i L ink ö ping U niv ers ity M ed ic al D is s ertatio n
N o . 15 8 9
I mmuno mo d ulato ry ef f ec ts o f pro b io tic
s upplementatio n d uring pregnanc y and inf anc y
in allergy prev entio n s tud ies
A nna Fo rs b erg
D epartment o f C linic al and E x perimental M ed ic ine Fac ulty o f M ed ic ine and H ealth Sc ienc es , L ink ö ping U niv ers ity
SE - 5 8 1 8 5 L ink ö ping, Sw ed en
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©Anna Forsberg
Front cover: Coloured scanning electron micrograph of Lactobacillus reuteri
(blue) binding to the mucus layer (green) of the human small intestine. Alistair
Walsham & Stephanie Schüller
Published articles I-II have been reprinted with permission of the copyright
holders
Printed in Sweden by Liu-Tryck, Linköping, Sweden, 2017
ISBN: 978-91-7685-443-3
iii Life isn’t about waiting for the storm to pass… It’s about learning to dance in the rain
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Populärvetenskaplig sammanfattning
Förekomsten av allergiska sjukdomar ökar och nya metoder för att förebygga allergier behöver utvecklas och införas kliniskt. Mer kunskap om immunologiska mekanismer är nödvändigt att införskaffa för att identifiera allergiförebyggande strategier. Vi har använt oss av probiotika (en mjölksyreproducerande bakterie kallad Lactobacillus reuteri) för att se om en ökad tillförsel av en normalflorebakterie kan påverka allergiutvecklingen hos barn. En extra tillförsel av probiotika skulle kunna tänkas öka stimuleringen av immunsystemet och påverka tarmfloran på ett hälsosamt sätt. Miljön under graviditet har visat sig vara betydelsefull för utvecklingen av barnets immunförsvar, då både epidemiologiska studier och experimentella djurmodeller visar att bakteriell exponering har allergiförebyggande effekt.
Vi har gett probiotika-tillskott till gravida mödrar från graviditetsvecka 36 och barnen har sedan under första levnadsåret fått samma bakterier som mamman eller placebo som kontrolltillskott.
Blodprover har samlats in från olika tidpunkter under barnets första två år, för att kunna analysera hur probiotika-tillförseln har påverkat immunsystemets utveckling. Vid två års ålder hade de barn som fått den aktiva substansen mindre allergiskt eksem än kontrollgruppen. Våra analyser av stimulerade celler visar att barn som fått probiotika har ett mer reglerat immunsvar. Detta har vi mätt genom att analysera signalsubstanser som olika celler i immunsystemet producerar när de stöter på allergener eller mikrobiella produkter. De barn som fått det aktiva tillskottet verkar ha ett mer reglerbart immunsvar än övriga barn. Vi observerade även att probiotika verkar kunna påverka det maskineri som styr hur våra gener läses, s.k. epigenetiska förändringar.
Vi har även påbörjat en uppföljningsstudie där studietillskottet, i form av samma probiotika samt ω-3 fettsyror, ges tidigare under graviditeten för att möjligtvis kunna ge en starkare allergiförebyggande effekt. Vi har observerat vissa immunmodulerande effekter av tillskotten på mammans immunsvar under graviditeten.
Sammanfattningsvis verkar probiotika påverka immunsystemet hos barn och gravida på flera olika sätt. Mer forskning behövs dock för att bättre förstå hur probiotika modulerar immunsvar så att mer effektiva allergiförebyggande strategier kan identifieras i framtiden.
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Abstract
The incidence of allergic diseases is increasing, possibly due to a reduced intensity and diversity of microbial stimulation. More knowledge is needed on the immunological mechanisms underlying the eczema preventive effect of pre- and postnatal probiotic supplementation. The pregnancy period seems to be of essential importance, since both epidemiological and experimental animal studies show the importance of microbial exposure during gestation on allergy prevention.
We have performed a study where the probiotic lactic acid producing bacteria Lactobacillus
reuteri was supplemented to pregnant women, at risk of having an allergic infant. The
pregnant mothers received the study product from gestational week 36 until delivery, and the infants then continued with the same product until one year of age. The probiotic, as compared with placebo, supplemented infants had less IgE-associated eczema at two years of age.
In order to investigate how the supplementation affected the immune system peripheral blood was collected and immune cells were stimulated with common allergens and TLR ligands. The probiotic treated group responded with a more regulated response to allergens and TLR2 ligands in comparison to the placebo supplemented group.
We also investigated how the probiotic supplementation affected the epigenetic methylation pattern in circulating T helper cells during infancy, observing the most pronounced effects at birth.
In a follow up study, supplementation was started earlier to possibly gain a stronger allergy preventive effect via changes in maternal immune regulation. Supplementation with
Lactobacillus reuteri and ω-3 fatty acids started at gestational week 20 and throughout
pregnancy. After 20 weeks of supplementation, some immunomodulatory effects among circulating activated regulatory T cells and a subpopulation of monocytes were noted. Several systemic immune modifying effects of pregnancy were observed.
In summary, probiotics show several immunomodulatory effects in infants and pregnant women. However, more research is needed to better understand the effects of the probiotic supplementation to aid future identification of more efficacious allergy preventive strategies.
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Table of content
Abbreviations ...1 List of papers ...3 Paper I ...3 Paper II ...3 Paper III ...3 Paper IV ...3Supplementary relevant publications ...4
Aims ...5 Specific aims ...5 Paper I ...5 Paper II ...5 Paper III ...5 Paper IV ...5 Introduction ...6
General aspects of allergic disease ...6
Prevalence of allergic disease and the atopic march ...6
Allergic manifestations ...7
Food allergy...7
Asthma and Allergic rhinoconjunctivits (ARC) ...8
Environment and microbial exposure ...8
The hygiene Hypothesis ...8
Farm studies ...9
Farm studies and epigenetics ...10
Animal models of microbial exposure pre- and postnatally ...10
Gut microbiota and associated lymphoid tissue ...11
Probiotics in allergy prevention ...11
Probiotics in clinical trials ...12
Immunomodulatory effects of probiotics ...20
Gut microbiota and probiotic supplementation ...20
Lactobacillus reuteri ...21
ω-3 fatty acids ...22
Immunological mechanisms ...24
Overview of the immune system ...24
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Dendritic cells...25
Monocytes ...25
NK cells ...26
Epithelial cells and innate lymphoid cells ...26
Adaptive immunity ...27 T helper cells ...27 Antigen presentation ...27 T helper 1 cells ...28 T helper 2 cells ...28 T helper 17 cells ...29 Regulatory T cells ...29
Chemokines and cytokines ...30
Mucosal Immunity ...31
Gut microbiota ...32
The allergic response ...32
The allergic reaction ...33
Allergens ...33
Pregnancy immunology ...34
Development of the immune system during childhood ...35
Immunological interactions between mother and child during pregnancy ...35
Breastfeeding ...36
Prenatal development of the immune system ...37
Innate immune development in infants ...37
Immune development in relation to allergy ...38
Environmental differences in immune maturation ...38
Postnatal development of the immune system ...39
Basic principles of epigenetics ...39
Epigenetic regulation during immune development ...39
Role of gut microbiota in immune maturation ...40
Material and Methods ...42
Description of the clinical trials ...42
The PROOM3-study ...44
Clinical definitions of allergic disease (Paper I-IV) ...45
Design and study population (I and II) ...46
Description of participants in the EILA and Illumina study ...48
viii
Experimental design ...51
Collection of samples and preparations (Paper I-III) ...51
In vitro cultures of PBMCs (Paper I-II) ...51
RNA extraction and Quantitative Real Time PCR (Paper I-II) ...52
Luminex optimization assay ...52
Coupling of antibodies to microspheres ...53
Luminex assay ...53
Measurement of cytokine and chemokine secretion by Luminex (paper II) ...53
Enzyme linked immunosorbent assay (ELISA) (paper I) ...54
Epigenetic Immune lineage Analysis to investigate immune regulatory loci in T helper cells (paper III) ...55
Illumina 450K for global methylation analysis in circulating peripheral CD4+ cells (paper III) ...56
Flow cytometry phenotyping of peripheral immune cell populations during pregnancy (paper IV) ...56
Statistical methods ...59
