ahtauttavaan
keuhkoputkitulehdukseen 6 vuoden ikään mennessä. Korkea ulosteen
kalprotektiinipitoisuus oli yhteydessä ulosteen Escherichia- bakteerien
alhaiseen määrään. Tuloksemme viittaavat siihen, että varhaislapsuuden
korkea suolitulehdus aiheuttaa kohonneen allergisten sairauksien riskin ja
että varhaisella suoliston mikrobikolonisaatiolla on pitkäaikaisia
terveysvaikutuksia.
Johtopäätös tämän väitöskirjan tuloksista on, että mikrobikuorma
varhaislapsuudessa muokkaa lapsen immuunivasteita ja allergisten
sairauksien kehittymistä. Mikrobialtistus vaikuttaa suoraan lapsen
immuunijärjestelmän kehittymiseen, mutta myös epäsuorasti äidinmaidon
koostumusta, kuten liukoista IgA-pitoisuutta, muuttaen. Mielenkiintoista
oli havaita, että varhainen suolitulehdus imeväisiässä näyttää liittyvän
allergisiin sairauksiin ja suolen kolonisaatiolla voi olla olennainen
vaikutus allergioiden kehittymiseen.
Avainsanat: Allergia, astma, atooppinen ihottuma, IgE- herkistyminen,maatilaympäristö, tulehdus, ulosteen kalprotektiini, vasta-aine.
7
CONTENTS
ABSTRACT ................................................................................. 1
TIIVISTELMÄ ................................................................................. 4
LIST OF ORIGINAL PUBLICATIONS ............................................................ 10
ABBREAVIATIONS ............................................................................... 11
1 INTRODUCTION ............................................................................... 12
2 REVIEW OF THE LITERATURE ........................................................... 14
2.1 Allergy………………………...…………………… .. ………………14
2.1.1 Mechanisms of allergy ..... …………………………………..15
2.1.2 IgE-sensitization………… ...... …………………………..…16
2.1.3 Tolerance…………………………… ..... ……...……………18
2.1.4 Asthma……..……………………… ..... ……………………18
2.1.4.1 Risk and protective factors of asthma ............... ..19
2.1.5 Atopic dermatitis…………………………………………. ... 21
2.1.5.1 Risk and protective factors of AD…... ................ 22
2.1.6 Breastfeeding and allergy development ..... …………………23
2.2 The hygiene hypothesis…………………………… ..... ……..………25
2.3 Farming environment……………………………… . ……….………27
2.4 Immune system……………………………… . ……………………..29
2.4.1 Innate immune system .... …………………………………...29
2.4.2 Adaptive immune system .... ………………………………...30
2.5 Gut immune system………………………………………… . ………34
2.5.1 Gut anatomy ..... ……………………………………………..34
2.5.2 Mucosal immune system and oral tolerance ..... …………….38
2.5.3 Fecal biomarkers of inflammation ..... …………………...….40
8
2.6 Gut normal flora………………………………………………...… …..42
2.6.1 Gut microbes in allergic diseases ...... ……………………….44
2.6.2 Breastfeeding and gut microbes in infants ……… ...... ……..46
2.6.3 Measurement of gut microbes…………………… ..... ……...47
3 AIMS OF THE STUDY ........................................................................... 48
4 SUBJECTS AND METHODS .................................................................. 50
4.1 Subjects………………………………………...……………… ……50
4.1.1 Families in the PASTURE study ...... ………………….……50
4.1.2 Health outcomes ...... …………………………………...…...51
4.1.3 Samples in Study I……………...………..………… ...... …..53
4.1.4 Samples in Study II…….…………………………… ...... ….53
4.1.5 Samples in Study III………….……………………… ...... …53
4.1.6 Samples in Study IV………………….…………… ..... ……53
4.2 Methods…………………………… .. ……………………………….56
4.2.1 Measurement of hs-CRP levels in serum (Study I) ............... 56
4.2.2 Measurement of TGF-β1 and sIgA in breast milk (Study II). 56
4.2.3 Measurement of IgA an IgG antibodies against cow’s milk
β-lactoglobulin and wheat gliadin in serum (Study III)........ 57
4.2.4 Measurement of fecal and breast milk calprotectin
(Study IV) ............................................................................ 58
4.2.5 Pyrosequencing (Study IV)………………… ... …………….59
4.2.6 Detection of IL-10 secreting cells (Study IV)………… .... …60
4.2.7 Statistical analyses…………… ... …………………..………61
9
5 RESULTS AND DISCUSSION……………………………… ...... …….65
5.1 Early-age inflammatory response and development of IgE-
sensitization (Study I)…………………………………………… ......65
5.2 Soluble immunoglobulin A in breast milk decreases the risk of atopic
dermatitis in the child (Study II)……………………………… . ….....73
5.3 Early-age antibody response to food antigens associates with IgE-
sensitization later in life (Study III)…………………………… ... …..80
5.4 High level of fecal calprotectin at 2 months as a marker of intestinal
inflammation predicts atopic dermatitis and asthma by age 6
(Study IV) .............................................................................. 88
6 CONCLUSIONS ............................................................................... 97
7 ACKNOWLEDGEMENTS ...................................................................... 99
8 REFERENCES ............................................................................. 102
10
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original articles referred to in the text by theirRoman numerals:
I. Mustonen K, Orivuori L, Keski-Nisula L, Hyvärinen A, PfefferlePI, Riedler J, Dalphin JC, Genuneit J, Lauener R, Roduit C,Braun-Fahrländer C, Weber J, Schaub B, von Mutius E,Pekkanen J, Vaarala O and the PASTURE Study Group.Inflammatory response and IgE sensitization at early age.Pediatric Allergy and Immunology 2013; 24(4): 395-401.1
II. Orivuori L*, Loss G*, Roduit C, Dalphin JC, Depner M,Genuneit J, Lauener R, Pekkanen J, Pfefferle P, Riedler J,Roponen M, Weber J, von Mutius E, Braun-Fahrländer C,Vaarala O and the PASTURE Study Group. Solubleimmunoglobulin A in breast milk is inversely associated withatopic dermatitis at early age: The PASTURE cohort study.Clinical and Experimental Allergy 2014; 44(1): 102-112.
III. Orivuori L, Mustonen K, Roduit C, Braun-Fahrländer C, DalphinJC, Genuneit J, Lauener R, Pfefferle P, Riedler J, Weber J, vonMutius E, Pekkanen J, Vaarala O and the PASTURE StudyGroup. Immunoglobulin A and immunoglobulin G antibodiesagainst β-lactoglobulin and gliadin at age 1 associate withimmunoglobulin E sensitization at age 6. Pediatric Allergy andImmunology 2014; 25(4): 329-337.
IV. Orivuori L, Mustonen K, de Goffau M.C, Hakala S, Monika P,Roduit C, Dalphin J-C, Genuneit J, Lauener R, Riedler J, WeberJ, von Mutius E, Pekkanen J, Harmsen H.J.M, Vaarala O and thePASTURE Study Group. High level of fecal calprotectin at 2months as a marker of intestinal inflammation predicts atopicdermatitis and asthma by age 6. Clinical and ExperimentalAllergy 2015; 45(5):928-939.
These articles are reproduced with the kind permission of their copyrightholders.1 This article was published earlier in Mustonen K (2014): The role ofinflammation and its environmental triggers in allergic diseases.* Both authors equally contributed to this study.
11
ABBREVIATIONS
AD Atopic dermatitisAMP Antimicrobial peptidesAPC Antigen presenting cellBLG β- lactoglobulinCI Confidence intervalCRP C-reactive proteinDC Dendritic cellEAACI European Academy of Allergy and Clinical ImmunologyEDTA Ethylenediaminetetraacetic acidELISA Enzyme-linked immunosorbent assayGALT Gut-associated lymphoid tissueGF Germ- freeHBD Human β- defensinHEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHLA Human leukocyte antigenHSA Human serum albuminhs-CRP High-sensitivity C-reactive proteinIBD Inflammatory bowel diseaseIFN-γ Interferon-γIg ImmunoglobulinIL InterleukinLP Lamina propriaLPS LipopolysaccharideM cell Microfold cellMHC Major histocompability complexNF-κB Nuclear factor kappa-light-chain-enhancer of activated B
cellsOPD Ortho-phenylenediamineOR Odds ratioPASTURE Protection against allergy: study in rural environmentsPBMC peripheral blood mononuclear cellsPRR Pattern recognition receptorsrRNA Ribosomal ribonucleic acidSCFA Short-chain fatty acidsSCORAD Scoring atopic dermatitisSFF Standard flowgram formatsIgA Soluble immunoglobulin ATGF-β Transforming growth factor-βTNF-α Tumor necrosis factor- αTh T helperTLR Toll-like receptorTreg Regulatory T cell
12
1 INTRODUCTION
Allergies have become a major public health problem in the last 50 years,
now affecting the lives of more than 60 million Europeans, and
approximately one billion people worldwide. Of the European population,
approximately 30% suffer from allergic rhinoconjunctivitis, 20% from
asthma, and 15% from allergic skin conditions. Many allergies only
manifest themselves during certain periods of life. For example, milk and
egg allergies are common in children but often diminish with age,
whereas allergic rhinitis has been estimated to affect up to 45% of 20-40
year old Europeans. Importantly, the European Academy of Allergy and
Clinical Immunology (EAACI) estimates that in less than 15 years more
than 50% of Europeans will suffer from at least one allergy.
Hygiene hypothesis states that excess environmental hygiene associates
with allergic diseases. Decreased contact to environmental microbes, i.e.
less childhood infections, can lead to alterations in the normal immune
and inflammatory responses that cause allergic reactions to non-harmful
environmental antigens. Children who grow up on farms have been
shown to be at a decreased risk of hay fever, asthma, and allergic
sensitization. The most allergy-protective environmental exposures are
the consumption of unprocessed farm milk and early-life contact with
livestock and their feed. Also, maternal contact with increasing number of
farm animals, the time spent in stables while pregnant, and having
siblings decrease the risk of allergies in the child. Environmental
exposures which decrease the risk of allergies are the most effective when
13
encountered before or soon after birth, and they are often associated with
increased environmental microbial load.
This thesis aimed to study the role of the farm environment and other
environmental factors associated with elevated microbial load in the
development of gut immune system and allergies with the goal of
identifying risk and protective factors of allergies.
14
2 REVIEW OF THE LITERATURE
2.1 AllergyAllergy is an adverse immunological reaction to a harmless
environmental factor, most often a protein. The protein causing allergic
response is called an allergen. Less frequently, allergens can be
carbohydrates or small chemical compounds, such as nickel. Allergic
reactions can be immediate or delayed. Atopy is a characteristic of a
person prone to developing immunoglobulin (Ig) E antibodies against
allergens, which is also called sensitization. Atopic reactivity is a risk
factor for allergic diseases, but not all atopic individuals develop allergic
diseases. While allergies can be treated with medication and sometimes
immunotherapy, no cure has been discovered.
Allergic rhinoconjunctivitis is a major allergic health problem, but the
symptoms vary in duration and can be difficult to define. The EAACI
estimates that many people with allergic rhinitis are undiagnosed. Food
allergies are common in children but often diminish with age.
Anaphylaxis is a severe, potentially life-threatening systemic
hypersensitivity reaction, which is characterized by life-threatening
airway, breathing, or circulatory problems and can be associated with skin
and mucosal changes. The onset of anaphylaxis is rapid and needs to be
treated with epinephrine (1). The allergic diseases discussed in this thesis
in more detail are asthma and atopic dermatitis, which have high
incidence and relatively well established criteria of diagnosis.
15
2.1.1 Mechanisms of allergy
In the 1960’s, Gell and Coombs classified hypersensitivity reactions into
four classes, which is suitable for an overview of the allergic mechanisms
(2). Allergic reaction can be immediate (most often IgE-mediated) or
delayed (non-IgE-mediated).
Hay fever and allergic asthma are examples of type 1 hypersensitivity in
which the reaction is immediate and can be anaphylactic. Allergic
symptoms in susceptible individuals occur after a receptor-bound IgE
molecule on the mast cell surface has recognized a cognate allergen and
the mast cells degranulate, releasing inflammatory mediators such as
histamine and leukotrienes. Less often, an immediate allergic reaction can
occur without IgE (type II reaction). IgM or IgG class antibodies
recognize a microbe or antigens on the target cell and the complement is
activated. This leads to the release of histamine, and leukocyte activation.
The destruction of cells and subsequent enzyme release leads to tissue
destruction and symptoms such as the vasculitides and certain drug
allergies.
Delayed immunocomplex-mediated allergic reaction (type III) can occur
when the allergen is bound to IgM or IgG antibodies in the serum. This
complex activates the complement system leading to histamine release.
The complement can also form complexes with IgM or IgG, which attach
to vessel endothelium causing inflammation and urticaria. Contact
allergies are delayed cell-mediated allergies (type IV), which may
develop after several weeks or years of exposure to the allergen. Contact
allergies are often caused by a small molecule called a hapten. Although
16
haptens can bind to IgM on B cells, they cannot be presented to T cells
and therefore do not directly trigger allergic reactions. To turn into an
allergen, the hapten must first bind to a protein. The hapten-protein
complex is then attached to a major histocompatibility complex (MHC)
on the surface of a Langerhans cell that then migrates to a lymph node
where the protein in the hapten-protein complex can induce a T cell
response. If tolerance does not develop, T cells are activated and relocated
to the site of contact, and allergic eczema may occur (3,4).
2.1.2 IgE-sensitization
The function of IgE is to defend the host against parasite worms and
certain protozoan parasites (5). IgE is the least abundant of the antibody
classes, constituting only 0.004% of total immunoglobulins in serum.
Moreover, IgE concentrations are age-dependent with very low levels in
young children that increase towards adolescence (6).
In atopic individuals, production of IgE antibodies against environmental
antigens such as animal dandruff or pollen is increased. The class switch
to IgE in B cells is induced in a T helper (Th) 2 cytokine environment.
The location of allergen-specific IgE production is not entirely clear, but
it has been suggested to occur in the particular allergy effector organ,
such as the lung (7). Specific IgE against an allergen can be measured in
serum samples. Skin prick tests using suspected allergens show
immediate response in IgE-sensitized patients or patients with IgE-
mediated allergy while non-IgE-mediated allergies may be detected as
delayed skin reactions.
17
Polysensitization can be divided into cross-sensitization, in which the
same IgE molecule binds to several allergens with common structural
features, and co-sensitization, where different IgE molecules bind to
allergens that do not necessarily have similar structures. Monosensitized
people are sensitized to only one allergen. Monosensitized children are
likely to developed polysensitization and co-sensitization later in
childhood (8). There is an overall association between sensitization in
skin prick tests and total IgE values but it is not known if the levels of IgE
can be used in defining the presence of clinical symptoms. The clinical
relevance of IgE-sensitization is established when the patient reports
having clear symptoms, or the symptoms are demonstrated in a direct
allergen challenge.
In most allergies, the synthesis of an allergen-specific IgE is needed.
