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.

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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

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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.

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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

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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.

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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.

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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.

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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).

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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

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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-

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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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