Results and Discussion ...60
Probiotics ...60
Functional characterization (cytokine and chemokine production/capacity) ...60
The influence of Lactobacillus reuteri on allergen responsiveness (paper I) ...60
TLR ligand responsiveness and expression (paper II) ...62
Expression of T helper lineage specific transcription factors and TLR2/4/9 (paper I-II) ...64
Allergic disease and probiotic supplementation showed independent associations with allergen and mitogen induced responses (paper I) ...64
Cytokine and chemokine responses to TLR ligands are associated with probiotic treatment independently of allergy development (paper I and II) ...65
Epigenetic modifications in infancy and after probiotic supplementation (paper III) ...65
Circulating peripheral immune populations phenotyped with flow cytometry during pregnancy and after supplementation with probiotics and ω-3 (paper IV) ...71
Supplementation with ω-3 and L. reuteri from gestational week 20 alters activated Treg (CD4+CD45RA-Foxp3++) number and percentage in peripheral blood after delivery (paper IV) ...71
Supplementation with ω-3 and L. reuteri from gestational week 20 alters the frequencies of CD14+CD16- monocytes in the pregnant women after giving birth ...74
Allergy ...75
Allergic disease is associated with high Th2 related chemokines (paper I) ...75
Allergic and non-allergic infant response to TLR ligand stimulation (paper II) ...76
Timing ...77
Timing of age dependent changes in peripheral immunity ...77
Age dependent changes in memory and naïve T cells (paper III) ...77
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Timing of peripheral immunity during pregnancy (paper IV) ...80
Systemic changes in subsets of lymphocytes (CD4+/8+/19+/56+) during pregnancy and compared to non-pregnant women ...80
Memory T helper cells (CD4+CD45RA-) decrease during pregnancy and naïve T helper cells (CD4+CD45RA+) increase ...83
T regulatory cell populations during pregnancy ...83
Th1, Th2 and Th17 subpopulations during pregnancy and in comparison with non-pregnant women ...87
Summary and conclusion ...89
Future perspectives ...89
Acknowledgment ...90
1
Abbreviations
α-IL-4R anti-IL-4-receptor AA arachidonic acid AD atopic dermatitis APC antigen-presenting cell ARC allergic rhino-conjuncivitis BSA bovine serum albumin CB cord blood
CBMC cord blood mononuclear cells CCL C-C motif ligand
CD cluster of differentiation Ct threshold cycle
CTLA-4 cytotoxic T lymphocyte antigen 4 CV coefficient of variance
CXCL CXC ligand CXCL3L CX3C ligand DC dendritic cell DHA docosahexaenoic acid DMSO dimethyl sulphoxide EBI3 Epstein-Barr virus induced 3 EPA eicosapentaenoic acid FCS fetal calf serum FcεRI Fcε receptor type 1 Foxp3 forkhead box p3 GATA-3 GATA binding protein 3 gw gestational week HLA human leukocyte antigen IFN-γ interferon-γ
Ig immunoglobulin IL interleukin
ILC2 type 2 innate lymphoid cells LTA Lipoteichoic acid
2
LPS Lipopolysaccharides
MHC major histocompatibility complex NK natural killer
PBMC peripheral blood mononuclear cells PBS phoshate buffered saline
PCR polymerase chain reaction PHA phytohemagglutinin RNA ribonucleic acid
RORC retinoic acid-related orphan receptor C RT-PCR reverse transcription polymerase chain reaction SA-PE streptavidin R-phycoerythrin conjugate SPT skin prick test
STAT signal transducer and activator of transcription Tbet T-box expressed in T cells
Tbx21 T-box transcription factor 21 TCR T cell receptor
TGFβ transforming growth factor-β
Th T-helper
TLR Toll-like receptor Treg regulatory T cells
3
List of papers
Paper I
Forsberg A, Abrahamsson TR, Björkstén B, Jenmalm MC,
Pre- and post-natal Lactobacillus reuteri supplementation decreases allergen responsiveness in infancy.
Clin Exp Allergy 2013; 43: 434-42.
Paper II
Forsberg A, Abrahamsson TR, Björkstén B, Jenmalm MC,
Pre- and postnatal administration of Lactobacillus reuteri decreases TLR2 responses in infants.
Clin Transl Allergy 2014; 4: 21.
Paper III
Forsberg A*, Huoman J*, Bhai Mehta R, Nilsson L, Abrahamsson TR., Ernerudh J, Jenmalm, MC.
Global and Immunoregulatory epigenetic patterning in circulating CD4+ T cells of infants participating in a probiotic prevention study
Manuscript
Paper IV
Forsberg A, Abrahamsson TR., Nilsson L, Ernerudh J, Duchén K, Jenmalm MC. Modulation of maternal immunity during pregnancy by probiotics and ω-3 fatty acids
4
Supplementary relevant publications
Tulic MK, Hodder M, Forsberg A, McCarthy S, Richman T, D'Vaz N, van den Biggelaar AH, Thornton CA, Prescott SL,
Differences in innate immune function between allergic and nonallergic children: New insights into immune ontogeny.
J Allergy Clin Immunol 2011; 127: 470-78 e1.
Abrahamsson TR, Sandberg Abelius M, Forsberg A, Björkstén B, Jenmalm MC,
A Th1/Th2 associated chemokine imbalance during infancy in children developing eczema, wheeze and sensitization.
Clin Exp Allergy 2011; 41: 1729-39.
Forsberg A, Bengtsson M, Eringfalt A, Ernerudh J, Mjosberg J, Jenmalm MC,
GATA binding protein 3(+) group 2 innate lymphoid cells are present in cord blood and in higher proportions in male than in female neonates.
J Allergy Clin Immunol 2014; 134: 228-30
Forsberg A, West CE, Prescott SL, Jenmalm MC,
Pre- and probiotics for allergy prevention: time to revisit recommendations? Clin Exp Allergy 2016; 46: 1506-21.
5
Aims
The general aim of this thesis was to increase our knowledge on immunoregulatory mechanisms after pre- and postnatal probiotic supplementation in an allergy prevention study where the probiotic addition was associated with decreased IgE-associated eczema at two years of age. In a follow-up study we aimed to investigate the effect of prolonged prenatal probiotic and ω-3 fatty acid supplementation on maternal immunity during pregnancy.
Specific aims
Paper I
The aim of paper I was to investigate the effect of probiotic supplementation on cytokine and chemokine producing capacity in response to allergens in infants included in the allergy prevention study.
Paper II
The aim of paper II was to further characterize the effect of probiotic supplementation on the longitudinal cytokine and chemokine producing capacity in response to bacterial moieties i.e. TLR2/4/9 ligands and to investigate the mRNA expression of these receptors.
Paper III
The aim of paper III was to explore local and global epigenetic modifications in circulating CD4+ cells during infancy after pre- and postnatal probiotic supplementation.
Paper IV
The aim of paper IV was to investigate how maternal peripheral immune cell populations, as assessed by flow cytometry phenotyping, might be modulated during pregnancy by
probiotic and ω-3 fatty acid supplementation, and what impact pregnancy itself has on these cell populations.
6
Introduction
This introduction will deal with the background to the use of probiotics in allergy-prevention trials, the potential immunological mechanisms of probiotic supplementation during pregnancy and in infancy to prevent allergy development. Allergic diseases have increased considerably during the latest decades and it is of great importance to try to identify efficacious prevention strategies and to put them into clinical practice.
General aspects of allergic disease
Prevalence of allergic disease and the atopic march
Allergic diseases are substantially increasing and represent a considerable socio-economic cost and a reduced quality of life. An estimated 20 % of the population worldwide is affected by allergic diseases, such as atopic dermatitis, food allergy, asthma, allergic rhinitis and conjunctivitis [1]. Allergic diseases manifest in several ways and an age-associated variation in allergy-related symptoms in childhood is typically seen in a population and is referred to as the “atopic march”. The first allergy-related symptoms are commonly eczema and food allergy, later followed by asthma and rhinoconjunctivitis. In addition to the age variation in allergic diseases, there seem to be a gender association as well. Boys have a higher incidence of allergic diseases than girls early in life [2, 3] and also a more Th2 deviated immunity with an increased susceptibility to infections [2, 3]. The stronger Th1 response in females, as evident by higher levels of inflammatory markers and more efficient infection clearance results in better protection against infection but also in increased susceptibility to autoimmune diseases [2, 3]. Allergy-related sex differences diminish at puberty, and at adult age no clear sex differences concerning allergy can be found [4]. There is a wide range in severity of the allergy related diseases. Symptoms range from very mild to disabling diseases such as severe treatment resistant asthma or eczema and life-threatening anaphylactic reactions.