Nevertheless, many people with allergen-specific IgE do not develop
allergic symptoms. For example, one Dutch study found that 43% of the
study subjects with serum-specific IgE to inhalant allergens did not
develop respiratory symptoms (9). There may be several factors causing
the differences in IgE-sensitized people who develop clinical allergies and
IgE-sensitized people who do not develop symptoms. The factors
affecting the course of clinical allergy may be genetic predisposition, total
serum IgE and specific IgE or IgG levels, mono vs. polysensitization, or
the balance of Th1/Th2 or T regulatory cells (Treg) (10).
Several studies have shown that growing up on a farm is associated with a
decreased risk of IgE-sensitization. Of the farm-related factors, early-life
18
and consistent contact to stables and the consumption of farm milk have
been associated with decreased risk of IgE-sensitization (11).
2.1.3 Tolerance
The development of immune tolerance is necessary for the maintenance
of homeostasis. The human body encounters numerous antigens that are
recognized by the immune system. Normally, the stimuli lead to
unresponsiveness, i.e. tolerance, which is maintained by alterations in
memory and allergen-specific T and B cell responses and raised
thresholds for basophil and mast cell activation. If the development of
tolerance fails, initially harmless environmental antigens can trigger a
hypersensitivity reaction, or lead to an allergic disease. Intolerance to
self-antigens can lead to autoimmune disorder. In turn, excessive
tolerance can lead to infection or cancer. Induction of tolerance with
repeatedly introduced doses of antigen has been performed in allergen-
specific immunotherapy (12).
2.1.4 Asthma
Asthma is a chronic inflammation of the respiratory system, causing
increased hyperreactivity to environmental stimuli. Long-term
inflammation can induce coughing, wheezing, mucus production, and
shortness of breath. Asthma is a multifactorial disease (13). Furthermore,
asthma can be atopic or non-atopic. A viral infection can lead to airway
obstruction in infants with transient wheezing and in small children with
non-atopic wheezing, while children who are IgE-sensitized and have
wheezing are more prone to developing chronic asthma. Stein et al. (14)
described three different childhood wheezing phenotypes that can co-exist
19
during childhood: “transient early wheezing” can occur in children up to
the age of 3 years; “non-atopic wheezing” can occur in toddlers and
during early school years with the highest prevalence of wheezing
between the ages of 3 and 6 years; “IgE-associated wheeze/asthma” may
exist at any age during childhood and is characterized by persistent
wheezing.
The diagnosis of asthma is based on airway obstruction measured with
altered airflow in pulmonary function tests, a physical examination, and a
medical history of the patient. In epidemiological studies, the data on the
diagnosis of asthma is usually received from questionnaires in which the
participant or guardian has answered whether a doctor has diagnosed
asthma or if they have had symptoms of asthma.
2.1.4.1 Risk and protective factors of asthma
The rapid increase in the prevalence of asthma cannot be explained by a
change in population genetics given the slow rate of genetic drift. Certain
environmental factors have been suggested to play a role in the
development of asthma. In order to affect to the development of asthma,
the exposure has to occur before the symptoms and affect the onset of
disease in conjunction with the genetic predisposition.
Microbial exposure plays a complex role in the development of asthma.
There is evidence showing that frequent infections related to low-hygiene
in childhood, having siblings, and day care attendance decrease the risk
allergies and asthma. The infectious agents that modulate disease
pathogenesis are thought to include respiratory viruses and non-
20
pathogenic environmental microbes, which support the Th1 type
immunity (15). The farming environment is thought to decrease the risk
of asthma due to the higher microbial load of environmental microbes
present on the farms (16). However, respiratory viruses are also
implicated in triggering asthma exacerbations in patients with established
asthma. Furthermore, inhaled endotoxin produced by Gram-negative
bacteria can cause strong pro-inflammatory reactions. Endotoxin has been
shown to be a risk factor of wheezing in the first year of life (17). The
severity of asthma has been shown to correlate with endotoxin levels in
the homes of patients with asthma (18). Interestingly, endotoxin levels
have been shown to be inversely associated with atopic sensitization and
atopic asthma, but not with non-atopic asthma (17). Therefore, endotoxin
seems to play two roles in asthma, with an atopy-protective effect while
still posing a risk for non-atopic asthma and wheezing (13).
There are several findings that suggest that parental smoking is associated
with childhood asthma and wheezing and this association is stronger with
wheezing among the non-atopic children than in the asthmatic children.
The severity of asthma is increased in children whose parents smoke (19).
However, it has been suggested that tobacco smoke is a co-factor in
triggering asthma symptoms (wheezing), rather than the cause of asthma
(13). Active smoking has been associated with the onset of asthma in
teenagers and adults (20,21). Studies performed in both animals and
humans have shown that tobacco smoke during pregnancy results in
reduced lung function in the newborn (22).
21
Air pollutants can reduce lung function and increase hospitalization rates
for asthma patients. Although air pollutants can worsen the symptoms,
there is no clear evidence showing that air pollutants lie behind the initial
development asthma (23).
The association of obesity and development of asthma is not entirely
clear. Body mass index has been shown to associate with asthma risk
whereas weight loss has been shown to improve lung function (24). On
the other hand, increased body mass index did not explain the increase in
the asthma incidence in a British population from 1982 to 1994 (25).
However, modern diet and lifestyle changes may influence both body
mass index and asthma, causing parallel increased trends in both
conditions (13).
2.1.5 Atopic dermatitis
Atopic dermatitis is a chronic inflammatory skin disease in persons most
often showing IgE-sensitization. Eczematous skin lesions and relapsing
progress characterize AD. Half of all AD cases begin in infancy.
Approximately 20% of infants in developed countries are affected by AD
(26). Approximately 25% of them continue to have symptoms throughout
their lives (3). It has been estimated that 30% of children with AD also
develop asthma. Patients with AD are prone to skin infections, which are
often caused by Staphlylococcus aureus or herpex simplex viruses
(27,28).
Ceramides are epidermal lipids that maintain the barrier function of the
skin. Ceramide levels are reduced in patients with AD, which leads to
22
increased transepidermal water loss (29). Altered tight junction formation
affects the skin barrier function in AD. In addition, mast cell activation
and the release of histamine may contribute to the defects of skin barrier
in AD.
Eczema is a chronic atopic or non-atopic inflammatory skin disease. In
the general population, including children and adults, up to 2/3 of eczema
patients do not show IgE-sensitization (30).
2.1.5.1 Risk and protective factors of AD
The etiology of AD is affected by the interaction of genetic
predisposition, environmental exposures and the immune system. The
lack of environmental microbial exposure and the altered composition of
skin microbiota may affect the development of the disease.
Results from the PASTURE study show that maternal contact to farm
animals and cats during pregnancy decreased the child’s risk of AD until
the age of 2 years. Also, elevated cord blood expression of innate
immunity toll-like receptors (TLR) 5 and 9 was associated with decreased
doctor diagnosis of AD (26). In a cross-sectional German study including
17 641 children aged 0 – 17 the prevalence of eczema diagnosis ever was
13.2%. The prevalence was positively associated with parental allergies ,
infection, or jaundice after birth. Having a dog as a pet or being a migrant
decreased the risk of eczema (31).
Skin microbiota composition is associated with the skin barrier function
(32). One factor that can affect the composition of skin microbiota is
aberrant cytokine production, which can lead to alterations in the
23
production of antimicrobial peptides (AMP) in the skin of AD patients.
This can contribute to the shift in the balance of skin microbial
communities and affect the onset of disease (33). The commensal skin
bacterium S. epidermis is capable of inducing TLR- mediated
inflammatory responses which suppress inflammation after skin injury
(34). In addition, S. epidermis can induce the production of AMPs from
keratinocytes and affect the functions of skin resident T lymphocytes
(35).
It has also been shown that gut microbial composition is associated with
the risk of AD, possibly by interacting indirectly via the gut immune
system. Early gut colonization with Clostridium cluster I was related to
increased risk of AD by the age of 2 years (36).
Filaggrin protein is involved in cornification. Mutations in the gene
coding for filaggrin associate with impaired skin barrier function and
have been associated with AD (37).
2.1.6 Breastfeeding and allergy development
Breast milk contains antimicrobial and anti-inflammatory components,
such as cytokines and antibodies. In addition, breast milk contains
bacteria and antigens derived from food. These components of breast milk
protect the infant from infections and educate the infant’s immune system
towards tolerance. There are contradictory findings on the effect of
breastfeeding to the development of allergic diseases in childhood.
Currently, there seems to be insufficient evidence to suggest that
breastfeeding protects from food allergy. There are both studies that show
24
that breastfeeding can protect from the development of AD and studies
that show no association (38,39). However, there is consistent evidence
that breastfeeding protects from childhood wheezing, possibly due to
breast milk components protecting from respiratory tract infections. Yet,
the effect of breastfeeding on asthma is more complex since wheezing
and childhood infections can affect the asthma risk. Breastfeeding has
been reported to associate with increased risk of asthma in late childhood
and adulthood (40). Nevertheless, the complex nature of asthma and
wheezing suggest that such associations are cautiously examined.
Conflicting findings in different studies can be due to differences in the
assessment of breastfeeding (e.g. exclusive vs. non-exclusive, or different
cut-offs used for the duration of breastfeeding), outcome definitions, or
not taking the genetic predisposition into account (41). The composition
of breast milk has been shown to differ between mothers with and without
allergies (42,43), which can also affect the findings.
Breast milk factors that have been suggested to associate with the
protective breastfeeding effect on allergic diseases are soluble IgA (sIgA),
soluble CD14, and transforming growth factor-β (TGF-β) (44-46).
Soluble IgA is the predominant immunoglobulin in breast milk. It
provides passive antimicrobial protection in the infant’s gut and can
protect from excess antigen intake and, thereby lower the risk of allergies.
TGF-β has an immunosuppressive role, such as the inhibition of
interferon-γ (IFN-γ) expression in CD4+ T cells, which may decrease the
risk of allergies. Soluble CD14 is a co-receptor for TLR4, which has a
significant role in lipopolysaccharide (LPS) recognition. TLR4 activation
25
may be important in infancy to strengthen the Th1 type immune
responses. However, not all allergy-protective findings of breastfeeding
and breast milk components are consistent, and the mechanisms remain
obscure. It is possible that the decreased risk of developing an allergic
disease associated with living in farming environment is mediated
through breast milk constituents, which have been shown to be different
in farm and non-farm mothers (47). Also, Estonian and Swedish mothers
have been shown to have differences in breast milk composition (48),
suggesting that environmental factors associated with elevated microbial
load affect the breast milk composition.
2.2 The hygiene hypothesisStrachan introduced the concept of the hygiene hypothesis in 1989 when
he reported that allergic rhinitis and family size were inversely correlated
(49). It was speculated that contact to other children increased infections
in early life, which led to a decreased risk of allergy. The theory has been
supported by several subsequent findings showing, e.g., that children who
were exposed to other children by attending day care at early age or had
older siblings had lower prevalence of atopy, asthma, and frequent
wheezing later in childhood (50,51). Although the associations of
environmental exposures and allergic diseases have been widely studied,
the specific factor or mechanism explaining the hygiene hypothesis
remains unknown. Yet, growing up in farming environment has been
repeatedly shown to be an allergy-protective factor (52).
26
The original message of the hygiene hypothesis was that decreased
exposure to environmental viruses, bacteria, and parasites leads to
insufficient activation of the Th1 immune response and the balance is
shifted towards Th2 response. More recent studies have shown that
Th1/Th2 imbalance is not the only reason for the increase in allergic
diseases in developed countries. For example, Th1 cytokines are also
involved in asthma and AD. There is also evidence showing that defects
in the Th1 pathway do not lead to increased risk of allergies, which
implies that Th1 pathway does not directly regulate Th2 responses in
allergy (53). Also, down-regulation of Th2-mediated inflammation does
not occur when Th1 polarized cells are applied to a site of inflammation
in an animal model. On the contrary, inflammation increases (54). In
addition, Th1/Th17-associated inflammatory diseases such as type 1
diabetes, multiple sclerosis, and Crohn’s disease have become more
prevalent in the same areas where allergies have increased (55,56) which
can be a signal of a defect in the patients’ ability to regulate immune
responses, leading to allergies or autoimmune diseases, rather than a mere
Th1/Th2 imbalance (57). The underlying cause behind such a defect,
whether it is the change in the environmental microbial load, a lack of
childhood infections, or some unknown factor in the western life style, is
yet to be defined.
The hygiene hypothesis can explain differences between the prevalence of
allergic diseases in developed and developing countries. Yet, the impact
of immunological responses is complex and not fully understood. Parasite
infections are common in developing countries where the prevalence of
27
allergies is low. It has been estimated that 2.5 billion people worldwide
are infected with helminth parasites (58). Helminths are able to modulate
the human immune system towards type 2 immune responses, such as the
secretion of Th2 type anti-inflammatory cytokines, but helminthes also
are strong inducers of Treg cells, which down-regulate the host immunity
against helminths. Epidemiological studies have shown that infections
with helminths are associated with low incidence of asthma, allergy and
autoimmunity. Animal studies have shown that helminths induce
regulatory immune responses which can suppress Th2 and Th1/Th17
responses that mediate allergy and autoimmunity, respectively (59).
The present immunological knowledge has changed the view of the
hygiene hypothesis so that the decreased microbial load is believed to
result in impaired activation of regulatory mechanisms, such as Tregs,
which further results in the impaired regulation of Th2 immunity and
allergic reactivity.
2.3 Farming environmentDuring the last decades, traditional agricultural lifestyle has become rare
in developed countries. While an urban life style demanded societal
changes, such as industrialized agriculture, the environmental biodiversity
and the microbial diversity decreased (60). It has been suggested that the
decrease in the environmental biodiversity has led to an increase in
allergic and autoimmune diseases in advanced societies (53).
28
Growing up in a farming environment has been shown to decrease the
risk of hay fever, asthma, and atopic sensitization (52). The exact farm-
associated factors behind the allergy-protective farm effect are unclear.
Nevertheless, it seems evident that prenatal or early-age exposures are
important. There are several separate farm-related factors, such as contact
to livestock and animal feed, and the consumption of unprocessed cow's
milk, that have been shown to associate with decreased risk of an allergic
disease or atopic sensitization in children (61). In addition, prenatal
exposure to animal sheds and hay has been reported to be inversely
associated with cord blood IgE levels against seasonal allergens (62).
In a study conducted in Eastern Finland, children who lived in families
with dogs, cats, or livestock had a lower risk of atopy, measured with skin
prick test at school age (63). Although, the farmers’ children had a
smaller risk of atopy, the effect of animal contact was independent of
farming status. Also, a dose-response effect was found: the risk of atopy
decreased with more frequent animal contact.
Farmers’ children are reported to use butter more often than margarine
when compared to non-farmers’ children. Also, the daily consumption of
farm milk or full milk is higher among farmers’ children. National
recommendations may affect the usage of raw milk products. In the
Finnish study (63), no association between atopy and farm milk
consumption was found, possibly due to the small number of children
consuming unprocessed milk. However, farm milk consumption has been
associated with decreased risk of allergies in several studies, and is
considered a distinctive farm exposure in decreasing the allergy risk
29
(61,64). A cross-sectional study by Perkin and Strachan showed that
current unpasteurized milk consumption was associated with less current
eczema symptoms, a 59% reduction in total IgE levels, and a 70%
decrease in skin prick test positivity in school-aged children (65). The
finding was also seen in children who did not live on farms.
Although there may be a wide range of factors that make up the allergy-
protective farm effect, microbial antigen contact in early life may be the
key factor in the modulation of the developing immune system. Microbes
derived from the environment, such as household pets, livestock and their
feed can modulate the normal flora and immunological responses via the
respiratory tract, the digestive tract, and the skin. Elevated bacterial
endotoxin levels measured in the mattress dust of school aged children
have been shown to associate with living on a farm, decreased risk of
asthma, atopic sensitization, and hay fever (66). In addition to a higher fat
and protein composition, raw milk contains a significantly higher total
bacteria count, more coliforms, E. coli, and coagulase-positive
staphlylococci than pasteurized milk (67). Digestion of the milk derived
bacteria or their components can alter the gut normal flora and lead to
beneficial modulation of the immune system.
2.4 Immune system
2.4.1 Innate immune system
The innate immune system consists of non-specific mechanisms that
enable an immediate response against pathogens. The mechanisms
30
include epithelial cell barrier and tight junctions, the secretion of mucus,
intestinal peristalsis, digestive enzymes, fever, and pH values not suitable
for microbes. Monocytes, neutrophils, macrophages, natural killer cells,
acute phase proteins, and the complement are a major part of the innate
immune response.
The innate immune response can be activated via signaling or non-
signaling pattern recognition receptors (PRRs). Signaling PPRs include
TLRs (68), which and are expressed on antigen-presenting cells (APCs)
such as macrophages and dendritic cells (DCs), and on the surface of
epithelial cells, monocytes and T cells. Microbial structures, such as LPS,
are recognized by TLRs. Signaling via TLRs can induce the production of
pro-inflammatory cytokines and chemokines (69). Non-signaling, soluble
PRRs include acute phase proteins and transmembrane proteins. The
acute phase protein CRP is produced in liver and released to circulation
where it can bind to invading pathogens leading to phagocytosis or
complement recognition. High levels of CRP measured in serum or
plasma is used as a marker of inflammation. Low-grade inflammation,
characterized by moderately increased levels of hs-CRP, has been
associated with decreased risk of eczema (70).
2.4.2 Adaptive immune system
Antigen-specific T and B lymphocytes mediate the adaptive immune
responses that are established in the secondary lymphoid tissues - spleen,
lymph nodes, and mucosa-associated lymphoid tissue. Adaptive immune
responses are specific to a particular pathogen and help to form long-
31
lasting immunologic memory against antigen structures. B cells are
activated to secrete immunoglobulins, whereas T cells are involved in
cell-mediated immune responses. T cells are divided into two broad
categories: CD4+ and CD8+ T cells. CD8+ cells are important in the
recognizing and killing of cells that are invaded by pathogens, whereas
CD4+ cells are important helper cells for the function of B cells, CD8+
cells, and macrophages. It may take days or weeks to develop an effective
adaptive immune response with antigen specific T and B cell populations.
T cell receptors are expressed on the surface of T cells. On CD4+ Th
cells, these receptors recognize antigens presented by class II major
histocompatibility complex (MHC) molecules, which are expressed on
APCs. The MHC in humans is called human leukocyte antigen (HLA).
CD4+ Th cells activate and regulate immune responses both by secreting
cytokines and by direct cell-cell interaction. Th cells mediate a broad
spectrum of immunological functions, including activating cytotoxic
CD8+ T cells, enhancing pathogen killing by macrophages, enhancing B
cell antibody secretion, and affecting apoptosis. Cytotoxic CD8+ T cells
recognize antigens presented by class I MHC molecules, which are
expressed on all somatic cells. After recognition of a presented pathogen
on the surface of a cell, cytotoxic CD8+ T cells proceed to kill the human
cells that have been infected by intracellular pathogens. For destruction,
cytotoxic CD8+ T cells use effector proteins such as perforin, granzyme
B, and apoptosis-inducing cytokines (71).
CD4+ Th cells can be divided into at least four subpopulations: Th1, Th2,
Th17, and Tregs (72-75). The composition of cytokines present at the
32
time of clonal expansion plays a large role in defining the polarization of
CD4+ Th cells into their subtypes. The integral cytokines regulating the
Th1 and Th2 polarization are IL-12 and IL-4, respectively. Th1 cells are
characterized by the release of IFN-γ, IL-2, tumor necrosis factor-α (TNF-
α), and lymphotoxin, which enhance cell-mediated immunity and
macrophage activation. Th1 cells are needed to mobilize CD8+ cells
against intracellular pathogens and have been linked to the development
of tissue destruction in autoimmune disease. Th2 cells mainly secrete B
cell activating cytokines such as IL-4, IL-5, and IL-13 (Figure 1) and play
a role in protecting against extracellular pathogens, such as parasites.
Dysregulated Th2 immunity is associated with the development of atopy
and allergic diseases (76).
Th17 cells secrete IL-17 and IL-22 cytokines and are important in
mucosal defence against fungi and bacterial infections. Activation of
Th17 cells leads to neutrophil recruitment and secretion of AMPs, such as
human β-defensin (HBD) 2, from epithelial cells. While Th17 cells are
the main source of IL-17, cytotoxic CD8+ T cells are also known to
secrete IL-17. Intriguingly, Th17 immunity is related to autoimmune and
inflammatory diseases such as type 1 diabetes, rheumatoid arthritis,
Crohn’s disease, and ulcerative colitis (77). The role of Th17 cells in
allergies is not entirely defined, however, up-regulated Th17 responses
have been detected in patients with allergic rhinitis or asthma (78).
Furthermore, Th17 mediated inflammation has been reported in AD,
which may play a role in tissue remodeling (79).
33
Tregs play a significant role in maintaining immunological homeostasis.
Tregs can be classified into subtypes that share some common features
including expression of Foxp3 and secretion of inhibitory cytokine IL-10
and/or TGF-β. Tregs regulate immune responses to allergens, self-
antigens, commensal microbes, pathogens, and tumors. Suppression of
effector T cells by Tregs can occur in two ways. In non-inflammatory
states, suppression aims to keep T cells in a naïve state and maintain self-
tolerance. This occurs by Treg-dependent deprivation of activation signals
from responder T cells. In inflammatory states, Tregs work to restore
local immune homeostasis by inactivating and killing responder T cells
and APCs, a process that can be mediated via IL-10 secretion (80). Tregs
may affect the pathogenesis of allergy, especially in the sensitization
phase. In healthy individuals, Th2 responses to allergens may be actively
prevented by Tregs, whereas in allergic patients, Treg functions may be
impaired (81).
In order to recognize antigens, B cells use antibodies as receptors. In their
naïve state, B cells express IgM and IgD on their surfaces. After
maturation, IgA, IgG, or IgE are expressed. Activated B cells proliferate
and develop into antibody-secreting plasma cells and can further
differentiate into memory cells. Both T and B cell mediated responses are
important, and presumably cross-regulated, in order to maintain a
functional adaptive immune response (82).
34
2.5 Gut immune system
2.5.1 Gut anatomy
The gut forms a significant barrier between the outside world and the
human body. The gut epithelium is often in direct contact with pathogens
and environmental antigens, which have the potential to provoke a
detrimental immune response. In order to function normally, the gut must
maintain both a robust immunity against pathogens as well as a tolerance
for the commensal flora and food antigens. In addition, the intestinal
mucosa has to allow fluid exchange and maintain a selective permeability
for the absorption of nutrients and secretion of waste (83).
Environmental agents and the cells of the immune system are in close
contact in the gut since the epithelium is generally only one cell layer
thick. On the lumen side, the epithelial cells have membrane protrusions
called microvilli, which increase the surface area of the cells and thereby
increase the surface area of the gut as well.
The goblet cells (Figure 1) are specialized epithelial cells that produce
mucin 2, a mucus forming molecule essential for colonic protection.
Mucus restricts relatively large particles, such as bacteria, from directly
contacting the epithelia. However, small constituents can penetrate the
mucus layer more easily. Animal studies have shown that mice with
mucin gene deficiency will develop intestinal inflammation and
eventually colitis (84). Microfold cells contain microfolds instead of
microvilli, and they have functions in antigen presentation.
35
The intestinal epithelium is protected by physical (mucus) and chemical
innate defence mechanisms which cooperate with the adaptive immune
system (83). In addition, the low pH of the stomach and proteolytic
enzymes secreted from the pancreas decrease the number of microbes
entering the gut, and also digest proteins to peptides, which are less prone
to activate immune responses.
The transepithelial transport of particles is made up of transcellular and
paracellular transport pathways. Paracellular pathway allows passive
movement of solutes across the epithelium. The solute flux is regulated
by tight junctions, which are the intercellular junctions between epithelial
cells. The tight junctions regulate the passage of solutes based on size.
Therefore, relatively small particles may passively pass across a tight
junction by a leaky pathway. The flux may be increased by cytokines
(85). Furthermore, small pores, possibly defined by claudin proteins in the
tight junction, allow the transport of particles based on their charge (86).
Claudin expression may also be regulated by cytokines. The transcellular
transport across the epithelium can be passive or active, and it is often
enabled by specific transport channels. Transcellular transport creates a
transepithelial concentration gradient that is sustained by tight junctions.
Without an intact tight junction barrier, diffusion would balance the
concentration between the two sides of the epithelium (83).
The intestinal epithelium plays an important role for both the innate and
adaptive immune systems, yet the mechanisms how the epithelium and
the mucosal immune system interact have not been fully elucidated.
36
Nevertheless, in response to infection, the tightly regulated nuclear factor
κB pathway becomes activated and further activates DCs, T cells, and
enterocytes (87).
37
Figure 1. The interaction between intestinal epithelial cells, antigen-presenting dendritic cells, and Th cellresponses. Dendritic cells react to pathogens and support the secretion of IL-12, IL-4, and IL-23 whichstimulate Th1, Th2, and Th17 responses, respectively. The secretion of calprotectin and/or HBD2 are inducedduring infection. (Adapted from Hundorfean et al. 2012).
38
2.5.2 Mucosal immune system and oral tolerance
The lamina propria (LP) is located between the surface epithelium and
muscularis mucosa of the gut. The gut-associated lymphoid tissue
(GALT) is located in the LP and it belongs to the mucosa-associated
lymphoid tissue. GALT consists of mesenteric lymph nodes, solitary
lymph follicles in ileum and colon, and Peyer’s patches, which are mainly
located in the submucosa of the small intestine and the appendix. In the
healthy gut, CD4+ and CD8+ T cells and IgA-producing plasma cells are
the most abundant cells in GALT. In addition, naïve B cells,
macrophages, and DCs can be found in GALT. As GALT is covered by
an epithelial cell layer, interaction with the environment is controlled by
barrier mechanisms (88).
Microfold cells (M cells) are specialized epithelial cells that reside in
isolated lymphoid follicles. Within the lymphoid follicles, they actively
transport foreign antigens and commensal bacteria to mucosal lymphoid
tissues in order to maintain the protective immune responses. Certain
pathogens use the microfold cells to enter the mucosa. It is suggested that
immature DCs endocytose antigens and pathogens after they have been
transported through M cells. DCs then present the antigens locally, or
migrate to the adjacent interfollicular region or to mesenteric lymph
nodes, mature, and present the antigens to T cells (88). DCs can also
catch bacteria by extending their dendrites into the intestinal lumen
(Figure 1).
39
The gastrointestinal tract is important for the development of immune
system, which has to recognize harmless antigens and tolerate them
without inducing severe inflammation, while being able to mount immune
responses against invading pathogens. Tolerance to food antigens is
induced via the small intestine and it generally affects systemic immune
responses. Failure to establish such a tolerance may lead to allergy, celiac
disease, or other immune-mediated diseases. Tolerance towards normal
flora occurs more locally in the large intestine. Failure to tolerate normal
microbiota may lead to inflammatory bowel disease (IBD) (89).
Mechanisms of tolerance include limiting the exposure to microbes with
mucus and IgA secretion (from plasma cells), and actively down-
regulating the immune responses through the control of PRR expression,
the secretion of inhibitory mediators, and the modulation of transcription
factors in intracellular signaling pathways (90).
Oral tolerance is mediated through the induction of regulatory T cells, T
cell anergy and clonal deletion. The effects of oral tolerance can be seen
in reductions in T cell proliferation and cytokine production. Antibody
levels in serum (such as IgE) in addition to mucosal IgA and T cell
responses may also be suppressed (89). The constitutive factor
determining the type of tolerance development is the dose of antigen
exposure. High doses of antigen favor the T cell anergy or deletion in the
gut and systemic antigen presentation, which can induce unresponsive T
cells. Low doses of antigen lead to active suppression. In the case of a
tolerogenic environment, antigen presentation by APCs results in the
40
production of antigen-specific regulatory T cells producing anti-
inflammatory IL-10 and/or TGF-β (91).
Animal studies have shown that tolerance is dependent on commensal gut
flora which can promote T cell hyporesponsiveness and down-regulate
antibody responses (92). The number of T cells in GALT of germ-free
(GF) mice has been shown to be lower when compared with the T cell
count in specific pathogen free mice, in which the situation corresponds
to an unsuccessful oral tolerance induction. Colonization of GF mice with
Bifidobacterium infantis and Escherichia coli restored the number of T
cells (93).
2.5.3 Fecal biomarkers of inflammation
Anti-microbial peptides (AMP) are important part of the mucosal innate
defence mechanisms. Defensins, lactoferrin, and cathelicidins are
common AMPs. In addition, microbial products such as short-chain fatty
acids (SCFA) and inflammatory proteins such as calprotectin are essential
in the shaping the gut inflammation (94).
AMPs distract microbes by forming micropores into their membranes.
Defensis and cathelicidins have chemotactic properties which they use to
attract cells such as T cells to the cite of microbial invasion. Lactoferrin
can inhibit pro-inflammatory cytokines and interact with bacteria by
binding iron. The production of SCFAs, such as butyrate, occurs when
microbes ferment carbohydrates. Butyrate has anti-inflammatory
properties and is an important factor in the regulation of gut permeability
(95). These regulators of intestinal inflammation can be used as fecal
41
biomarkers of gut inflammation and some of them can help to delay the
need for biopsy.
The most prominent human defensins are HBD1 in the colonic intestine
and α-defensin in the small intestine. The secretion of additonal defensins,
such as HBD2 are increased during infection. Levels of fecal HBD2 have
been reported to be elevated in ulcerative colitis (96) yet decreased in
Crohn’s disease which can be due to the different mechanisms of
regulating the mucosal innate defence system in the two diseases (97).
The levels of fecal calprotectin and lactoferrin are increased in IBD (98).
Of the markers mentioned above, all but SCFA can be measured with
enzyme-linked immunosorbent assay (ELISA) (94). In this thesis,
calprotectin was used as a biomarker for intestinal inflammation.