To further add complexity, early life events occurring during critical windows of immune development can have long-term impact on immune-mediated diseases [5]. The immune status at birth may have a role in this also. Of importance is the maternal immunity which influence the fetus in several proposed ways [6].
7 Although a prevention strategy has effect on one allergic manifestation, it may fail to reduce the development of another. Sensitization and IgE-associated eczema in infancy seem to be the most reliable predictors for subsequent respiratory allergic diseases in school age.
Allergic manifestations
Eczema, food allergy and asthma are the most common allergic manifestations in the first years of life. The cumulative incidence of infant eczema is approximately 20-30 % in an unselected population and 40-50 % in infants with a family history of allergic disease. Eczema is defined as pruritic, chronic or chronically relapsing non-infectious dermatitis with typical
features and distribution. The criteria by Hanifin and Rajka and modified by Seymour for
infants are the most commonly employed in allergy research [7]. The pathogenesis of eczema
is multifactorial, a loss of function in the skin-barrier seem to play a critical role in the development of eczema. These defects in skin-barrier likely results from a combination of factors including deficiency in skin barrier proteins, the lack of certain protease inhibitors and lipid abnormalities. Loss of function mutations in the protein fillaggrin are also considered a major risk factor [8].
Food allergy
While many foods can cause an allergy reaction, however, the most common food allergens are cow´s milk and egg allergy in infants [9]. Common features for allergens involved in infant food allergies are heat, acid, and protease stable water-soluble glycoproteins 10 to 70 kD in size. The allergenicity is possible to modify by heating or preparing foods and thereby modifying conformational epitopes, i.e. reduce (cooked egg) or enhance (roasted peanut) [10]. The term food allergy can be further split into IgE and non-IgE mediated reactions. IgE mediated food allergy begins in the first 1-2 years of life with sensitisation. For an allergic reaction to occur, re-exposure is needed [11]. The symptoms of IgE mediated food allergy include angioedema, urticaria, rashes, flushing, anaphylaxis, bronchospasm, rhinoconjunctivitis and immediate gastrointestinal symptoms. The non-IgE mediated immune reactions that can arise in the gastro-intestinal tract are not so well defined and more difficult to recognize.
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Asthma and Allergic rhinoconjunctivits (ARC)
Asthma is a chronic inflammatory disease in the lower respiratory tract involving mast cells, eosinophils and T lymphocytes. Asthma is characterized by repeated attacks, induced by allergen, often furred animals, or viral infections, of wheeze and cough [12]. About 10% of children in a high risk cohort in Sweden developed IgE mediated asthma [13], also called allergic asthma. Without concomitant sensitisation or asthma in the family, infants with non-allergic asthma do not have an increased risk of asthma at 7 years of age according to the German Multicentre Allergy study [14] Rhinoconjunctivitis mediated by allergens, most often birch, is uncommon in the first two years of life but the incidence increases with age and is about 20 % in the teen-age years [15].
In summary, allergic asthma and rhinoconjunctivitis are not as common as eczema during the first two years of life. Wheeze is a frequent symptom in early childhood but it is commonly triggered by infections and does not always predispose for future asthma.
Environment and microbial exposure
The hygiene hypothesis
Allergy-related diseases were rare a hundred years ago and there has been a substantial increase in prevalence the last half of the century [16]. Even if heredity is a significant risk factor for allergy-related diseases, especially if the mother is allergic [17], the large and rapid increase cannot be explained by genetics alone. Therefore, lifestyle-related and environmental exposures are believed to interact with our genes in the development of allergy-related disease and a “Westernized” lifestyle has contributed to a substantial part. Large geographical variations in prevalence of allergy-related diseases have been demonstrated, with higher occurrence in Westernized countries [18-20]. In 1989 a researcher named Strachan discovered that there was an association between siblings, family size and hay fever [21]. Furthermore, changing climate, living conditions and urbanization have led to a biodiversity loss both at the macro and micro level [22]. Studies have shown alterations in the microbiota composition and a general microbial deprivation which characterize people
9 living in urban affluent environments, further enhanced by physical inactivity and a Western diet poor in fresh fruit and vegetables, which may act in synergy with dysbiosis of the gut
microbiota, immune dysregulation and impaired immune tolerance.Microbial exposure in
early life may educate the developing immune system, driving postnatal maturation of immune regulation as discussed in [23]. The author also suggested that the theory should be referred to as “microbial deprivation hypothesis” since the exposure to a wealth of commensal, non-pathogenic microorganisms early in life is suggested to be of benefit for proper immune maturation [24].
Farm studies
Children that lived on farms had fewer allergies than children born in urban and non-farming rural areas [24-27], as evident with a long follow up time [28]. Additionally, such environment are of course associated with an enriched microbial burden evident in several ways, children exposed to several types of animals and consumption of unprocessed raw cow milk, seem to mediate a protective effect reviewed in [25]. In fact, living with a pet during childhood may also be protective against asthma [29]. In the Protection Against Allergy: Study in Rural Environments (PASTURE) birth cohort study, 1133 pregnant women were recruited in rural areas of Austria, Finland, France, Germany, and Switzerland, demonstrating that farming-related exposures, such as raw farm milk consumption and exposure to animals, that were previously reported to decrease the risk for allergic outcomes, were associated with a change in gene expression of innate immunity receptors in early life [30]. Raw milk contains a lot of Lactobacilli strains and have a different fat composition than processed milk. Exposures to farming areas and consumption of raw milk have been associated with the up regulation of certain receptors associated with innate immunity in school-aged children [31-33]. If the mother is exposed to farm environment, in particular during pregnancy, development of allergic diseases seems to be attenuated [34, 35]. Therefore, microbial exposure in early life
i.e. pre- and postnatal, may educate the developing immune system, driving postnatal
10
Farm studies and epigenetics
At least some of the protective effects of farm living may be mediated by epigenetic mechanisms. Thus, farm living has been associated with increased demethylation of the FOXP3 promoter in Treg cells. This is supposed to reflect a more efficient suppressive function in Tregs. Farm living in the same study was also associated with higher numbers of FOXP3
positive cells in cord blood mononuclear cells (CBMCs) [36].In addition, a subsample from the
PASTURE study showed hypomethylation in different regions of the ORMDL1 and signal transducer and activator of transcription 6 (STAT6) genes (genes highly associated with asthma) while regions in RAD50 and IL13 were hypermethylated in cord blood from children from farm environment [37].
Animal models of microbial exposure pre- and postnatally
The epidemiological studies are supported by animal models, demonstrating that microbial exposure during gestation can prevent allergic responses in the offspring [38, 39]. The beneficial effect of exposure to microbes have been further explored, primarily in animal models, to try to pinpoint what the mechanism on immune tolerance and protection of allergic disease might be. Of importance is the maternal environment, suggesting that maternal immunity may be transferred or at least influence the offspring. In experimental murine models the treatment of the mother with lipopolysaccharide [39-41] or the commensal Acinetobacter lwoffii [38] during gestation attenuates allergic sensitisation and airway inflammation in the offspring. The main receptors for bacterial products are the Toll like receptors (TLRs), which seem to be essential for the allergy protective effect of bacterial exposure during pregnancy [38]. Not only the maternal environment seem to be of importance, but the continued enhanced postnatal microbial exposure may be required for optimal allergy protection, however [42]. A reduced microbial pressure could result in insufficient induction of T cells with regulatory and/or Th1-like properties, which counteract allergy-inducing Th2 responses [25, 36, 43, 44]. Allergic diseases are known to be dependent on Th2 responses to allergens and microbial stimulation may be one way to deviate a skewed Th2 associated immunity to a more Th1/Treg associated response, as will be discussed later.