In addition to IBD, increased fecal calprotectin levels have been found in
celiac disease, rheumatoid arthritis (99-101) and in children with cow’s
milk allergy (102). Calprotectin is a cytosolic protein with the ability to
bind calcium and zinc ions (103-105). It is released during neutrophil
activation and monocyte-endothelial cell adhesion. The levels of fecal
calprotectin correlate with neutrophil migration into the gut lumen (101).
Monocytes and mucosal epithelial cells can also express calprotectin
(106).
Infants have higher fecal calprotectin levels when compared to children
and adults (104,107,108). This is likely due to the high gut permeability
in infancy (109), which leads to a high antigenic load and stimulation of
the inflammatory response in the gut immune system. The decrease in the
fecal calprotectin levels after infancy may reflect the maturation of the gut
42
(“gut closure”) and also the alleviation of inflammation. Colonization of
the gut by microbial flora may also contribute to intestinal inflammation
during infancy. The influence of bacteria on calprotectin levels was
reported in a study where antibiotic treatment correlated negatively with
calprotectin levels in low birth weight infants (110).
2.6 Gut normal flora
The co-evolution of humans and their commensal microbes has resulted
in humans’ dependence on their microbiota for normal immune function.
It seems that the normal function of the immune system and metabolism
are both strongly affected by the gut microbiota. The body has a 10-fold
number of microbial cells compared to eukaryotic cells. The gut is
specifically rich in normal bacterial flora. The number of microbial cells
in the human intestine can reach up to 1014 (111). In addition to affecting
the immune system, the gut bacteria use nutrients and poorly digestive
carbohydrates from food to produce metabolites (such as butyrate) which
can be used by the human host.
The composition of the gut microbiome is unique in each person and
dependent on life style factors such as dietary habits. The phylogenetic
microbial composition is different when compared between people living
in different countries (112). Nevertheless, the most prominent bacterial
phyla in adults are the Gram-positive Firmicutes and Actinobacteria, and
the Gram-negative Proteobacteria and Bacteroidetes (113). A 16S
ribosomal RNA gene (rRNA) sequence-based method showed that a 90%
43
majority of the gut bacteria belong to phylas Firmicutes or Bacteroidetes
(114).
At birth, the infant’s gastrointestinal tract is inhabited by bacteria derived
from the birth canal of the mother, including anaerobic bacteria. Infants
born with cesarean section acquire their first microbes from the hospital
environment in which anaerobes do not survive. They are also colonized
by bacteria from the skin, such as Staphylococci. In general, facultative
(aerotolerant) bacteria such as coliforms, enterobacteria, and lactobacilli
dominate the gut microbiota for the first years until anaerobes such as
Bifidobacterium, Bacteroides, Eubacterium and Clostridium establish
their colonization in the gut (114). Nevertheless, it was shown that infants
born by cesarean section have a lower ratio of anaerobic to facultative gut
bacteria when measured by one year of age (115). The Bacteroides,
bifidobacteria and E. coli colonization occurred later and in less
frequency in children who were born by caesarean section (116). After
delivery, skin-to-skin contact, breastfeeding, introduction of solid foods,
and the environmental conditions begin to modulate the microflora, which
is established during approximately the 3 first years of life and then
begins to resemble the adult microbiota. In a study comprised of more
than 500 individuals, children were shown to have a greater variety in
interpersonal composition of gut microbes than adults. Bacterial diversity
has been shown to increase with age (112).
Aberrancies in the ratio of microbial groups have been associated to the
development of certain disorders such as obesity, allergies, and type 1
diabetes (95,117). Bacteroidetes have been shown to be more abundant in
44
diabetic children, whereas the microbiota of healthy children seems to be
more in balance and contain more butyrate-producing bacteria (118). In
obesity, the relative proportion of Bacteroidetes is decreased, and the
proportion of Firmicutes is increased (117).
2.6.1 Gut microbes in allergic diseases
Similar to the environmental bacteria diversity, the diversity of the gut
microbial flora has altered and become poorer in the developed countries.
The diversity of gut microbiota was lower in children who later become
allergic (119). In addition to a higher diversity of gut microbes, children
from developing countries have been shown to be colonized earlier by E.
coli and have a higher turnover of E. coli strains (120) which may push
the development of the immune system towards a phenotype which is
tolerogenic against potential allergens.
Several epidemiological studies have shown differences of gut microbes
between atopic and healthy children. The quantity and colonization of
bifidobacteria has been shown to be lower in allergic children than in
their healthy peers. In addition, allergic infants are colonized by
Bifidobacterium species normally associated with adults, such as B.
adolescentis (114). It can be questioned which comes first, the allergic
predisposition altering the microbiota, or the alteration in microbiota
affecting the development of allergies. However, it has been shown that
skin-prick positive children had more clostridia at 12 months of age and
tended to have less bifidobacteria in feces at 3 weeks of age, when
compared to non-atopic children (121).
45
Species of Lactobacillus and Bifidocterium are commonly used as
probiotics. When probiotic bacteria has been given to the infants or their
mothers, the colonization seems to not be persistent after the end of
supplementation. Regardless of that, several studies have shown
probiotics to possess allergy-preventive effects, although, in most cases
only during the first 2 years of life. The incidence of atopic eczema was
decreased in high risk populations when probiotics were given to the
mothers before delivery and to the infants immediately after birth
(122,123). Also, probiotics were effective in eczema prevention in high-
risk children when the supplementation was started during pregnancy and
continued during breastfeeding (124). It has also been shown that the
altered bacterial colonization in children born with caesarean section
could be modified by probiotics, which led to fewer IgE- associated
allergies by the age of 5 years (125). It appears that the early timing and
extensive use of probiotic supplementation, and possibly the broader
range of bacteria, are essential since many studies have also failed to
show allergy-prevention in the child. There are also several studies in
which no effect of the probiotic treatment in the prevention or treatment
of allergies has been observed (126,127) and there is often high
heterogenity between studies. However, the immunological changes
induced by probiotics in the infants, such as incrase in hs-CRP or changes
in the cytokine profile, support the view that gut microbiota is an
important regulator of the immune system.
46
2.6.2 Breastfeeding and gut microbes in infants
Gut normal flora composition is altered by diet. Breast milk contains a
high concentration of fat, nutrients, anti-microbial agents, anti-
inflammatory agents, and bacteria, such as bifidobacteria and lactic acid
bacteria (114,128). It has been suggested that some of the bacteria in
breast milk may be derived from the mother’s gut via dendritic cells and
macrophages. In animal models it has been shown that bacterial
translocation from the gut to mesenteric lymph nodes and mammary
glands happened during late pregnancy and lactation (129). Therefore,
alteration of the mothers gut microbiota may be beneficial to the infant, as
did the above mentioned findings show, maternal probiotic
supplementation in addition to the supplemention given to the infant
prevented atopic eczema.
Several studies have shown that bifidobacteria dominate the gut
microbiota in breastfed babies during the first year of life (112).
Although formula milk is prepared to resemble the nutritional
composition of breast milk, it lacks maternal antibodies such as IgA as
well as active cytokines. Pre- and probiotics are often added to formula
for the modification and establishment of beneficial normal flora.
Nevertheless, the gut microbiota is different between formulafed and
breastfed infants. Formulafed infants have higher levels and frequencies
of facultative bacteria such as Bacteroides and clostridia, and lower
numbers and frequencies of bifidobacteria than breastfed infants.
Formulafed infants have been reported to have higher levels of fecal
SCFAs when compared to breastfed infants. It may be due to the different
47
bacterial composition, and therefore the different ability to ferment
carbohydrates (130).
2.6.3 Measurement of gut microbes
Measuring microbes in feces is the most straightforward way to define the
gut microflora. Only approximately half of the gut microbes can be
cultured (116). Modern techniques used for the microbial definition
include quantitative PCR, DNA fingerprinting, metagenomics,
phylogenetic microarrays and sequencing. These methods give different
degrees of taxonomic information. The use of different methods can also
cause difficulties in comparing the results of studies (131). It can be
questioned how well microbes excreted into feces represent the actual
microenvironments in different parts of the gut. Nevertheless, fecal
samples are widely used for microbial and inflammation marker analysis
since they are relatively easy to collect and process for laboratory
measurements.
48
3 AIMS OF THE STUDY
Allergies are a growing health burden in Western societies. The incidence
of allergic diseases, such as asthma, has also been increasing in
developing countries in recent years (132). The increased incidence of
allergic diseases and atopy is believed to associate with the more hygienic
environment and the lack of certain infections – mainly bacterial
infections. The aim of the studies presented in this thesis was to examine
the development of immune responses, IgE-sensitization, and allergic
disease in children from farming and non-farming environments, and the
study subjects were participants of the PASTURE birth cohort study.
1. The aim of the first study was to determine whether low-grade
inflammation, detected as elevated hs-CRP levels at early age, protects
from IgE-sensitization or allergic diseases later in life. The association of
farming factors related to microbial exposure and hs-CRP levels were
also investigated.
2. The aim of the second study was to characterize whether breast milk
sIgA or TGF-β1 levels associated with asthma, AD, or IgE-sensitization
in children. The duration of breastfeeding and the association of farm
related factors to the levels of breast milk components were also
evaluated.
49
3. The aim of the third study was to determine whether a farming
environment affected the development of mucosal tolerance defined by
the levels of IgA and IgG antibodies against cow’s milk BLG and whey
gliadin, and whether these antibodies to food antigens associated with
allergic diseases or IgE-sensitization later in life.
4. The aim of the fourth study was to evaluate the association of early-age
intestinal inflammation, detected as fecal calprotectin, with the later
development of allergic diseases in children from farming and non-
farming environments. In addition, we studied the effect of gut microbes
on the fecal calprotectin concentrations.
50
4 SUBJECTS AND METHODS
4.1 Subjects4.1.1 Families in the PASTURE study
Samples for the studies presented in this thesis were acquired from
children and their mothers who participated in the PASTURE birth cohort
study conducted in rural areas of Austria, Finland, France, Germany, and
Switzerland. Women were recruited to the study during pregnancy. They
were defined as farmers if they worked or lived on family-run farms
where any livestock was kept. The reference group consisted of women
from the same rural areas who did not live on a farm. Exclusion criteria
were maternal age less than 18 years, no livestock at the farm, premature
delivery, a genetic disorder in the infant, an insufficient knowledge of the
language spoken in the country, and no telephone connection.
Originally, 1133 mothers agreed to participate. Questionnaires were
administered by interview to the mother of the child within the third
trimester of pregnancy. Questionnaires were answered again when the
child was 2, 12, 18, and 24 months of age and then every year up to the
age of 6 years. Questions were based on previous studies (133-136) and
assessed respiratory and other health conditions of the mother,
environmental exposures and potential confounders. In addition, the
mothers kept a weekly diary from the third month of the child’s life to
their first birthday. The duration of breastfeeding and the introduction of
complementary foods were also recorded.
51
4.1.2 Health outcomes
Specific immunoglobulin E (sIgE) against 19 common allergens was
measured in sera at the ages of 1, 4, and 6 years using the Allergy Screen
Test Panel for Atopy (Mediwiss Analytic, Moers, Germany) (137) The
inhalation allergens measured were house dust mites (Dermatophagoides
pteronyssinus and D. farinae), pollen (alder, birch, European hazel, grass
pollen mixture, mugwort, rye, and plantain), horse, cat, and dog dander,
and the mold Alternaria alternata. The food allergens measured were
cow’s milk, hen’s egg, peanut, carrot, hazelnut, and wheat. Any allergic
sensitization was defined as a positive result to at least one of the
measured allergens. The cut-offs used for sensitization at different age-
points were sIgE concentration of 0.35 kU/L or greater at the age of 1
year, 0.70 kU/L or greater at the age of 4 years, or 0.70 or alternatively
3.50 kU/L or greater at the age of 6 years. Inhalation or food atopy was
defined as a positive result to any of the 13 inhalation or 6 food allergens
tested, respectively.
Data on doctor-diagnosed AD and asthma (also defined as more than one
episode of asthmatic, obstructive, or wheezy bronchitis which describe
the same condition) were obtained from questionnaires and used in
defining the diseases at specific age points in each study.
Study I: Parents reported if a doctor had diagnosed AD or asthma at least
once, or asthmatic bronchitis at least twice during the follow-up. Children
with no report of a doctor’s diagnosis of AD or asthma/had only one or no
report of asthmatic bronchitis in the 1 year questionnaire but who
developed the disease between ages 1 and 4 years were defined as having
52
AD or asthma, respectively. Children who had AD or asthma/more than
one report of asthmatic bronchitis before the measurement of hs-CRP
values (at the age of 1 year) were excluded from analyses. Atopic
sensitization was defined at ages 1 and 4 years.
Study II: Children were defined as having cumulative AD up to the age
of 2 or 4 years if the parents reported that the child had AD diagnosed by
a doctor at least once up to 2 or 4 years of age, respectively, or if the child
had a positive scoring index result for AD, i.e. SCORAD score (> 0) at
the age of 1 year. Atopy at age 4 and 6 was defined as having sIgE
antibodies against at least one of the measured allergens. Asthma at the
age of 4 years was defined as a doctor’s diagnosis of asthma or wheezy
bronchitis more than once during the last 12 months. Asthma at the age of
6 years was defined as doctor’s diagnosis of asthma or wheezy bronchitis
after between the ages of 3 and 6 years.
Study III: AD was defined as the first doctor diagnosis of the disease
during the 6 years of follow-up. Asthma was defined as the first doctor’s
diagnosis of asthma and/or second doctor’s diagnosis of asthmatic
(obstructive) bronchitis during the follow-up. Children with no reports of
diagnosis in the 1 year questionnaire but who developed AD or asthma
between the ages of 1 and 6 years were defined as having the disease.
Children who already had AD or asthma/asthmatic bronchitis (more than
once) before the measurement of IgA and IgG antibodies at the age of 1
year were excluded from analyses. Specific IgE was measured at the ages
of 1 and 6 year.
53
Study IV: Children were defined as having AD up to the age of 6 years
when the parents reported in the questionnaires that the child had at least
one AD diagnosis during the 6-year follow-up. Atopy at 6 years of age
was defined as a positive result for specific IgE antibodies (cut-off
0.70kU/L) against at least one of the measured allergens. Asthma by the
age of 6 years was defined as a doctor’s diagnosis of asthma at least once
or asthmatic bronchitis more than once during the 6 years.
4.1.3 Samples in Study I
Serum samples were collected at the age of 1 year for measurement of hs-
CRP. Both hs-CRP and sIgE values at the age of 1 year were available
from 634 children. Both hs-CRP values at the age of 1 year and sIgE at
the age of 4 years were available from 448 children (Figure 2).
4.1.4 Samples in Study II
Breast milk samples were collected from 622 mothers two months after
delivery. Austrian samples were not applicable since they were not sent to
Finland for measurements. In total, 610 samples were analysed for both
sIgA and TGF-β1 (Figure 2).
4.1.5 Samples in Study III
Serum samples were collected at the age of 1 year for measurement of
IgA and IgG against cows’ milk BLG (N=639) and IgA and IgG against
wheat gliadin (N=636) (Figure 2).
4.1.6 Samples in Study IV
Fecal samples were collected at the age of 2 months and calprotectin was
measured in 758 samples. French samples had been stored improperly and
54
were therefore excluded. Furthermore, French samples were collected
using different tubes and collection sticks than in other countries. Since
the sample material was powder-like and clearly appeared to be different
from the samples from other countries, a decision was made not to use
them.
Gut microbiota was analysed in a subgroup of 120 infants. The children
were included for the selection if they were exclusively or partially
breastfed at the age 2 months, if the fecal calprotectin level was above the
detection limit, if there was data available for AD and asthma at the age
of 6 years, and if the amount of fecal sample was sufficient for extraction
of DNA. Case samples (N=40) were selected starting from the first
applicable sample with fecal calprotectin in the 90th percentile (fecal
calprotectin values ranging from 517.6 to 1542.0 µg/g). Control samples
were defined as samples with fecal calprotectin concentration below the
90th percentile. For each study centre, two control samples were randomly
selected for each case sample (N=80). To study the correlation between
fecal and breast milk calprotectin levels, calprotectin concentrations were
measured in 115 breast milk samples from the Finnish mothers 2 months
after giving birth (Figure 2).
The ethical committees of all study centers approved the study, and
written informed consent was obtained from the children’s parents for
questionnaires and samples.
55
Figure 2. Samples in the PASTURE study and in studies I, II, III, and IV of this thesis.
56
4.2 Methods
4.2.1 Measurement of hs-CRP levels in serum (Study I)
Serum samples were stored at -20° C. They were thawed for analysis in
Study I. High-sensitivity CRP concentrations in serum samples were
measured using ELISA (BenderMedSystems human CRP, Vienna,
Austria) according to the manufacturer’s instructions. The hs-CRP ELISA
is used for research purposes to measure low levels of CRP, not for
diagnostic procedures. The sensitivity of the assay is 3.0x10-6 mg/l.
4.2.2 Measurement of TGF-β1 and sIgA in breast milk (Study II)
Breast milk samples were stored at -20° C. The samples were thawed and
centrifuged at 10 000 g for 10 minutes at +4° C. The fat layer was
removed and the clear supernatants were stored at -75° C. The
supernatants were thawed for analysis in Study II.
TGF-β1 values were measured from breast milk samples (supernatants)
using a commercial ELISA method (Quantikine Human TGF-β1
Immunoassay, Abingdon, UK). Prior to the measurements, latent TGF-β1
was activated using hydrochloric acid. The samples were subsequently
neutralized with sodium hydroxide containing 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES).
Values of sIgA were measured using ELISA modified from Lehtonen et
al. (138). The primary coating antibody was rabbit antihuman IgA
(DakoCytomation, Glostrup, Denmark) diluted 1:1000 in carbonate
buffer. The plates were incubated at +4° C overnight and blocked with
57
1% BSA in PBS for 1 h at +37° C. The plates were washed with 0.5%
Tween 20 in PBS. The conjugate was peroxidase-conjugated rabbit anti-
human IgA (DakoCytomation) 1:5000 in PBS. The substrate was Ortho-
Phenylenediamine (OPD) (Kem-En-Tec-laboratories, Taastrup,
Denmark). The reaction was stopped using 1 molar H2SO4 and optical
density was read at 492 nm. A standard with known amounts of human
IgA (Caltag laboratories, Burlingame, CA, USA) was used to create a
control curve in each plate, from which the immunoglobulin
concentrations were calculated.
4.2.3 Measurement of IgA an IgG antibodies against cow’s milk β-
lactoglobulin and wheat gliadin in serum (Study III)
Serum samples were stored at -20° C. They were thawed for analysis in
Study III. IgA and IgG antibodies against cow's milk BLG were measured
using enhanced binding microtitre plates (Thermo Scientific, Roskilde,
Denmark) which were coated with 0.1 μg of BLG/well (Sigma, St Louis,
MO, USA). The antibody used for measuring IgA was biotinylated anti-
human IgA (Vector Lab, Burlingame, CA, USA) and the conjugate was
AP-streptavidin (Zymed, San Francisco, CA, USA). Fc fragment-specific
alkaline phosphatase-conjugated AffiniPure rabbit anti-human IgG
(Jackson Immunoresearch, West Grove, PA, USA) and Fast p-nitrophenyl
phosphate tablet (Sigma) were used for measuring IgG.
IgA and IgG against wheat gliadin were measured using ELISA modified
from Ludvigsson et al. (139). The coating of ELISA plates (MaxiSorp
Surface; Nunc-Immunoplate, Roskilde, Denmark) was performed with 1
58
μg of gliadin/well (Sigma). Biotinylated polyclonal goat anti-human IgA
(α-chain 62-8440) or IgG (γ-chain 62-7440) (Sigma) was used as
antibody, and AP-streptavidin (Zymed) as conjugate.
Residual coatings were performed with 1% HSA, serum samples were
diluted in 0.2% HSA-0.05% TWEEN20-PBS, and 0.05% TWEEN20/PBS
was used for washing the plates. Optical densities were read at 405 nm.
Standard curves were created using serial dilutions of pooled plasma
obtained from children with high levels of IgA or IgG antibodies against
BLG or gliadin. The results were presented as arbitrary units, which were
calculated from the standard curve on each plate. Detection limits were
2.5 or 15 arbitrary units for IgA or IgG antibodies against BLG,
respectively, and 10 or 6 units for IgA or IgG antibodies against gliadin,
respectively.
4.2.4 Measurement of fecal and breast milk calprotectin (Study IV)
Fecal samples in Study IV were stored at -20° C. The Calpro Extraction
Device (Calpro AS, Lysaker, Norway) was used for extracting samples. A
beaker in the Calpro Extraction Device was filled with thawed and
homogenized stool sample (∼100 mg). The extraction tube was filled
with 4.9 mL extraction buffer and vortexed for 30 s. The samples were
mixed using a shaker at 1000 rpm for 3 min or until only solid particles
remained, and centrifuged for 10 minutes at 10 000 g at room
temperature. Supernatants were collected, stored at -20° C, and
subsequently thawed for analysis in Study IV.
59
Fecal calprotectin levels were determined using ELISA (Calpro AS,
Lysaker, Norway) according to the manufacturer’s instructions. Fecal
extracts were diluted 1:50 in sample dilution buffer. The optical density
was read at 405 nm. Samples below the lowest measurable concentration
(39.2 μg/g) were given an arbitrary value of 19.6 μg/g.
The breast milk samples from the Finnish mothers were previously
processed in Study II. The supernatants were diluted 1:10 and analyzed
for calprotectin using ELISA (Calpro AS, Lysaker, Norway).
4.2.5 Pyrosequencing (Study IV)
In Study IV, total DNA was extracted from approximately 0.25 g fecal
sample using the repeated bead beating method modified from Yu et al.
(140). A Precellys 24 (Bertin Technologies, Montigny le Bretonneux,
France) was used for bead beating at 5.5 ms−1 in three rounds of 1 min.
The incubation temperature after the bead beating was increased to 95°C.
Protein precipitation with 260 μL ammonium acetate and elution of DNA
from the purification columns were conducted twice. Columns from the
QiaAmp Stool kit were replaced by columns from the QIAamp DNA
Mini Kit (Qiagen, Hilden, Germany).
16S rRNA genes were amplified from each sample using a primer set
corresponding to primers 27F-degS (141) and 534-R (142) that targets the
V1, V2, and V3 hypervariable regions of the 16S rRNA. 27-degS was
chosen because it seems to provide a more complete assessment of the
abundance of actinobacteria (141). Pyrosequencing of the PCR products
60
was performed with Roche FLX Genome Sequencer at DNAvision
(Liège, Belgium) according to their standard protocol (143).
Pyrosequencing produced 1,766,995 reads of 16S rDNA. Sequences were
assigned to samples according to sample-specific barcodes. SFF files
were converted into FASTA files and FASTA quality files, which
contained an average (± SD) of 14,725 ± 5,018 reads per sample. The
RDP pyrosequencing pipeline (144) (RDP 10 database, update 17) was
then used to check the FASTA sequence files for the same criteria as
previously described (143) and to confirm that the average experimental
quality score was at least 20. After the quality check, the FASTA files
contained an average (± SD) of 11,285 ± 4,102 high-quality reads.
The RDP classifier 2.01 (145) was used for the taxonomy assignment
(phylum, family, and genus level). ARB software and SSU reference
database (SSURef_111_SILVA_04_08_12) were used to identify the
species level (146,147). Only sequences of cultured and identified isolates
were used from the database. A “PT-server” database was built from
these sequences, and used to find the closest match for each of our high-
quality sequences imported from the FASTA files. Sequences from
different strains of the same species were grouped together. All identified
sequences, together representing 99.1 ± 1.04% of all high-quality FASTA
reads per sample, were included for statistical analysis.
4.2.6 Detection of IL-10 secreting cells (Study IV)
To study the effect of LPS on IL-10 secretion, peripheral blood
mononuclear cells (PBMC) from 3 healthy adult blood donors were
isolated using Ficoll-Paque solution (GE Healthcare, Uppsala, Sweden)
61
and stimulated with LPS from E. coli 0128:B12 (1µg/ml) for 12 hours in
CO2 at 37Cº. The concentration used was 106 cells/ml in 200 µl of media.
A negative control was PBMCs incubated in media (RPMI 1640 + 5%
human AB-serum+ 2mM glutamine+ Gentamycin). PBMCs were then
collected and washed with 0.5% BSA-PBS containing 2mM EDTA.
Identification of IL-10 secreting cells was performed using IL-10
secretion assay (Miltenyi Biotech, Germany). In short, washed PBMCs
were labelled with a catch reagent consisting of a cell surface specific
antibody (anti-CD45) conjugated with an anti-IL-10 antibody. The cells
were incubated in warm medium for the cytokine secretion to occur.
Secreted IL-10 was detected with a secondary PE-labelled anti-IL-10
antibody. FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes,
NJ, USA) was used for the detection of IL-10 secreting cells and their
characterization by side and forward scatter properties.
4.2.7 Statistical analyses
In Study I, statistical analyses were performed with PASW Statistics
18.0 (SPSS Inc., Chicago, IL, USA). The hs-CRP values (or the Log-
transformed values) were not normally distributed. The risk of AD and
asthma, and the risk of developing IgE-sensitization were analyzed by
logistic multivariate regression. The hs-CRP values were categorized into
quartiles. Odds ratios (OR) were adjusted for farming status, study center,
mother's history of allergic disease (No/Yes), and potential confounders,
which were at least marginally significantly (P<0.10) associated with hs-
CRP values in the univariate analysis, that is gender, number of siblings,
and day care with other children and siblings (No/Yes).
62
In the association analyses between hs-CRP values and environmental
factors, results were presented as median values with interquartile ranges.
Mann–Whitney U-test and the Kruskal–Wallis test were used for the
calculations of P-values for the comparisons between different categories
in the univariate analysis. For logistic multivariate regression analysis, the
hs-CRP values were divided into two groups using the 75th percentile
cut-off (0.19 mg/l). ORs were adjusted for farming status, study center,
and potential confounders.
In Study II, statistical analyses were performed using STATA/SE 12.1
(STATACorp, College Station, TX, USA), and P-values <0.05 were
considered statistically significant.
The concentrations of TGF-β1 and sIgA were categorized into quintiles
(Q1-Q5). Differences in maternal variables and the duration of
breastfeeding were tested using Pearson's chi-square test and the results
were expressed as P-values. Pearson's coefficient was used to express
correlations of continuous variables. TGF-β1 and sIgA values were log-
transformed for showing approximate normal distribution, and expressed
as geometric means and 95% confidence intervals. Environmental
exposures occurring up to the age of 2 months were related to TGF-β1
and sIgA levels by linear regression and expressed as geometric mean
ratios and 95% confidence intervals (CI). Factors that showed a
significant association with TGF-β1 or sIgA levels in univariate models
were entered in multivariate linear regression models.
63
In Study III, statistical analyses were performed with SAS 9.3 for
Windows (SAS, Cary, NC, USA). The association between levels of IgA
and IgG antibodies against BLG or gliadin and AD or asthma between the
ages of 1 and 6 years were analyzed by discrete time hazard models.
Logistic regression was used for analyzing the association between levels
of antibodies and IgE-sensitization.
The levels of IgA and IgG antibodies against BLG and IgG antibodies
against gliadin were categorized into quartiles (Q1-Q4) using 25th, 50th,
and 75th percentile cut-off points. IgA antibodies against gliadin were
categorized into three groups since the distribution was skewed.
The results were presented as ORs (95% CI) adjusted for farming status,
study centre, gender, mother's history of allergic disease, farm milk
consumption, and potential confounders, which were at least marginally
significantly (P<0.10) associated with BLG or gliadin concentrations in
the univariate analysis. In addition, the associations between antibody
concentrations and IgE-sensitization at the age of 6 years were also
adjusted for IgE-sensitization at the age of 1 year.
The results were given as median values with interquartile ranges in the
analyses of associations between IgA and IgG antibodies and
environmental and farming factors. P-values were calculated using the
Mann–Whitney U and the Kruskal–Wallis tests, and were considered
statistically significant below the value 0.05. For logistic multivariate
regression analysis, the 75th percentile cut-off point was used. ORs were
adjusted for farming status, study center, gender, farm milk consumption,
64
and potential confounders, which were at least marginally significantly
(P<0.10) associated with the antibody levels in the univariate analysis,
i.e., maternal smoking during pregnancy, duration of breastfeeding, age of
introduction of formula milk, and pets at home at the age of 1 year.
In Study IV, statistical analyses considering the calprotectin data were
performed with PASW Statistics 18.0 (SPSS Inc., Chicago, IL, USA).
The calprotectin values (or the Log-transformed values) were not
normally distributed. The results were presented as median values with
interquartile ranges. P-values for the comparisons between the categories
in univariate analysis were calculated with the Mann-Whitney U- and the
Kruskal-Wallis- tests. P-values <0.05 were considered statistically
significant.
Logistic multivariate regression was used to analyze the risk of AD and
asthma until the age of 6 years and the risk of IgE-sensitization at the age
of 6 years. A 90th percentile cut-off point was used to categorize the
calprotectin concentrations into two groups. All ORs were adjusted for
farming status, gender, and mother’s history of allergic disease, and with
potential confounders, which were at least marginally significantly
(P<0.10) associated with calprotectin in the univariate analysis.
Pyrosequencing data was analyzed with SPSS 20 (SPSS Inc, Chicago, IL,
USA). Principal component analysis was performed to visualize the
difference between atypical (diarrhea) samples and normal feces samples.
FlowJo software (Tree Star, Inc., Ashland, OR, USA) was used to analyze
flow cytometry data.