11 Gut microbiota and associated lymphoid tissue
There are several important sites where antigens encounters occur, but since the gastrointestinal tract is the largest immunological organ in the body and the gut mucosa has
a surface of approximately 400 m2, this area is of essential importance [45]. The surface
epithelium of the mucosa is constantly exposed to myriads of microbes and dietary constituents. This is a very important site for immune regulation and encounters with microbes and almost 70 % of the human lymphoid tissue is located there [45, 46]. The adult
human intestinal canal is inhabited by an estimated 1014 microbes, mainly bacteria and mainly
in the colon [47]. Earlier, the estimation of microbes was ten times the estimated number of human cells in our body, but was recently challenged and estimated to be the approximately the same number [48]. The gut microbiota has several beneficial functions for the host, including fermentation of indigestible carbohydrates into short chain fatty acids, immune system maturation and protection from invasion of exogenous microbes. There are several theories regarding the importance of the exposure to microbes. One of them, the microbial deprivation hypothesis states that an alteration of the microbial exposure may affect the maturation of the immune system, resulting in failure of clinical tolerance development to harmless antigens, and finally, in the development of allergy [23].
The gut microbiota is important in the maturation of the immune system, which is well documented in germ free animal models. These animals fail in developing normal maturation and in particular the regulatory network essential for controlling exaggerated immune responses, leading to disease states such as allergies and autoimmunity (reviewed in [49]). Development of allergic diseases in infants have been preceded by differences in composition and diversity of their gut microbiota [50-62].
Probiotics in allergy prevention
Probiotics are defined as “live microorganisms which when ingested in adequate amounts confer a beneficial effect on the host” [63]. Host interactions with microbial organisms from the environment [21] and at mucosal sites, such as the gut [50, 64, 65] have been proposed to be important for correcting the dysbiotic drift caused by modern living. Probiotics have been proposed to have immunomodulatory effects [66] and promote gut microbiota diversity
12
although evidence for this is scarce in allergy prevention studies (as reviewed in [67]). The intestine provides a unique environment for the development of both immunity and tolerance. Probiotic supplementation during pregnancy and early childhood could possibly provide microbial stimulation needed for normal development of immune regulatory capacity by providing a source of TLR-ligand exposure [26].
Probiotics in clinical trials
Current evidence of using probiotics for allergy prevention in pregnant women, breast-feeding mothers and/or children were summarized in [68] concluding that the most effect is observed when supplementation is given pre- and postnatal. A total of 29 trials fulfilled the criteria set up for the analysis [68]. Unfortunately, there are some factors that make the certainty of evidence low, the heterogeneity among studies (see Table Overview of Probiotic trials). There is a need for high quality studies in this area. The conclusion of this which is also stated by the World Allergy Organization (WAO) [69], is that probiotics given to pregnant women, breast-feeding mothers and/or infants reduced the risk of eczema in infants. Currently available evidence does not indicate that probiotics prevent the development of other allergies. The studies are summarized in [67] as well as investigated immune- and gut modulatory effect. However, there is a plea for future studies that consider the weakness identified regarding the risk of bias and indirectness of the evidence. To have in mind, there are several problems when advising that needs to be solved, which strain, doses and timing to use?
However, other international expert bodies including EAACI, the American Academy of Pediatrics, ESPGHAN, NIAID and FAO/WHO [70-74] do not generally recommend probiotics for allergy prevention at this time.
13 Tab le I. S um mary of pr obi oti c t rials , S uppl em ent ar y Tab le fro m [67 ]: Ov er view o f pr obio tic tr ia ls Stud y po pul ati on a nd p rob ioti c i nte rv en tion Ef fe ct on e cz em a Ef fe ct on se ns iti za tio n Ef fe ct on re sp ira tor y sy m ptom s Ef fe ct on lun g func tion m ea sur es Im m un om od ul at or y e ffe ct s Ef fe ct s on g ut m icr ob iota MA TE RNA L A DMI NI ST RA TI ON O NL Y Hu urre e t a l, 20 08 [75 ] M at ern al a lle rg ic d ise ase L. rh amn os us GG an d B. lac tis Bb -1 2 1x 10 10 CF U dai ly fr om fi rs t tr im es te r an d th en to b rea st feed in g m ot he r u nt il ce ssa tio n of e xc lu siv e bre ast fe ed in g No Long ter m ou tc om es n ot rep or ted Not repor ted Not rep or ted No t r ep or ted Huu rr e 2 00 8; T he br ea st m ilk T GF -β 2, s CD 14, IF N-γ, TN F, IL -1 0, IL -6, IL -4 a nd I L-2 l eve ls we re m ea su re d from s am pl es t ak en i m m ed ia te ly a fte r b irt h a nd 1 m on th a fte r d el ive ry . T he c ol os tr um T GF -β 2 le ve ls te nde d t o be hi ghe r i n t he pr obi ot ic tha n t he pl ac ebo gr ou p, wh ile t he ot he r m ed ia tor s we re n ot a ffe ct ed [75 ]. No t r ep or ted Dot te ru d et a l, 20 10 [76 ] a nd Si m ps on et a l, 20 15 [77 ] Un selec ted - ab ou t 2 /3 wit h f am ily hist ory o f a lle rg ic dise ase L. rh amn os us GG, L . ac ido phi lus LA 5, a nd B. la ct is Bb -1 2 (5 x 1 0 10 CF U of e ac h dai ly ) fr om 3 6 w ee ks ge st at io n a nd t he n to br eas tfe ed in g m ot he r for 3 m on th s Re du ce d cu m ula tiv e inci de nce o f ec ze m a a t 2 an d 6 ye ar s No No No t r ep or ted No t r ep or ted Do tte rud 20 15 ; I nfa nt s to ol s am pl es fr om 1 0 da ys , 3 m on th s, 1 y ea r a nd 2 y ea rs , w er e ana ly se d by qP CR a nd 1 6S r RNA g ene de ep se que nc ing o n t he I llum ina M iS eq pl at fo rm . Onl y t he LG G ba ct er ia co lo ni ze d t he chi ldr en a t 10 d ay s a nd a t 3 m on th s of a ge . T he re we re n o sig nif ica nt di ffe re nc es in t he a bunda nc e o f t he pr ob iot ic b ac te ria b et we en th e g rou ps a t 1 a nd 2 ye ar s of a ge , n or for th e b ac te ria l c la ss es a nd ge ne ra , a lpha a nd be ta di ve rs ity [78 ]. Bo yl e et al , 201 1 [79 ] An y fir st d egr ee re la tiv e wit h all erg ic dise ase L. rh amn os us G G 1. 8 x 1 0 10 C FU d ail y fro m 3 6 we ek s g est at io n un til d el iv ery - no p os tn at al a dmi ni st ra tion to m ot he r No at 1 2 m on th s Lo ng ter m ou tc om es n ot rep or ted No No No t r ep or ted Bo yl e 20 08 ; C BM Cs fro m 7 3 n eo na te s w ere c ul tu re d w ith he at -k ill ed LGG, o va lbum in ( OV A) o r w itho ut st im ul us . LGG tr ea tm ent o f pr eg na nt w om en di d no t in flu en ce C D4 + T c el l p rol ife ra tion , F ox P3 e xp re ss ion , DC phe no ty pe o r c yt ok ine s ec re tio n i n CB M Cs cul tur ed w ith he at -k ill ed LGG o r O VA [80 ]. Bo yl e 20 11 ; C BM Cs fro m 7 3 n eo na te s w ere e xa m in ed fo r D C a nd T re g num be rs a nd LT A a nd LP S i nduc ed pr oduc tio n o f T GF -β , I L-10, IL -12 p40 , IL -13, IF N-γ and TN F. P re na ta l p rob iot ic tr ea tm en t wa s n ot a ss oc ia te d La ht ine n 20 09 ; In ve sti ga te d inf ant f aeca l sa m pl es fr om 7 a nd 9 0 da ys us ing qP CR a nd T -RF LP . A t 9 0 d ay s of a ge , i nf an ts w hos e m ot he rs re ce ive d L GG we re m or e of te n c ol on ize d wi th sp ec ie s b el on gi ng t o t he B l ong um g ro up. Bif id ob ac te ria l s pe cie s c olo niz at io n a t 7 d ay s o r Bi fido ba ct er ium le ve ls di d n ot d iff er b et we en the 2 g ro ups [81 ]. Ism ai l 2 01 2; us ing T -R FLP a na ly sis sho w ed tha t pr en at al L GG f ai le d t o m od ul at e d ive rs ity of
14 w ith a ny c ha ng e i n c or d bl oo d i m m une m ar ke rs o r cy to ki ne s ec re tio n. Bre as t m ilk s am pl es f ro m t he pr ob iot ic gr ou p h ad lowe r l eve ls of tot al Ig A a t d ay 28 and l ow er s CD1 4 at da y 7 , w hi le br ea st m ilk T GF -β1 le ve ls we re n ot a ffe ct ed [79 ]. ea rly in fa nt gu t m icr ob io ta d es pi te p rom ot in g a be ne fic ia l b ifid ob ac te ria p ro file [82 ]. Rau tav a et al , 20 12 [83 ] M at ern al a lle rg ic d ise ase L. rh amn os us LP R a nd B. lo ngu m B L99 9 or L . par ac as ei a nd B. lo ngum B L9 – e ach pr ob ioti c at a dai ly d os e of 1x 1 0 9 C FU from tw o m on th s b ef or e de liv er y an d dur in g two m on th s t o b re ast fe ed in g m ot her Re du cti on of ec ze m a a t 2 ye ar s i n bo th pr obi ot ic gr ou ps Lo ng ter m ou tc om es n ot rep or ted No Not rep or ted No t r ep or ted No t r ep or ted No t r ep or ted PE RI NA TA L A DMI NI ST RA TI ON TO MO TH ER AND /O R C HI LD Ka llio m äk i e t al , 20 01 [84 ] a nd Ka llio m äk i e t al , 20 07 [85 ] An y first d eg re e re la tiv e w ith a lle rg ic dise ase L. rh amn os us G G 1x 10 10 C FU da ily g iv en to m oth er s 2 -4 w eek s b ef or e del iv er y an d th en to br eas tfe ed in g m oth er s or di re ct ly to inf ant , f or 6 m ont hs Re du cti on of ec ze m a a t 2 ye ars wh ich rem ai ned a t 7 yea rs No No No Ra ut av a 2 00 2; P ro bi ot ic a dm ini st ra tio n i nc re as ed t he am ount o f a nt i-i nf la mma to ry TGF -β2 in t he m ilk a t th re e m on th s i n m ot he rs r ec ei vi ng p rob iot ics a s ana ly se d by ELI SA . I nf ant s w ith e le va te d I gE i n c or d bl ood b en ef ite d m os t of t he p rob iot ic suppl em ent at io n [86 ]. Gui em on de 2 006 ; A t 5 d ay s o f a ge , i nfa nts wh os e m ot he rs re ce ive d L. rh am no sus G G sh owe d a si gn ifi ca nt ly h ig he r oc cu rr en ce of B. br ev e an d l owe r of B . a do le sc en tis tha n t ho se from th e pl ac eb o gr ou p, a s d et er m in ed b y PCR . I n a ddi tio n, L. rham no sus G G co ns um pt io n inc re as ed t he bi fido ba ct er ia l div er sit y a t 3 w ee ks in in fa nt s [ 87 ]. Abr aha m sso n et al , 20 07 [88 ] a nd Abr aha m sso n et al , 20 13 [89 ] An y first d eg re e re la tiv e w ith a lle rg ic dise ase No re du cti on o f ec ze m a, b ut re du cti on of Ig E-asso cia te d ec ze m a i n th e pr obi ot ic gr ou p at 2 y ear s No No No d iff er en ces bet w een th e gr oups w he n ev al uate d by sp iro m et ry re ve rsi bi lit y te st Abr aha m ss on 2 011 ; Th er e we re n o s ig ni fic an t di ffe re nc e i n c he m ok in e l eve ls b et we en th e L. reu teri and t he pl ac ebo -tr ea te d g rou p, a t C B, 6 -, 12 - a nd 2 4 m on th s. T he p re se nc e of L . reu teri in s tool t he fi rs t we ek of li fe wa s a ss oc ia te d wi th lowe r C CL 22 a nd CCL1 7 a nd hi ghe r CX CL1 1 l ev el s a t 6 m ont hs o f a ge [90 ]. Abr aha m ss on 2 00 9; The pr ev al enc e of L reu te ri w as hi ghe r du ring t he f irs t y ea r o f l ife in t he st oo l s am pl es f ro m i nf ant s i n t he pr obi ot ic gr ou p. T he h ig he st p re va le nc e wa s r ec or de d a t 5 to 6 d ay s of a ge (8 2% in th e t re at ed vs 2 0% i n th e p la ce bo g rou p) . S up pl em en ta tion a ffe ct ed ne ith er t he p re va le nc e n or t he c ou nt s of bi fid ob ac te ria or C d iff icile , e xc ep t f or h ig he r co unt s o f bi fido ba ct er ia in the tr ea te d gr oup a t
15 L. re ut er i 1 x 10 8 C FU d ail y 2-4 we ek s be for e de liv er y an d th en to i nfan t for 1 2 m on th s No di ffe re nc e bet w een th e tw o gr ou ps at 7 ye ars fo llo w up an d F eN O le ve ls at 7 y ear s Bö ttc he r 20 08 ; P ro bi ot ic s uppl em ent at io n w as as soc ia te d wi th l ow l eve ls of T GF -β 2 and sli ght ly in cr ea se d l ev el s of I L-10 i n c ol os tr um . I nf ant s re ce iv in g b re as t m ilk w ith lo w T GF -β 2 le ve ls we re le ss lik ely t o b ec om e s en sit ize d d ur in g t he ir firs t 2 y r o f lif e. T he le ve ls of tot al Ig A, S Ig A, T GF -β 1, T NF , s CD 14, an d N a/ K r at ios in b re as t m ilk we re n ot a ffe ct ed b y L. reu teri in ta ke [91 ]. Fo rs be rg 2 01 3; P rob iot ic t re at m en t wa s a ss oc ia te d wi th low c at -induc ed I L-5 a nd I L-13 r es pon se s a t 6 m ont hs , w ith a s im ila r t re nd f or IL -5 a t 1 2 m on th s. Ca t-i nduc ed I FN -γ r es pon se s w er e al so lowe r af te r pr obi ot ic t ha n a fte r pl ac ebo tr ea tm ent a t 2 4 m ont hs , w ith s im ila r f in di ng s f or IL -10 a t bi rt h a nd a t 12 m ont hs . A t 2 4 m ont hs , bi rc h i ndu ce d CC L2 2 l eve ls we re lo we r i n t he p rob iot ic t ha n i n t he p la ce bo g rou p [92 ]. Fo rs be rg 2 01 4; Pr obi ot ic s uppl em ent at io n w as as so cia te d w ith de cr ea se d LT A i nduc ed CCL4 , CX CL 8, IL -1 β and IL -6 re sp on se s a t 1 2 m on th s a nd d ec re as ed CCL4 a nd I L-1β se cr eti on a t 2 4 m on th s. TLR 2 and T LR 4 m RN A e xp re ss ion a nd r es pon se s t o L PS w er e not af fe ct ed b y p rob iot ic tr ea tm en t [ 93 ]. 2 y ea rs . A t 12 m on th s t he p re va le nc e of L reu teri w as l ow er in b re ast -fe d t ha n f or m ul a-fe d i nf ant . L . r eu ter i w as i so la te d f ro m 1 2% a nd 2% of t he c ol os tr um s am pl es in t he p rob iot ic an d pl ac eb o g rou p, re sp ec tive ly [94 ]. Ab ra ha m ss on 201 2; P rob iot ic suppl em ent at io n di d no t a ffe ct g ut m icr ob iot a di ve rs ity a t 1 a nd 1 2 m on th s, as d et er m in ed b y 16S rD NA 4 54 -py ro se que nc ing [55 ]. Ku kko ne n et al , 2 007 [95 ] an d Ku itun en et al , 20 09 [96 ] An y first d eg re e re la tiv e w ith a lle rg ic dise ase M ix of L. rh am no su s G G an d LC 70 5 (bo th 5 x 10 9) a nd B. b re ve B b9 9 a nd Pr opr io ni bac te rium fr eud enr ei chi i s sp. sh er ma ni JS (b ot h 2 x 10 9) pl us pr ebi ot ic ga la ct oo ligo sa cch ar ide s; gi ve n tw ice dai ly to m oth er 2 -4 w eek s b ef or e de liv er y an d t he n to in fan t fo r 6 m on th s Ec ze m a red uc tion in th e pr obi ot ic gr ou p at 2 y ear s No ec ze m a re du cti on a t fi ve yea rs No No No d iff er en ces in F eN O le ve ls bet w een th e gr oups a t 5 ye ars in a ran dom ize d subpo pul at io n Kuk ko ne n 20 06; In t he p ro bi ot ic co m pa re d w ith t he pl ac ebo g ro up, pr ot ec tiv e a nt ibo dy co nc ent ra tio ns to Ha em ophi lus inf lu enz ae t ype b ( Hi b) o cc ur re d m or e fre qu en tly a t 6 m on th s, a nd g eo m et ric m ea n I gG titr es to H ib te nd ed to b e h igh er . I gG t itr es to di pht he ria a nd t et anus w er e s im ila r i n t he 2 g ro ups [97 ]. M ar sc ha n 2 00 8; Inf ant s r ec ei vi ng pr obi ot ic ba ct er ia ha d hi ghe r pl as m a l ev el s o f CR P, to ta l I gA , to ta l I gE , and I L-10 tha n i nf ant s i n t he pl ac ebo g ro up [98 ]. Kui tu ne n 2 00 9; P ro bi ot ic s uppl em en ta tio n c aus ed a gut m uc os al inf la m m at io n w ith de cr ea se d H b v al ue s at 6 m ont hs , but Hb a nd t he o the r he m at ol og ic v al ue s w ere si m ila r a t 2 y ea rs in th e 2 g ro up s [ 99 ]. Kuk ko ne n 2 00 7; F aeca l co un ts of a ll th e suppl em ent ed m icr obe s w er e s ig nif ica nt ly hi ghe r a t 3 a nd 6 m ont hs . A t 2 ye ar s, n o di ffe re nc es we re ob se rve d b et w ee n s tu dy gr oups i n f ae ca l ba ct er ia l c olo niz at io n u sin g ag ar cul tur ing a nd PCR [95 ]. Kuk ko ne n 2 009 ; Fa ec al I gA , α1 -A T, T NF a nd ca lp rot ec tin wa s m ea su re d a t t he a ge of 3 a nd 6 m on th s. P rob iot ics te nd ed to a ug m en t f ae ca l Ig A a nd s ig ni fic ant ly i nc re as ed f ae ca l a 1-AT . Hi gh i nt es tina l I gA a ss oc ia te d w ith r edu ce d al le rg y ri sk [10 2] .