65
5 RESULTS AND DISCUSSION
5.1 Early-age inflammatory response and development of
IgE-sensitization (Study I)
The acute-phase protein CRP is a marker of inflammation. Low-grade
inflammation can therefore be measured using a hs-CRP ELISA method,
which was used in Study I. Previous studies have shown that treatment
with probiotics induced increased hs-CRP levels and this was associated
with a decreased risk of allergic diseases (148,149). These findings
suggest that inflammation triggered by environmental microbes could
mediate protection from the development of allergic immune responses in
children. Based on these findings, our aim was to test whether low-grade
inflammation, measured as elevated levels of hs-CRP at the age of 1 year,
provides protection from IgE-sensitization and allergic diseases later in
childhood. Because the presence of atopy at the age of 1 year could affect
hs-CRP levels, we stratified the children into IgE-sensitized and non-
sensitized children at the age of 1 year.
All children had hs-CRP values below 5 mg/l. Among the children who
were non-sensitized at the age of 1 year, elevated hs-CRP values
associated with decreased risk of IgE-sensitization at the age of 4.5 years.
That is, children who had the concentration of hs-CRP in the highest
quartile had a significantly lower risk of sensitization to any allergen
66
when compared to those with hs-CRP concentrations in the lowest
quartile (aOR 95% CI: 0.48 (0.24-0.95), also presented in Figure 4).
There was an inverse, dose-dependent trend among the children who were
non-sensitized at the age of 1 year, i.e. the higher the hs-CRP value at the
age of 1 year, the lower the risk for IgE-sensitization at the age of 4.5
years (P=0.060). There was a similar trend in the whole cohort of children
(P=0.077). No association of hs-CRP and later sensitization was seen in
the children who were already sensitized at the age of 1 year.
No significant association of hs-CRP and allergic diseases was found
either in the stratified group or in the whole cohort. However, the number
of children who developed AD and asthma was low, making it difficult to
draw conclusions regarding the relationship between hs-CRP values and
allergic diseases.
Since there is evidence that a farming environment protects against
allergic diseases (52,150), we evaluated whether farm-associated
environmental factors indicating increased microbial load were associated
with low-grade inflammation as measured by hs-CRP levels. No
associations were seen between environmental factors and hs-CRP levels
among non-sensitized children (Table 1). Therefore, this study found that
a farming environment did not show significant effects in inducing
systemic low-grade inflammation.
The early IgE-response to allergens does not necessarily imply allergic
disease, but may signal a physiological response to environmental
antigens in infants (151). When sensitization was observed separately
67
from hs-CRP levels, the children who had developed IgE-sensitization at
the age of 1 year had a significantly higher risk of allergic sensitization at
the age of 4.5 years (aOR 95% CI: 2.01 (1.39–2.90), P<0.001) and tended
to have a higher risk of AD and asthma/asthmatic bronchitis (aOR 95%
CI: 1.57 (0.96–2.56), P=0.072 and aOR 95% CI 1.55 (0.97–2.48),
P=0.069, respectively) between the ages of 1 and 4 years when compared
to the non-sensitized children.
Thus, the children who had developed IgE-sensitization at the age of 1
year represented the high allergy risk population. In these IgE-sensitized
children, having dogs or both dogs and cats at home, drinking boiled or
both unboiled and boiled farm milk, or having a mother who had visited
stables weekly during pregnancy or during the last 10 months implied
significantly lower hs-CRP values compared with children who did not
encounter these exposures. These exposures are considered to associate
with high microbial load. Thus, it was interesting that an association with
low, rather than the expected high, hs-CRP values was found in the IgE-
sensitized children.
68
Non-sensitized at age 1 year IgE-sensitized at age 1 yearN hs-CRP* aOR# N hs-CRP* aOR#
Farm milk consumption of the childNo 311 0.06 (0.04/0.14) 1 114 0.07 (0.04/0.30) 1Only boiled 67 0.06 (0.04/0.26) 1.14 (0.58-2.22) 29 0.06 (0.04/0.08) 0.16 (0.04-0.60)Both unboiled and boiled 34 0.06 (0.03/0.37) 1.16 (0.48-2.81) 18 0.06 (0.04/0.14) 0.18 (0.04-0.85)Only unboiled 41 0.07 (0.05/0.20) 1.29 (0.57-2.91) 12 0.05 (0.03/0.18) 0.41 (0.09-1.97)P-value 0.928 0.581 0.283 0.026Mother staying in stable (weekly) during pregnancyNo 180 0.06 (0.04/0.13) 1 60 0.07 (0.05/0.40) 1Yes 279 0.07 (0.04/0.20) 1.27 (0.69-2.33) 115 0.06 (0.04/0.29) 0.24 (0.07-0.79)P-value 0.183 0.444 0.129 0.019Mother's staying in stable weekly in last 10 monthsNo 257 0.06 (0.04/0.14) 1 86 0.07 (0.04/0.28) 1Yes 202 0.07 (0.04/0.19) 0.88 (0.32-2.46) 89 0.06 (0.04/0.20) 0.13 (0.02-0.96)P-value 0.322 0.808 0.675 0.045Having pets at home (1year of age)No cats or dogs 197 0.06 (0.04/0.13) 1 64 0.07 (0.04/0.40) 1Only cat/cats 123 0.08 (0.04/0.23) 1.40 (0.76-2.58) 44 0.07 (0.04/0.30) 0.68 (0.24-1.93)Only dog/dogs 43 0.06 (0.05/0.10) 1.11 (0.44-2.76) 21 0.06 (0.05/0.10) 0.15 (0.04-0.62)Both cat(s) and dog(s) 89 0.07 (0.04/0.19) 1.33 (0.65-2.74) 44 0.06 (0.03/0.12) 0.22 (0.07-0.72)P-value 0.22 0.548 0.336 0.003*hs-CRP values are presented as median values with interquartile range. P-value estimated by Mann-Whitney or Kruskal-Wallis test.# hs-CRP values are presented in two groups according to 75th cut-off points. The comparison between groups are shown values as odds ratios (OR) with their 95% confidence intervals(CI). P-values are obtained from the trend test (Wald) in logistic regression models. Odds ratios are adjusted for gender, country, farming, siblings and day care with other children thansiblings.Statistically significant adjusted aORs are marked in bold
Table 1. The associations between environmental exposures and hs-CRP levels measured at the age of 1year in non-sensitized and IgE-sensitized children.
69
Microbial exposure may play an important role in the establishment of
effective regulatory networks during the development of the immune
system in infancy. In environments with a higher microbial load, episodes
of inflammation are more common (Figure 3). Nevertheless, adults living
in developing countries have lower baseline hs-CRP levels when
compared to adults living in more hygienic countries (152). Repeated
activation and deactivation of inflammation may promote the
development of more efficient regulatory pathways, which can effectively
turn inflammation on and off when needed. In the case of a successful
establishment of the regulatory pathways, inflammatory stimuli in
adulthood are processed similarly, i.e. inflammatory responses increase
quickly, and anti-inflammatory processes control the responses. In
hygienic environments, the low microbial exposure can cause the
inflammatory reactions in infancy to be less often activated and
deactivated which may lead to a more pro-inflammatory phenotype.
When inflammatory stimuli are encountered later in adulthood,
inflammation can be activated but the deactivation by anti-inflammatory
regulatory mechanisms is not sufficient to prevent the development of
excessive or chronic inflammation (153). Similarly, in IgE-sensitized
children, it is possible that continuous exposure to microbial stimuli has
resulted in more tightly regulated inflammatory pathways and this is seen
in the low hs-CRP values in the children exposed to high microbial load.
70
Figure 3. A hypothesis of the association between environmentalmicrobial exposure in infancy and regulation of inflammation inadulthood. The arrow from infancy through adulthood represents time.The upper section represents environments with higher microbialexposures; the lower describes highly hygienic environments and lowinfection. (Adapted from McDade 2012.)
It is also possible that the level of hs-CRP reflects not only the microbial
exposure, but also the individual ability to respond to diverse
inflammatory stimuli. The children with early IgE-sensitization, who also
showed a high risk of allergies later in their life, may not have developed
regulation of the inflammatory response due to the repeated stimuli but
originally reacted to the microbial exposure by an impaired inflammatory
response. In the absence of proper inflammatory response, which would
71
be protective from the IgE-sensitization, they were more susceptible to
IgE-sensitization. It is intriguing to hypothesize that a proper
inflammatory response to microbial stimuli is needed for the regulation of
allergic immune response and IgE-production. This view is supported by
our findings showing that 1) increased hs-CRP levels at one year of life
decreased the risk of IgE-sensitization in the children who were not
sensitized at the time of hs-CRP measurement, i.e. 1 year of life; and 2) in
the IgE-sensitized children decreased hs-CRP values are seen in those
children exposed to microbial stimuli.
Therefore, the hs-CRP concentration as a marker of low-grade
inflammation could at least be partially explained by diverse responders
(good vs. poor, Figure 4), and not necessarily only by environmental
microbial exposure. An association between impaired epithelial cell
response to environmental antigens and asthma has been reported (154).
Therefore, the poor response of the innate immune system, which is also
reflected in an impaired induction of hs-CRP, could be a determinant for
allergen-specific immune response later in life. Our findings are in
accordance with these observations and suggest that the induction of
inflammation plays a significant role in the controlling of IgE-responses.
However, the people with poor inflammatory responses may benefit from
treatments that enhance inflammation, such as shown for certain probiotic
preparations (148), especially in environments where the inflammatory
pressure is low. The impaired capability for inflammatory response may
be sufficient protection against allergies in environments with high
72
microbial load, but could lead to allergic immune responses in highly
hygienic environments.
The impaired inflammatory response to environmental triggers of
inflammation may be a fundamental characteristic of the hosts' immune
response, which may predispose to allergy development. In the children
who had an allergic response at 1 year of age, it could be a factor
associated with genetic or epigenetic variation. The production of acute
phase proteins, such as CRP, is controlled by several cytokines released
during the inflammatory process such as IL-1 and IL-6, which involve
STAT3 and NF-κB signaling (155).
The poor response to known inflammatory determinants of the host at an
early age appeared to be a key factor in controlling IgE-sensitization later
in life. Our results suggest that the role of innate immunity and
inflammation in the regulation of allergic immune responses and thus
predisposition to IgE-mediated allergy is important. Our results support
the earlier findings of the protective effect of low-grade inflammation in
the control of IgE-mediated allergic diseases (148).
73
Figure 4. Summary of findings and discussion in Study I. Non-sensitizedand IgE-sensitized children at the age of 1 year show divergentassociations between hs-CRP concentrations at the age of 1 year and IgE-sensitization at the age of 4.5 years. “Good responders” indicate childrenwho can, as response to microbial load, induce hs-CRP production whichassociates with decreased risk of IgE-sensitization. “Poor responders”indicate children who have impaired ability to produce hs-CRP.
5.2 Soluble immunoglobulin A in breast milk decreases the
risk of atopic dermatitis in the child (Study II)
The previous studies of the effects of breastfeeding on allergy
development have shown discrepant findings. The aim of our study was
to observe the effect of breast milk components sIgA and TGF-β1, the
duration of breastfeeding, and the estimated total amount of sIgA or TGF-
β1 (termed the “dose” of ingested component) during the first year of life
to the development of IgE-sensitization, asthma, and AD in the child. The
main immunoglobulin in breast milk is sIgA, which protects the infant
from infections and may also prevent excessive uptake of foreign antigens
across the mucosa, therefore possibly decreasing the risk of allergic
sensitization (156). The cytokine TGF-β1 regulates cell growth through
74
exerting both stimulatory and inhibitory effects on a variety of cell types.
TGF-β activates Tregs by inducing the expression of forkhead box protein
3 (157). TGF-β increases the secretion of IgA in B cells by inducing class
switch (158,159). IgA works in conjunction with TGF-β and other
cytokines which are suggested to affect oral tolerance development (160).
In addition, it has been shown in animal models that mice with TGF-β1
deficiency develop inflammatory autoimmune diseases (161).
Our results showed that sIgA levels in breast milk measured 2 months
after delivery were inversely related to AD up to the ages of 2 and 4 years
(Table 2). The association was strongest up to the age of 2 years, and
followed a dose response (P-value 0.005 for adjusted linear trend). After
adjusting for all potential confounders, the findings remained significant.
The infants who were breastfed for a shorter period of time tended to have
an increased risk of AD.
75
Table 2. The adjusted association of sIgA concentrations in breast milk 2months after delivery and atopic dermatitis in the child up to the ages of 2and 4 years. Quantile 5 represents the highest levels of sIgA.
Atopic Dermatitis
IgA in Quantiles
(Q)
N Up to age 2 years
aOR (95% CI)
Up to age 4 years
aOR (95% CI)
Q1 124 1.00 1.00
Q2 123 0.67 (0.35-1.29) 0.69 (0.37-1.31)
Q3 123 0.44 (0.22-0.88) 0.62 (0.32-1.18)
Q4 123 0.43 (0.22-0.85) 0.46 (0.24-0.88)
Q5 117 0.41 (0.20-0.85) 0.60 (0.31-1.17)
Statistically significant adjusted aORs are marked in bold. Logistic regression modelsadjusted for center, sex, farming, maternal history of allergies, and food score in the firstyear of life.
The dose of breast milk sIgA or TGF- β1 ingested by an infant during the
first year of life was estimated by multiplying the breast milk sIgA or
TGF- β1 level by the duration of total breastfeeding. The dose of sIgA
was inversely associated with AD up to the ages of 2 and 4 years (aOR
95% CI: 0.74 (0.55–0.99) and 0.73 (0.55–0.96), respectively). Figure 5
shows the adjusted inverse association of sIgA dose and AD up to the age
of 2 years. The dose of TGF-β1 was not significantly associated with AD
up to the ages of 2 or 4 years (aOR 95% CI: 0.86 (0.65–1.14) or 0.83
(0.63–1.08), respectively).
76
Although we only had one measurement at 2 months, the concentrations
are unlikely to change during lactation. It has been shown that the values
of breast milk sIgA drop 10 days after birth and then remain relatively
stable (162). The levels of TGF-β have been shown to be relatively
constant at least for the first 3 months after birth (163). The effect of sIgA
dose to AD remained significant despite the possible variations in the
number of feeding times per day. Soluble IgA or TGF-β1 levels in breast
milk were not related to asthma or IgE-sensitization.
Figure 5. Association of the dose of sIgA during the first year of life andthe probability for atopic dermatitis up to 2 years of age. Adjustedassociation adjusted for center, sex, farming, maternal history of allergies,and food score in the first year of life.
77
The duration of breastfeeding was not associated with farming or any
other environmental factor tested. Mothers with predisposition for allergic
diseases tended to breastfeed for a longer period of time (over 6 months)
when compared to mother with no predisposition. Children who had not
been breastfed did not have an increased risk of developing AD, asthma
or IgE-sensitization. The children who had maternal history of allergic
diseases were at a higher risk of asthma at 6 years of age if they were
breastfed (exclusively or partially) for an extended period of time. This
was evident when the lowest and the highest quartile of breastfeeding
duration were compared (aOR for the interquartile range of duration of
breastfeeding; 95% CI: 2.34 (1.05–5.21)). The levels of TGF-β1 and sIgA
were significantly higher in the breast milk of mothers who went on to
breastfeed for a shorter duration of time (P-value for adjusted linear trend
<0.001).