16 Kui tu ne n 2 012 ; P rob iot ic s up pl em en ta tion wa s asso cia te d w ith le ss I gA t o ca se in a nd m or e I L-10 in m at ur e B M (3 m ont h s am pl es ) but no t i n c ol os tr um , and w ith r edu ce d co lo st ra l but no t m at ur e BM T GF -β2 le ve ls. P rob iot ic su pp le m en ta tion d id n ot a ffe ct col os tr um or m at ur e BM tot al Ig A l eve ls, n or le ve ls of Ig A ant ibo di es to CM , B LG a nd OV A [10 0] . Sa vi la th i 2 01 5; Co lo st rum a nd 3 m ont h B M s CD1 4, hum an ne ut ro phi l pe pt ide (H NP ) 1 –3 a nd β-de fe ns in 2 ( HB D2 ) l ev el s we re n ot a ffe ct ed b y p rob iot ic suppl em ent at io n [10 1] . Ko pp et al , 200 8 [1 03 ] An y fir st d eg re e re la tiv e wit h a lle rg ic dise ase L. rh am no su s GG 1x1 0 10 C FU da ily gi ve n to m oth er s 4 -6 w eek s b ef or e deli ver y an d th en to br eas tfe ed in g m oth er for 3 m on th s or to i nfan t for 6 m on th s No at 2 y ear s Lo ng ter m ou tc om es n ot rep or ted No No No t r ep or ted Ko pp 2 008 ; CB M C a nd P BM C o f t he c or re spo ndi ng m ot he r w er e i sol at ed fr om co rd b loo d a nd p er ip he ra l bl oo d ( n= 68 ). Ce lls w er e s tim ul at ed w ith I L-2, β -la ct og lo bul in o r LGG a nd I FN -γ , I L-10 a nd IL -13 i n t he supe rna ta nt s w er e m ea sur ed w ith ELI SA . LGG induc ed IL -10 a nd IF N-γ se cre tio n in v itro , b ut inde pe nde nt ly of pr obi ot ic suppl em ent at io n [10 4] . No t r ep or ted W ick en s e t al , 20 08 [1 05 ] an d W ick en s et al , 20 13 [106 ] An y first d eg re e re la tiv e w ith a lle rg ic dise ase L. rh amn os us HN 00 1 or B . l ac tis H N01 9 1x 10 10 CF U dai ly fr om 2 -5 we ek s b ef or e de liv er y an d t he n to in fan t d ire ctl y for 2 yea rs Ec ze m a re du ct io n i n the L. rh amn os us gr oup a t 2 ye ar s w hi ch re m ai ne d un til 6 y ea rs No be ne fit of B. la ct is Lo w er cu m ula tiv e se nsit isa tio n in the gr oup rec eiv in g L. rham no sus at 6 y ear s No be ne fit of B. la ct is No No d iff er en ces bet w een th e gr oups w he n ev al uate d by sp iro m et ry re ve rsi bi lit y te st an d F eN O le ve ls at 6 y ear s Pr es co tt 20 08 ; N eon at es of m ot he rs wh o r ec ei ve d L. rham no sus but no t B. lact is ha d hi ghe r C B I FN -γ le ve ls, co mp ar ed w ith t he pl ac ebo g ro up. C ol os tr um T GF -β1 le ve ls we re in cr ea se d af te r B. lac tis suppl em ent at io n, w ith a s im ila r t en de nc y f or L. r ham no sus . I nc re as ed col os tr um Ig A l eve ls we re ob se rve d a fte r b ot h B. lact is and L. r ham no sus a dm ini st ra tio n. Ne ona ta l pl as m a sC D14 le ve ls we re l owe r i n t he B. l act is gro up co m pa re d w ith t he pl ac ebo g ro up [1 07 ]. Wi ck en s 20 08 ; L . r ha m no sus (7 1. 5% ) w as m ore lik ely th an B. lact is (2 2. 6%) to b e p re se nt in th e fa ec es a t 3 m on th s, a lth ou gh d et ec tion r at es w ere si m ila r b y 24 m on th s [ 105 ]. Ni er s e t a l, 20 09 [1 08 ] a nd Go risse n et a l, 20 14 [10 9] Re du ce d cu m ula tiv e inci de nce o f No No No t r ep or ted Ni er s 2 00 9; R educ ed a nt i-CD 2/ CD2 8 induc ed I L-5 an d IL -1 3 l eve ls we re fou nd in wh ol e b lood c ul tu re s a t 3 m ont hs o f a ge in t he pr obi ot ic c om pa re d w ith t he pl ac ebo g ro up. T he in v itro ly m ph oc yt e p rol ife ra tive Ni er s 2 00 9; U sin g T -R FLP , qP CR a nd DGGE, L . la ct is and B. bi fidu m but no t B. lac tis w ere m ore ea sil y d et ec ta bl e in t he p ro bi ot ic com pa re d
17 Al le rg ic dise ase o f e ith er p ar ent a nd in at le ast o ne sib lin g La ct oco ccu s l act is W 58 , B . l ac tis W 52 and B. bi fid um W2 3 1 x 10 9 C FU e ach da ily si x w eek s b ef or e del iv er y a nd th en di re ctl y to in fa nt for 1 2 m on th s ec ze m a i n th e fir st th ree m on th s o f l ife No di ffe re nc e at 6 ye ar s re spo ns e t o e ithe r a nt i CD 2/ CD 28 o r P HA di d no t di ffe r b etw ee n t he g ro ups [10 8] . w ith t he pl ac ebo g ro up a t 3 m on ths o f a ge [10 8] . Rut te n 2 01 5; On ly m in or a nd s hor t t er m di ffe re nc es i n com pos iti on of m icr ob iot a be twe en th e p rob iot ic a nd p la ce bo g rou p we re fo und us ing 1 6S –2 3S r DN A i nt er sp ac e re gi on ba se d p rof ili ng . G ut m icr ob iot a d ev el op m en t co nt inue d be tw ee n t wo a nd s ix y ea rs , the n appr oa chi ng a m or e adul t-lik e co m po sit io n [11 0] . Ki m e t al , 20 10 [11 1] An y first d eg re e re la tiv e w ith a lle rg ic dise ase B. bi fidum BG N4 , B . l ac tis AD 01 1, a nd L. ac ido phi lus AD 03 1( 1. 6 x 1 0 9 C FU of e ac h da ily) 4-8 w ee ks b ef or e deli ver y, 3 m ont hs to br ea st fe edi ng m ot he r a nd th en to in fan t f rom 4 to 6 m on th s Re du ce d cu m ula tiv e inci de nce a nd pr ev alen ce o f ec ze m a a t 1 2 m on th s Lo ng ter m ou tc om es n ot rep or ted Not repor ted Not rep or ted No t r ep or ted No t r ep or ted No t r ep or ted Ou et a l, 20 12 [112 ] M at ern al a lle rg ic d ise ase L. rh amn os us GG 1 x 10 10 CF U dai ly fr om se con d tr im es te r an d th en 6 m on th s to m ot her if b rea st feed in g or d irec tly to in fan t No Long ter m ou tc om es n ot rep or ted No No No t r ep or ted Ou 2 012 ; T he re w er e n o sig ni fic an t d iff er en ce s i n pla sm a Ig E le ve ls in th e ch ild re n a t 6, 1 8, a nd 36 m on th s of a ge , wh ile m ea n p la sm a I gE wa s h ig he r i n the co rd bl oo d of the LG G g ro up. M at er na l pl as m a IL -13, IL -1 0, IF N-γ, CX CL1 0, a nd TGF -β le ve ls we re n ot affe cte d [11 2] . No t r ep or ted Al len e t a l, 20 14 [1 13 ] An y first d eg re e re la tiv e w ith a lle rg ic dise ase No re du cti on o f ec ze m a, b ut a re du cti on of Ig E-asso cia te d ec ze m a a t 2 Re du ce d cu m ula tiv e fre que nc y o f SPT react iv ity at No No t r ep or ted No t r ep or ted No t r ep or ted