Although there was no association with farming, the levels of breast milk
sIgA were associated with several environmental factors in unadjusted
analyses including time spent in a barn (more than 5 hours per week; aOR
95% CI: 1.21 (1.06–1.39)) and contact to one or two species of farm
animals (aOR 95% CI: 1.10 (1.01–1.20)) during pregnancy.
The association was regarded as strong when it remained significant in
adjusted analyses. After mutual adjustment, sIgA levels were elevated in
the breast milk of mothers who had been in contact with cats during
pregnancy (aOR 95% CI: 1.01 (1.01–1.20), smoked during pregnancy
(1.22 (1.04-1.43)) or at the time of sample collection (1.40 (1.17-1.69)),
and in the mothers who already had one child (1.11 (1.01-1.22)). These
78
factors could represent increased microbial exposure in the environment
leading to the stimulation of the maternal immune system and increased
sIgA in the breast milk. It is therefore possible that sIgA levels in breast
milk are a signal of the environmental microbial load, which modifies the
development of allergic diseases, such as AD. Asthma and breast milk
sIgA did not show any association, suggesting a specific effect on AD.
The mechanism mediating the association of sIgA and AD is not known.
Soluble IgA-mediated passive antimicrobial protection could influence
gut normal flora colonization or affect antigen transport across the
mucosa (40). Soluble IgA might also be a signal of other milk
components that affect the development of adaptive immune responses or
are involved in creating a microenvironment that promotes Treg
development (164).
While we reported an inverse association with AD and sIgA in breast
milk, there are other studies that did not find IgA levels in breast milk to
be associated with atopic diseases such as AD (165,166). It has been
shown that elevated levels of breast milk sIgA, measured three weeks
after delivery, were associated with a reduced risk of asthmatic symptoms
in the first year of life (45). A study by Savilahti et al. showed that low
cow’s milk specific IgA levels in the colostrum associated with a
significant risk of developing atopic symptoms and IgE-sensitization at
the age of 4 years if the infant was exclusively breastfed for longer than
3.5 months (44). Interestingly, we observed that long breastfeeding was
associated with an increased risk of asthma in children of mothers with
allergic history. Matheson et al. also reported of studies where
79
breastfeeding was associated with an increased risk of asthma later in
childhood (40), as discussed earlier in this thesis. Nevertheless, there are
studies showing prolonged breastfeeding to be either protective or a risk
factor of AD (39,167). We did not see any associations between TGF-β1
and atopic outcomes in the child which can be due to the low
concentrations of breast milk TGF-β1 in this cohort (168).
Mothers who smoked during pregnancy had increased levels of breast
milk sIgA and TGF-β1. Cigarette smoke contains toxins and microbial
cell components, which can promote inflammation at mucosal surfaces,
promote airway remodeling, and influence immunity through unknown
mechanisms. It has been suggested that cigarette smoke promotes allergic
inflammation by enhancing the activation of DCs, which transport inhaled
antigens to lymph nodes (169). In addition, smokers may have more
infections (170), which can contribute to the increased microbial load,
thereby increasing the levels of sIgA in breast milk. Similar mechanisms
might be behind the increased levels of breast milk TGF-β1 in mothers
who smoke.
We found that elevated levels of sIgA or TGF-β1 in breast milk indicated
a short duration of breastfeeding, which has previously been reported in
colostrum by Savilahti et al. (171). Animal studies have shown bacteria-
induced mastitis to increase the levels of TGF-β1 in breast milk
(172,173). TGF-β has also been shown to regulate mammary gland
development by inhibiting alveolar and ductal development in breast
tissue (174). Mastitis is often caused by Staphylococci. The subclinical
infection is common in dairy cows, with estimated prevalence of 5.5-
80
27.1% (175). Acute mastitis is reported to occur approximately in 10% of
breastfeeding mothers (176). As the subclinical mastitis can occur without
symptoms, the prevalence in humans is not well known. In the present
study, the increased TGF-β1 and sIgA levels in breast milk can indicate
microbial stimulus and the association with short breastfeeding may be
due to subclinical inflammation in the breast tissue. Interestingly, an
inverse association of sIgA and AD was observed regardless of the
simultaneous association of elevated sIgA and shorter duration of
breastfeeding.
Our results suggest that high sIgA levels in breast milk reduce the risk of
developing AD at early age and, therefore support the protective effects
of breastfeeding on atopic diseases and emphasize that the effect is
dependent on breast milk composition.
5.3 Early-age antibody response to food antigens associates
with IgE-sensitization later in life (Study III)
Dietary antigen exposure is necessary for the development of a functional
immune response and the development of oral tolerance in the gut.
Intestinal permeability is higher in infants when compared to adults and
children, which can be necessary for the development of tolerance.
Excessive intestinal permeability or inflammation may interfere with the
development of oral tolerance and lead to increased antibody response.
Cow's milk, hen’s egg, and wheat are among the most common allergens
in children. We used serum IgA and IgG antibodies against cow’s milk
81
BLG and wheat gliadin (at the age of 1 year) as markers of mucosal
tolerance development and studied their association with environmental
factors, IgE-sensitization, and the development of asthma and AD.
Our results show that increased levels of IgA or IgG antibodies against
BLG were associated with IgE-sensitization at 1 year of age (Table 3).
Children who had IgA antibody concentrations against BLG in the
highest quartile or who had increased IgG antibodies against BLG were at
a significantly increased risk of being sensitized to at least one of the
measured allergens (Fig. 6 A and B) and food allergens at the age of 6
year (Table 4).
Increased levels of IgA or IgG antibodies against gliadin were associated
with IgE-sensitization at the age of 1 year (Table 3). Children who had
increased IgG antibody levels against gliadin were at a higher risk of
being sensitized to at least one of the allergens (Fig. 6 C), inhalant
allergens (IgG in Q4: aOR 95% CI: 2.15 (1.16–3.98)), and food allergens
at the age of 6 years (Table 4).
We did not find consistent associations between the antibody levels and
asthma or AD. No association with hs-CRP at the age of 1 year and IgA
or IgG against BLG or gliadin was found in 640 sample pairs (data not
shown).
82
Figure 6. A) The association of IgA levels against cow’s milk β-lactoglobulin at 1 year of age and IgE-sensitization to any allergen at 6years of age. B) The levels of IgG against β-lactoglobulin at 1 year of ageand the risk of IgE-sensitization to any allergen at 6 years of age. C) Thelevels of IgG against gliadin at 1 year of age and IgE-sensitization to anyallergen at 6 years of age. Antibody levels expressed as quartiles, Q4being the highest.
83
Table 3. Association of IgA and IgG antibodies against milk ß-lactoglobulin or wheat gliadin at the age of 1 year and IgE-sensitization atthe age of 1 year.
IgA(BLG)
Sensitizationto at least one
allergen atage 1y N(%)
aOR (95% CI)1 IgG(BLG)
Sensitizationto at least one
allergen atage 1y N(%)
aOR (95% CI)1
Q1 29 (18.2) 1 Q1 36 (22.6) 1
Q2 44 (27.5) 1.80 (1.03-3.16) Q2 38 (23.8) 1.09 (0.63-1.87)
Q3 47 (29.4) 2.28 (1.28-4.06) Q3 43 (27.0) 1.38 (0.79-2.40)
Q4 56 (35.2) 2.97 (1.64-5.37) Q4 59 (36.9) 2.36 (1.33-4.19)
IgA(Gliadin)
IgG(Gliadin)
Q1+Q2 74 (22.8) 1 Q1 35 (22.0) 1
Q2 40 (25.2) 1.37 (0.79-2.38)
Q3 45 (29.4) 1.34 (0.85-2.11) Q3 42 (26.4) 1.52 (0.89-2.62)
Q4 56 (35.4) 1.86 (1.20-2.90) Q4 58 (36.7) 2.29 (1.34-3.91)
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Table 4. Predictive value of IgA and IgG antibodies against milk β-lactoglobulin or wheat gliadin at the age of 1 year and IgE-sensitization toat least one food allergen at the age of 6 years.
IgA(BLG)
Sensitizationto at least
food allergenat age 6y
N(%)
aOR (95% CI)1 IgG(BLG)
Sensitizationto at least onefood allergen
at age 6yN(%)
aOR (95% CI)1
Q1 31 (24.8) 1 Q1 26 (22.0) 1
Q2 37 (30.8) 1.58 (0.85-2.91) Q2 42 (34.7) 2.07 (1.12-3.82)
Q3 38 (34.2) 1.71 (0.90-3.25) Q3 35 (31.8) 1.95 (1.00-3.78)
Q4 51 (49.5) 3.64 (1.84-7.17) Q4 54 (49.1) 4.85 (2.41-9.76)
IgA(Gliadin)
IgG(Gliadin)
Q1+Q2 72 (30.5) 1 Q1 21 (19.6) 1
Q2 43 (35.0) 2.06 (1.09-3.89)
Q3 36 (35.3) 1.18 (0.70-1.99) Q3 38 (32.5) 1.75 (0.92-3.33)
Q4 48 (40.3) 1.53 (0.92-2.53) Q4 54 (49.1) 4.13 (2.16-7.91)
Tables 3 and 4: Statistically significant adjusted ORs (aOR) are marked in bold.1Results are presented as odds ratios (OR) and their 95% confidence intervals (CI). ORs
are adjusted for farmer, center, gender, maternal smoking during pregnancy,breastfeeding, farm milk consumption, pets at home, mother's history of allergic disease,atopy at the age of 1 year, and age of formula milk introduction.
85
Specific immunoglobulin E (sIgE) was measured against 19 common
allergens. There were some possible weaknesses in the test panel. Some
allergens may not be feasible for the screening of atopy since the level of
exposure varies between persons, i.e. farmers’ children have more contact
to cows when compared to children who life in towns. Specific IgE
against cow was not measured in this study. However, IgE against horse
was measured in the test panel even though horse is a common farm
animal. In addition, variations in the quantity of certain allergens, such as
plant pollen and house dust mite, in different parts of Europe might also
affect the findings. It has been suggested that epidemiological studies
should use allergens that all of the study subjects are exposed to in order
to measure the genetic predisposition to develop IgE-sensitization (177).
Nevertheless, the panel of tested allergens was large and the country of
the child was taken into account in adjusted analyses. In addition to BLG
and gliadin, it would have been interesting to measure IgA and IgG
antibodies against hen’s egg ovalbumin, which is a common food allergen
in children.
The levels of IgA and IgG antibodies against BLG varied between the
study centers (P<0.001). Children who were introduced to formula milk
before 14.6 weeks had elevated levels of IgA or IgG against BLG at the
age of 1 year. (Table 5). The infants who were breastfed for longer than 6
months tended to have lower IgA and IgG antibody levels against BLG
than children who were not breastfed or who were breastfed for less than
6 months. A farming environment did not show association with antibody
levels. If the child consumed unboiled farm milk, the family had cats or
86
dogs at home, or if the mother smoked during pregnancy, there was a
tendency of association with increased levels of IgA and IgG antibodies
against BLG.
There are discrepant findings on breast milk promoting gut barrier
maturation (178,179). Breastfeeding may affect tolerance induction in the
infant by delivering antibodies and antigens derived from the maternal
diet. Long breastfeeding can lead to decreased antibody production
against BLG either by improving gut maturation or by postponing
introduction of cow's milk to the diet. Interestingly, children who
consumed formula milk during their first year of life had lower levels of
IgA against gliadin when compared to children who did not receive
formula milk (Table 6). This may be due to differences in the composition
of breast milk and formula milk.
There is evidence that increased intestinal permeability can result from
allergic inflammation in the gut (180,181). It has also been shown that
altered intestinal permeability can remain while the patient is on an
elimination diet, and that the symptoms correlate with measured gut
permeability (182). It is possible that environmental factors affect gut
permeability by causing intestinal inflammation, which can lead to food
allergies in people at high risk of allergies. Therefore, gut permeability
would not be the primary cause of allergies (183). Animal models have
shown that increased intestinal permeability can lead to increased antigen
uptake and IgE-sensitization (184). Therefore, our reported association of
increased IgA and IgG antibody response to gliadin and BLG with IgE-
87
sensitization can be an indication of increased intestinal inflammation and
permeability.
Table 5. Association between serum IgA and IgG antibodies against milkβ-lactoglobulin at the age of 1 year and the age of formula introduction todiet.
Table 6. Association between serum IgA and IgG antibodies againstwheat gliadin at the age of 1 year and the age of formula introduction todiet.
Tables 5 and 6: Statistically significant adjusted ORs (aOR) are marked in bold.aResults
are presented with median values and interquartile ranges. P-values are estimated byKruskal–Wallis test.
bP-values are estimated by Pearson chi-square test.
cIgA and IgG
values are presented in two groups according to their 75th cut-off points (Q4), andcomparison between two groups are shown values as ORs and their 95% confidenceintervals (CI). Odds ratios are adjusted for farmer, center, gender, maternal smokingduring pregnancy, breastfeeding, farm milk consumption, pets at home, and age offormula introduction.
Age of formulaintroduction to N(%)of N(%)of
diet (wk) N IgA (AU)a IgA in Q4b aOR (95% CI)c N IgG (AU)a IgG in Q4b aOR (95% CI)c
No milk formula 47 16.89 (10.00–43.41) 21 (44.7) 1 47 117.59 (33.04–283.93) 15 (31.9) 1
<14.6 279 10.00 (10.00–18.55) 60 (21.5) 0.39 (0.18–0.86) 279 108.27 (22.68–227.16) 65 (23.3) 0.86 (0.39–1.92)
14.6 ≤ and <22.1 112 10.00 (10.00–19.19) 24 (21.4) 0.36 (0.17–0.78) 112 94.79 (28.96–226.64) 26 (23.2) 0.78 (0.36–1.72)
≥22.1 190 10.00 (10.00–22.70) 51 (26.8) 0.46 (0.23–0.90) 190 86.68 (26.80–275.85) 49 (25.8) 0.79 (0.38–1.62)
P-value 0.032 0.905
Age of formulaintroduction to N(%)of N(%)of
diet (wk) N IgA (AU)a (BLG) IgA in Q4b aOR (95% CI)c N IgG (AU)a (BLG) IgG in Q4b aOR (95% CI)c
No milk formula 48 4.10 (2.50–16.60) 4 (8.3) 1 48 46.76 (29.93–88.31) 5 (10.4) 1
<14.6 280 36.08 (9.87–90.03) 106 (37.9) 5.16 (1.65–16.12) 280 325.35 (103.83–747.59) 112 (40.0) 3.68 (1.27–10.68)
14.6 ≤ and <22.1 112 14.79 (4.20–49.92) 24 (21.4) 2.96 (0.94–9.29) 112 145.70 (50.65–298.57) 17 (15.2) 1.32 (0.44–4.02)
≥22.1 191 7.87 (3.90–27.80) 23 (12.0) 1.41 (0.45–4.37) 191 72.86 (31.51–230.00) 24 (12.6) 1.16 (0.40–3.33)
P-value <0.001 <0.001
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We were not able to measure gut permeability in children who were 1
year of age. Therefore, we cannot correlate antibody levels and gut
permeability in this cohort. However, we reported that having pets at
home and drinking of unboiled milk were associated with increased levels
of dietary antibodies. Unboiled milk and animal contact associate with
high environmental bacterial load, which can indicate that intestinal
microbes enhance the antibody response.
IgG antibodies against foods have been shown to associate with IgE
antibody development against inhalant allergens in children with and
without allergy risk (185,186). Our results are in agreement with these
previous studies and show that enhanced antibody response to cow's milk
or wheat antigens can reflect altered intestinal immune response, which
associates with later sensitization to allergens. The findings in our study
suggest that mucosal antibody response in early life and IgE-sensitization
later in childhood are regulated by similar factors such as gut microbiota
and/or permeability. Since we did not see associations with mucosal
antibody response and allergic diseases later in life, it seems that other
factors are more significant in regulating the development of clinical
allergy.
5.4 High level of fecal calprotectin at 2 months as a markerof intestinal inflammation predicts atopic dermatitis andasthma by age 6 (Study IV).Alterations in gut microbiota and intestinal inflammation have been
linked to immune-mediated diseases (187). We used fecal calprotectin as
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a marker of early intestinal inflammation. We observed an association
between fecal calprotectin and asthma or AD development by the age of 6
years. There were 758 fecal samples from 2 month old children available
for the measurement of fecal calprotectin. The composition of fecal
microbiota using 16S rRNA pyrosequencing was analyzed in 120 infants.
In addition, the effect of E. coli LPS on the anti-inflammatory response in
human monocytes was also studied in three adults outside the PASTURE
cohort.
Farmers’ children had higher fecal calprotectin levels when compared to
non-farmer’s children (P=0.003). Also, increased levels of fecal
calprotectin were found in children with one or more siblings when
compared to the children with no siblings (P<0.001), and in breastfed
children (exclusively and partially breastfed) when compared to non-
breastfed children (P<0.001). Infants of non-smoking mothers had higher
levels of fecal calprotectin when compared to the infants of mothers who
smoked during pregnancy or who abstained from smoking during
pregnancy (P=0.003). Because fecal calprotectin was associated with
breastfeeding, we measured the levels of calprotectin in breast milk
samples from 115 Finnish mothers. There was no correlation between
levels of calprotectin in breast milk and feces (R=0.104 and P-value
0.271).
Since the distribution of fecal calprotectin levels was skewed, we studied
the importance of very high fecal calprotectin levels (above the 90th
percentile, i.e. 517.6 µg/g), which indicate a high degree of intestinal
inflammation. The children who had very high fecal calprotectin levels
90
were at an increased risk of developing AD and asthma/asthmatic
bronchitis by the age of 6 years when compared to the children who had
fecal calprotectin levels below the 90th percentile (Table 7).
We analyzed the composition of the gut microbiota in a selected subgroup
of children with high fecal calprotectin (above the 90th percentile) and
lower fecal calprotectin (below the 90th percentile) in order to study the
associations of fecal microbiota and fecal calprotectin. The selection
criteria are described in the study methods. Infants with very high fecal
calprotectin levels (above the 90th percentile) had a significantly lower
percentage of Escherichia in their fecal microbiota when compared to
infants with fecal calprotectin levels below the 90th percentile (P=0.019)
(Figure 8). Children with fecal calprotectin levels below 200µg/g had
significantly lower Lactobacillaceae numbers than children with fecal
calprotectin levels in between 200µg/g and the 90th percentile (517µg/g)
(P=0.016) or than children with fecal calprotectin above the 90th
percentile (P=0.011). However, the amount of Lactobacillaceae did not
significantly associate with the very high levels of fecal calprotectin (the
90th percentile) when compared to fecal calprotectin values below the 90th
percentile (P=0.125) (Figure 9).
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Table 7. Predictive value of fecal calprotectin at 2 months of age and development of atopic dermatitis,asthma/asthmatic bronchitis, and IgE-sensitization by the age of 6 years.
Fecal
calprotectin
(percentile)
Atopic
dermatitis
N (%)
aOR (95% CI) Asthma/asthm
atic bronchitis
N (%)
aOR (95% CI) Sensitization to at
least one allergen at
age 6y N (%)
aOR (95% CI)
<90 125 1 106 1 217 1
>90 19 2.02 (1.06-3.85) 18 2.41 (1.25-4.64) 29 1.50 (0.81-2.77)
P-value 0.066 0.033 0.022 0.009 0.182 0.197
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Figure 8. The association between fecal calprotectin concentrations andthe abundance of Escherichia in feces. Both parameters were measured at2 months of age.
Figure 9. The association between fecal calprotectin concentrations andthe abundance of Lactobacilliceae in feces. Both parameters weremeasured at 2 months of age.
93
The percentage of IL-10 secreting cells from monocytes was increased
after stimulation with LPS from E. coli (Figure 10). In person 1, the
frequency of IL-10 secreting cells from monocytes was 3.7 times higher
after LPS stimulation; in person 2, 6.9 times higher; and in person 3, 2.7
times higher when compared to the non-stimulated control sample.
Figure 10. The effect of E. coli derived LPS on the percentage of IL-10secreting cells in 3 adult blood donors.
Although there was no linear association of fecal calprotectin levels and
allergic disease, we found remarkably high levels of fecal calprotectin to
predict asthma and AD by the age of 6 years. These results indicate long
term effects of early gut immune system alterations on allergic diseases.
Our results suggest that a farming environment induces intestinal
inflammation, which can be caused by increased environmental microbial
load. However, the very high fecal calprotectin levels were not explained
by farming. Therefore, our finding does not contradict studies that show
farming to be protective against allergies. In addition, we showed that
fecal calprotectin did not directly derive from breast milk. Yet, the
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children who were exclusively breastfed had the highest levels of fecal
calprotectin, which implies that breastfeeding leads to a higher microbial
exposure than bottlefeeding. Breast milk bacteria (188) may lead to
increased fecal calprotectin levels by inducing neutrophil infiltration due
to the stimulation of the gut immune system. The growth of intestinal
microbes can also be modulated by breast milk factors such as
oligosaccharides.
Gut permeability and fecal calprotectin levels are high in infants. Until
recently, there have not been established cut-off levels to define elevated
fecal calprotectin levels as a marker of disease in infants and young
children. In 2014, Oord et al. (189) defined three cut-off levels based on
the 97.5% percentiles of fecal calprotectin in different age groups: 538
µg/g (1 < 6 months), 214 µg/g (6 months < 3 years) and 75 µg/g (3 < 4
years). The 90th percentile cut-off (517.6 µg/g) used in our study for 2
month old infants is comparable to the limits defined by Oord et al. A
reference value of 50 μg/g calprotectin in feces is used for children from 4
to 17 years of age and healthy adults in commercial ELISA tests (190). In
un-published data, we found that the PASTURE cohort children had
significantly lower levels of fecal calprotectin at 1 year of age when
compared to the levels at the age of 2 months. Other studies have shown
similar results (104,108).
In Study IV, the distribution of fecal calprotectin levels was skewed and
some of the infants had very high levels of calprotectin without a known
history of intestinal disease. Therefore, we decided to use the 90th
percentile cut-off, and found an association between high fecal
95
calprotectin and elevated risk of developing asthma/asthmatic bronchitis
and AD by the age of 6 years.
The gut microbiota findings in Study IV suggest down-regulation of
intestinal inflammation in infants who are colonized with Escherichia.
Continuous exposure to LPS has been shown to cause tolerance and
down-regulation of the TLR-4 signaling pathway (191). Therefore, the
early colonization of the gut with Escherichia may result in a down-
regulation of TLR-4 signaling and alleviation of the inflammatory
response, which was detected as lower levels of fecal calprotectin in our
study. TLR-4 is also expressed by Tregs (192), and early colonization
with Escherichia could alleviate inflammation via the initiation of
regulatory mechanisms. In addition, several studies demonstrate that
TLR-4 signaling attenuates allergic diseases and asthma (193-195). Our
findings in three adult blood donors showed that the production of IL-10
in monocytes was triggered by E. coli derived LPS and are in agreement
with previous studies (196). Thus, LPS induced IL-10 production could
offer an explanation for the decreased intestinal inflammation associated
with early Escherichia colonization in Study IV.
We reported that lower fecal calprotectin levels in infants were associated
with low numbers of Lactobacillaceae, which implies that early
colonization with Lactobacillaceae contributes to the levels of intestinal
inflammation. Lactobacilli have been shown to induce the pro-
inflammatory IL-12 and IFN- ɣ production in murine DC cultures (197)
and in human blood mononuclear cells (198,199). In addition, some
Lactobacilli can produce hydrogen-peroxide which may contribute to the
96
induction of intestinal inflammation (200). Although it has been shown
that the low-grade inflammation caused by a mixture of probiotics
(including L. rhamnosus GG and L. rhamnosus LC705) could be
beneficial against allergies (70), we observed an association between
early high degree intestinal inflammation and later development of
asthma. However, we did not find the fecal calprotectin values above the
90th percentile to significantly associate with Lactobacillaceae
abundances, suggesting that Lactobacillaceae alone do not cause the very
high inflammation.
Our findings suggest that early intestinal inflammation can indicate both
skin related inflammation and respiratory tract related inflammation later
in life. The colonization pattern appeared to be an important regulator of
the intestinal inflammation. Our finding that early colonization with
Escherichia alleviated the intestinal inflammation may have implications
for the design of probiotic treatments for infants. Our findings also
suggest that early colonization is associated with long-term health effects.
97
6 CONCLUSIONS
The following conclusion can be drawn from the results presented in this
thesis:
I. The findings suggest that low-grade inflammation at an early age
associates with decreased allergic sensitization later in life and that poor
inflammatory response could predispose for IgE-sensitization.
II. The study showed an inverse association between breast milk sIgA
levels and the risk of AD later in childhood. The results support the
protective effects of breastfeeding on AD development and suggest that
the effect is dependent on breast milk composition.
III. The results suggest that enhanced antibody responses to wheat or
cow’s milk antigens reflect aberrancies in mucosal tolerance such as
altered microbiota and/or increased gut permeability, which is later seen
as sensitization to allergens.
IV. The present study showed that very high intestinal inflammation in
infancy is a risk factor for the development of allergic diseases later in
life. The results also suggest that early intestinal colonization regulates
intestinal inflammation.
Children who live in a farming environment are generally exposed to a
higher number and diversity of environmental microbes, and they have
been reported to have fewer allergies. The results presented in this thesis
98
showed that farmers’ children had increased levels of fecal calprotectin
indicating increased intestinal inflammation. Although a farm
environment did not constantly associate with altered immune response in
the present studies, we reported several associations between factors
reflecting elevated environmental microbial load and altered immune
response in the child or in the mother. In addition, low-grade
inflammation was shown to have a protective role against IgE-
sensitization.
The findings presented in this thesis suggest that gut alterations and
bacterial colonization play a significant role in the development of the
immune system. Our study provides new a perspective on allergy-
development by showing that very high intestinal inflammation in early
infancy associates with increased risk of allergic diseases. These results
contribute to the growing understanding of the interaction between early
life microbial exposure and immune system developments. Further
developments in this field might shed new light on potential avenues for
the prevention or treatment of allergic diseases.
99
7 ACKNOWLEDGEMENTS
The work in this thesis was carried out between 2008 and 2015 first in the
Department of Viral Diseases and Immunology at the former National
Public Health Institute, continued in the Department of Vaccines and
Immune Protection at the National Institute of Health and Welfare, and
finalized in the Scientific Laboratory of Children’s Hospital, University
of Helsinki and Helsinki University Hospital.
I wish to thank Professor Pekka Puska, the former head of the National
Institute of Health and Welfare and Professor Juhani Eskola, the present
director of the National Institute of Health and Welfare. I would also like
to thank the former heads of the Department of Vaccines and Immune
Protection, Professor Terhi Kilpi and Professor Outi Vaarala, and the
head of the Scientific Laboratory, Professor Mikael Knip, for providing
excellent research facilities.
I am thankful to Professor Erika von Mutius, the coordinator of the
PASTURE project, and all the members of the PASTURE study group in
Finland and abroad. I owe my gratitude to the parents and children
participating in the PASTURE study. Without their willingness to
participate over the years this type of research would not be possible.
I am deeply grateful for my supervisor, Professor Outi Vaarala, for
teaching me how to be a scientist. Her guidance, positive attitude and
encouragement have been invaluable.
100
I want to thank Professor Juha Pekkanen for introducing me to the
PASTURE study group and providing guidance in my work.
I sincerely thank the official reviewers of this thesis, Docent Petri
Kulmala and MD, PhD Marita Paassilta for their review and comments
for improving my thesis.
I would like to thank PhD Jarno Honkanen, the former head of Immune
Response Unit, for the encouragement and support over the years.
I am thankful for the advice considering the PASTURE project that I have
received from Docent Anne Hyvärinen, Anne Karvonen, and Maria
Valkonen. I want to thank all the co-authors for the collaboration. I owe a
special thanks to Kirsi Mustonen, Marcus de Goffau, and Georg Loss for
performing the statistical analyses in the publications, and the willingness
to answer my numerous e-mails. I would like to thank Professor Hermie
Harmsen for collaboration and for allowing me to visit his laboratory, and
Marcus for teaching me. I want to thank Saara Hakala for the calprotectin
measurements, Pirjo Mäki for the serum sample measurements, and
Monika Paasela for the flow cytometry analyses.
I would like to thank all the former and present colleagues in our
laboratory: Jarno Honkanen for the inspiring discussions and support
when writing has felt challenging, Terhi Ruohtula and Linnea Hartwall
for sharing not just an office but good ideas as well, Harri Salo for the
friendly advice over the years, Saara Hakala and Anna Kallio for the
memorable moments in the laboratory and for staying in contact after
leaving for new opportunities, Kristiina Luopajärvi and Veera Hölttä for
101
the advice and enjoyable discussions over the coffee breaks, Hanna
Laitinen, Janne Nieminen, Paula Klemetti, Nina Ekström, Merit Melin,
Arja Vuorela for their direct and indirect contribution to this thesis. I
would also like to express my deep gratitude to Tarja Alander for the
administrative assistance and friendship over the years, Maria Kiikeri,
Sinikka Tsupari, Pirjo Mäki, Anneli Suomela, Kaisa Jousimies, Camilla
Virta, Hannele Lehtonen, Teija Jaakkola, and Leena Saarinen for
technical assistance and companionship in our laboratory.
My gratitude goes to all my friends for listening and giving support,
especially to Zanki and Liisa.
I would like to express my gratitude to my mother Hannele. Without her
help over the years, I could not have finished this thesis. I would also like
to warmly thank my brother Eero, Simo, Pekka, Sara, and my
grandparents Sinikka, Jussi, and Ritva for the encouragement. I especially
thank my son Anton who has given me so many other things to think
about.
This study was financially supported by the Sigrid Juselius Foundation.
Helsinki, July 2015
Laura Orivuori
102
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