18 L. sa liv aris C UL6 1, L. p ar ac as ei C UL0 8, B . an im ali s s sp la ct is CU L3 4 and B. bi fidu m CU L2 0, 10 10 CF U dai ly in tot al star tin g 2-4 w eek s b ef or e del iv er y a nd th en to th e in fan t for si x m on th s ye ar s of ag e i n th e pr ob ioti c gr oup 2 ye ar s i n th e pr ob ioti c gr oup PO ST NA TA L A DMI NI ST RA TI ON Tay lo r e t a l, 20 07 [11 4] a nd Je nse n et a l, 20 12 [11 5] M at ern al a lle rg ic d ise ase L. ac ido phi lus (L AVRI -A 1) 3 x 10 8 C FU gi ve n w ithi n 4 8 ho ur s, and the n fo r s ix m on th s, d ire ct ly to in fan t No re du cti on a t 1 ye ar n or at th e or 5 y ea r fol low -up No Sensi tisa tio n m or e co m m on in th e pr ob ioti c gr ou p at 1 ye ar , but no t at th e l ate r fol low -ups No No t r ep or ted Ta yl or 200 6; Inf an t c yt ok in e ( IL -5, IL -6, IL -10, IL -13, TNF o r T GF -β ) r es pon se s to T T, H DM , O VA , B LG , S EB an d P HA we re m ea su re d a t 6 m on th s of a ge . P rob iot ic suppl em ent at io n w as a ss oc ia te d w ith r edu ce d pr oduc tio n o f I L-5 a nd T GF -β i n re sp on se t o SE B st im ula tio n, lo w er IL -1 0 re sp on se s t o T T va cc in e ant ig en a nd r educ ed T NF a nd I L-10 re sp on se s t o H DM al le rg en s[ 11 6] . Ta yl or 20 06 ; M on on uc le ar ce ll s am pl es we re av ai la bl e f ro m 1 18 i nf ant s a nd s tim ul at ed us ing lig ands fo r T LR 2 a nd T LR 4/ CD 14 , f indi ng no e ffe ct s o f th e p rob iot ic s up pl em en ta tion on cy tok in e r es pon se s. Ci rc ul at ing DC s ubs et f re qu enc ie s a nd a nt ig en pr es en tin g c ap ac ity we re si m ila r b et we en th e g rou ps [11 7] . Ta yl or 2 007 ; In fa nt r egu la to ry T -c el l f unc tio n w as ex am ine d a t 6 m ont hs . P ro bi ot ic s up pl em ent at io n di d no t affe ct th e p ro po rti on o f c irc ul ati ng CD 4+ CD 25 +CT LA 4+ c el ls o r F ox P3 m RNA e xpr es sio n [11 8] . Ta yl or 20 07 ; A t 1 m on th o f a ge , i nfa nts in th e pr ob iot ic g rou p w er e a lm os t t wi ce a s l ik el y t o sh ow c ul tu ra bl e l eve ls of La ct ob ac ill us sp ec ie s. By 6 m on th s of a ge , t he r at e o f La ct oba cil lus co lo niz at io n w as s ig nif ica nt ly h ig he r in t he pr obi ot ic t ha n t he pl ac ebo g ro up. T he ra te s o f col on iza tion wi th B ifi dob ac te riu m n ot a ffe ct ed by th e t re at m en t [ 11 4] . So h et al , 20 09 [1 19 ] an d Lo o et al , 20 14 [1 20 ] An y fir st d eg re e r el at iv e w ith a lle rg ic dise ase , L. rh amn ou s LP R 1 x 10 9 C FU and B. lo ngum (B L9 99 ) 6 x 10 8 C FU da ily to in fa nt (in in fan t for m ul a) fo r 6 m on th s No re du cti on a t 2 or 5 y ea rs No No No t r ep or ted So h 20 10 ; Co m pa re d w ith pl ac ebo , p ro bi ot ic suppl em ent at io n i m pr ov ed he pa tit is B ( He pB ) s urf ac e ant ibo dy re spo ns es a t 12 m on th s i n s ubj ec ts re ce iv ing m on ova le nt d os es of H ep B va cc in e a t 0 , 1 m on th a nd a DT Pa –He pB c om bi na tio n v ac cine a t 6 m ont hs , b ut not th os e wh o r ec ei ve d 3 m on ova le nt d os es [121 ]. No t r ep or ted
19 We st e t a l, 20 09 [1 22 ] We st e t a l, 20 13 [41] M ixe d ( 2/ 3 wit h a t le ast o ne fi rst g ra de re la tiv e wit h a lle rg ic d ise ase ) L. par ac as ei ss p par ac as ei F19 1 x 10 9 CF U d ai ly to in fan t ( in in fan t c er eal ) dur in g w ea ni ng fro m 4 -1 3 m ont hs Re du ce d cu m ula tiv e inci de nce o f ec ze m a a t 1 3 m on th s No di ffe re nc e at 8 ye ar s No No No d iff er en ces bet w een th e gr oups w he n ev al uate d by sp iro m et ry re ve rsi bi lit y te st an d F eN O le ve ls at 8 y ear s We st 20 08 ; A nt ibo dy c onc ent ra tio ns to Hi b c aps ul ar po ly sa cc ha ride (H ibP S) , di pht he ria t ox in ( D) a nd te ta nus t ox oi d ( T) be fo re a nd a fte r th e s ec on d a nd th ird d ose s w as m ea su re d. LF 19 e nha nc ed a nt ibo dy co nc ent ra tio ns t o D a nd T , e spe cia lly i n inf ant s br ea st fe d l es s t ha n 6 m ont hs . Co nv er se ly , br ea st fe edi ng dur at io n i nf lue nc ed t he a nt i-H ib PS con ce nt ra tion s, wi th n o e ffe ct b y L F1 9 [ 123 ] We st 20 09 ; A t 1 3 m on th s o f a ge , th e a nti -CD 3/ CD2 8 induc ed I FN -γ /IL4 m RNA r at io w as hi ghe r in the pr obi ot ic c om pa re d w ith the pl ac ebo g ro up [122 ]. We st 20 12 ; A t 1 3 m ont hs o f a ge , t he a nt i-CD 3/ CD2 8 induc ed I FN -γ /IL -2 a nd I L-17 A/I L-2 m RN A ra tio s w er e hi ghe r i n the pr obi ot ic t ha n the pl ac ec o g ro up, a s w as the T T i nduc ed IL1 7A e xpr es sio n. No di ffe re nc es w er e ob se rve d b et we en t he t wo g rou ps a t 5 .5 m on th s [12 4] . We st 20 08 : U sin g RA PD -P CR, L. par ac as ei ssp pa rac as ei F 19 wa s d et ec te d i n st oo l i n 90 % o f t he inf ant s i n th e pr ob iot ic gr oup at 6 m ont hs a nd the fr eque nc y re m ai ne d hi gh t hr oug ho ut the int er ve nt io n [12 3] .
20
Immunomodulatory effects of probiotics
Probiotics may have systemic effects and may modify breastmilk composition [75, 79, 86, 91, 100, 101, 107] since the entero-mammary pathway reflect nutritional, metabolic and immunological processes in the gut and thereby the mammary glands [125]. In summary, no consistent effects on TGF-β1, TGF-β2 and IgA levels in breastmilk have been observed. The effect of probiotics on peripheral immune response include several theories, including enhanced immune maturation, increased T helper 1 (Th1) associated immunity, but also induction of T regulatory cells (Tregs) and thereby increased peripheral tolerance. Effects on both the innate and adaptive immunity have been investigated, see Table I and [67]. In summary, there are some indications of reduced cytokine and chemokine responses to certain stimuli may be associated with probiotic supplementation during pregnancy and/or infancy. For example, reduced anti-CD2/CD28-induced IL-5 and IL-13 levels in whole blood cultures were noted at 3 months of age after pre- and postnatal supplementation with a mixture of B. bifidum, B. lactis and L. lactis as compared with placebo [108]. The same pattern with reduced responses to polyclonal stimuli with Staphylococcal Enterotoxin B (SEB) (lower IL-5 and TGF-β levels) and house dust mite (HDM) allergens (lower tumour necrosis factor (TNF) and IL-10) at 6 months was found after postnatal L. acidophilus as compared with placebo administration [116]. However, all studies have slightly different designs and time points for sample collection, in addition to the variation in probiotic strains and treatment duration.
Gut microbiota and probiotic supplementation
Probiotics have been proposed to have a beneficial effect on gut microbiota composition and the probiotic strain may be transiently detected during the supplementation period in most studies (Table 1)[67]. However, gut microbial diversity promoting effects early in life have not been observed generally, Table I. Although, when comparing the results from the different studies it is central to acknowledge how varying methodologies may affect the findings. Traditional culture based methods are difficult to equate with the next generation sequencing tools that are available today. There is some proof for a bifidogenic effect of probiotic supplementation [87, 126], although this has not been consistently observed [108, 114]. Also,
21 the probiotic strain has been detected in faeces during but not after the administration period in several studies. Long term effects remain to be investigated, as few such studies have been performed. The effects on gut microbiota composition seem to depend on choice of strain and treatment duration, which is consistent with the reported strain-specific differences also for immunomodulatory and clinical outcomes, Table I.
Lactobacillus reuteri
More and more evidence highlight the importance of choice of probiotic strain when conducting clinical trial in allergy prevention [127] and also in other areas were probiotics have had beneficial effects, such as preventing necrotizing enterocolitis (NEC) in preterm infants [128]. Lactobacillus reuteri is an obligate heterofermentative [129] Gram positive rod that has been isolated from the GI tract in several mammals, including humans, as well as from different food products [130-132]. In addition to glycerol, L. reuteri produce the antimicrobial metabolite reuterin during anaerobic conditions (272). The strain L. reuteri ATCC 55730 is consider safe as no adverse events have been recorded [67].
In conclusion, although promising effects of probiotics in eczema prevention has been observed, further research is required to be able to translate the WAO recommendations into practice guidelines. As specific advice, at the present, on choice of strains, dose, timing, mode of administration and duration is not possible to give due to the great heterogeneity between studies performed so far. To be able to do so more knowledge on immune modulating effects after supplementation is needed.
22
ω-3 fatty acids
T h e nutritio nal c o ntent o f th e d iet h as c h anged in th e W es terniz ed w o rld [ 133] , epid emio lo gic al d ata s h o w an inc reas e o f - 6 f atty ac id s intak e and a d ec reas e in th e intak e o f - 3 f atty ac id s [ 134] . Sev eral s tud ies [ 135 - 142] ind ic ate th at f is h ric h in f atty ac id s h as a pro tec tiv e ef f ec t o n th e d ev elo pment o f allergic d is eas e [ 143, 144] . E s pec ially th e - 3 P U FA s lino leic ac id s , eic o s apentaeno ic ac id ( E P A ) and d o c o s ah ex aeno ic ac id ( D H A ) and th e - 6 P U FA s lino leic ac id ( L A ) and arac h id o nic ac id ( A A ) , are k ey f atty ac id s , Figure 1. - 3 f atty ac id s are c o ns id ered to b e anti- inf lammato ry and - 6 pro - inf lammato ry. T h e main link b etw een n-6 P U FA s and inf lammato ry pro c es s es is th ro ugh A A and its metab o lites . B ec aus e o f th eir anti-inf lammato ry pro perties , n- 3 P U FA s , partic ularly E P A and D H A , migh t prev ent th e d ev elo pment o f as th ma b y antago niz ing th e ef f ec ts o f A A . T h e rec epto r o f impo rtanc e may b e G P R 120 w h ic h - 3 s ignal mainly th ro ugh [ 145 ] . H uman s tud ies ind ic ate th at s upplementatio n w ith - 3 P U FA s migh t h av e b enef ic ial ef f ec t in patients w ith allergic as th ma and ato pic d ermatitis [ 133] , and als o d ec reas e th e allergic res po ns e to allergens in inf ants [ 138 ] . I n a rand o miz ed c linic al trial s upplementatio n w ith - 3 f atty ac id s ( 1.6 g E P A and 1.1 g D H A o r plac eb o ) d aily d uring pregnanc y w eek 25 to av erage 3- 4 mo nth s o f b reas tf eed ing
Figure 1. I nf lammato ry path w ays , A A : arac h id o nic ac id , E P A : eic o s apentaeno ic ac id , D H A : d o c o s ah ex aeno ic ac id
23 resulted in lower prevalence of food allergy and IgE- associated eczema in the treated group during the first year of life [146]. The cumulative incidence of IgE-associated disease was also decreased at two years in the treated group, although no obvious effect on the clinical symptoms of allergic diseases. Furthermore, higher mother and infant intake of DHA and EPA were associated with lower prevalence of allergic disease [147]. The effect of supplementation on the peripheral immune system was also investigated, in non-allergic infants the circulating CXCL11 levels were higher as well as the titers of IgG to diphtheria compared to non-allergic infants [148]. During pregnancy, the proportions of EPA and DHA in plasma/serum phospholipids increased in the ω-3-supplemented group, whereas AA decreased during intervention. Also, lipopolysaccharide-induced prostaglandin E2 secretion decreased in a majority of the ω-3-supplemented, with more pronounced effects among non-atopic than non-atopic mothers [149].
In conclusion, a higher intake of ω-3 may have an allergy preventive effect by possibly antagonize the effect of AA. The synergistic effect of supplementation with both ω-3 and probiotics for allergy prevention would be of interest to evaluate.
24
Immunological mechanisms
Overview of the immune system
Innate immunity
The immune system is traditionally divided into innate and adaptive immunity, Figure 2 [46]. The first line of defense is the innate immunity which responds rapidly to common components of microorganisms such as bacteria, viruses, parasites and fungi, structures preserved during evolution called pathogen associated molecular patterns (PAMPs). The innate immune system includes physical barriers of the mucosa, the epithelial cell layer, as well as cells responding immediately with phagocytosis of microorganisms, extinction of infected cells and cooperation with adaptive immunity. The primary sensors, pattern recognition receptors (PRRs) recognize these PAMPs. The PRRs are expressed on various cells of the immune system such as monocytes, macrophages, DCs, NK cells and innate lymphoid cells as well as mucosal epithelial and endothelial cells. Examples of PRRs are Toll-like receptors (TLR), NOD-like receptors (NLR), RIG-1-like receptors (RLR), β-glucan receptors and
