Download Licentiate-thesis from the Department of Immunology, Wenner-Gren Institute, Stockholm University, Sweden

Licentiate-thesis from the Department of Immunology, Wenner-Gren Institute, Stockholm
University, Sweden
Early infant gut flora and neutral oligosaccharides in
colostrum in relation to allergy development in children.
Ylva Margareta Sjögren
Stockholm 2007
2
Licentiate-thesis from the Department of Immunology, Wenner-Gren Institute, Stockholm
University, Sweden
Early infant gut flora and neutral oligosaccharides in
colostrum in relation to allergy development in children.
Ylva Margareta Sjögren
Stockholm 2007
3
“If you always must know what´s right you will end up among the ones who
always are wrong” –Ola Salo
4
SUMMARY
Today, atopic allergy is the most common chronic disease among children in the developed
world. The increase in allergy prevalence during the past decades in these countries might be
associated with lower microbial exposure. The gut flora, consisting of approximately 800
different species of bacteria, has been postulated to be important for the development of a
fully functional immune system. Essentially, these bacteria are in constant contact with the
gut flora associated lymphoid tissue, the largest lymphoid tissue of the human body.
Following birth, the sterile gut of the newborn is immediately colonised by various bacterial
species. Actually, alterations in the infant gut flora have been associated with allergy
development.
Human milk is the major food in infancy and could thus influence the composition of the
infant gut flora. Immunomodulatory components in human milk might differ between mothers
and could therefore explain the contradictory results seen regarding breastfeeding and allergy
development. Oligosaccharides, the third most abundant solid component in human milk,
survive the passage through the stomach and are utilised by the gut microbiota. We analysed
nine abundant neutral oligosaccharides in colostrum samples from allergic and non-allergic
women and related to subsequent allergy development in their children. We found a
considerable variation in the concentration of neutral oligosaccharides in colostrum, which
was not to be explained by the allergic status of the women. Neither was the consumption of
neutral colostrum oligosaccharides related to the allergy development in children.
Relevant bacterial species in early faecal samples were analysed, with Real-time PCR, and
related to allergy development in children followed up to five years of age. Infants who
harboured Lactobacilli (L.) group I (L. rhamnosus, L. paracasei, L. casei) at 1 week of age
and Bifidobacterium adolescentis at 1 month of age developed allergic disease less frequently
during their first five years than infants who did not harbour these bacteria at the same time
(p=0.004 and p=0.008 respectively).
In conclusion, the work presented in this thesis implies the importance of a diverse gut flora
early in life for the development of a fully functional immune system. However, consumption
of colostrum with high amounts of neutral oligosaccharides does not protect against early
allergy development.
5
LIST OF PAPERS
This thesis is based on the following original articles, referred to in the text by their Roman
numerals:
I
Sjögren Y. M, Duchén K, Lindh F, Björkstén B, Sverremark-Ekström E.
Neutral oligosaccharides in colostrum in relation to maternal allergy and allergy
development in children up to 18 months of age. Pediatr Allergy Immunol
2007;18(1):20-26.
II
Sjögren Y. M, Jenmalm M. C, Björkstén B, Sverremark-Ekström E. Altered
early infant gut microbiota at species level in children developing allergy during
the first five years of life. Manuscript
6
ABBREVIATIONS.................................................................................................................... 8
INTRODUCTION...................................................................................................................... 9
INNATE AND ADAPTIVE IMMUNITY ............................................................................ 9
THE GUT ASSOCIATED IMMUNE SYSTEM................................................................. 10
Oral tolerance .................................................................................................................. 11
ALLERGY ........................................................................................................................... 13
Factors influencing allergy development......................................................................... 14
The Hygiene hypothesis.................................................................................................... 15
THE COMMENSAL GUT FLORA .................................................................................... 17
Factors influencing the composition of the gut flora ....................................................... 18
Infant gut flora ................................................................................................................. 19
Bacterial fermentation, prebiotics and probiotics ........................................................... 19
Gut flora and the immune system..................................................................................... 20
Gut flora and allergy........................................................................................................ 21
IMMUNOLOGY OF HUMAN MILK ................................................................................ 23
Breast feeding in relation to allergy development ........................................................... 24
Human milk oligosaccharides.......................................................................................... 25
PRESENT STUDY .................................................................................................................. 26
AIMS.................................................................................................................................... 26
MATERIAL AND METHODS ........................................................................................... 27
Study population study I................................................................................................... 27
Study population study II.................................................................................................. 28
RESULTS AND DISCUSSION .......................................................................................... 29
Study I............................................................................................................................... 29
Study II ............................................................................................................................. 30
Clinical relevance ............................................................................................................ 32
CONCLUDING REMARKS ............................................................................................... 34
ONGOING AND FUTURE PROJECTS................................................................................. 34
• STIMULATION OF CORD BLOOD MONONUCLEAR CELLS AND
PERIPHERAL BLOOD MONONUCLEAR CELLS WITH COMMENSAL
BACTERIAL DNA.................................................................................................. 34
• HUMAN MILK OLIGOSACCHARIDES IN RELATION TO INFANT GUT
FLORA AND ALLERGY DEVELOPMENT IN CHILDREN AT FIVE YEARS
OF AGE.................................................................................................................... 35
ACKNOWLEDGEMENTS ..................................................................................................... 36
REFERENCES......................................................................................................................... 37
7
ABBREVIATIONS
2´-FL
3-FL
APC
B.
CBMC
CD
Cl.
DC
E.
GALT
GF
HMO
HPLC
IFNγ
Ig
IL
ILF
L.
LDFT
LNDFH I
LNFP I
LNFP II
LNFP III
LNnT
LNT
LPS
MAMP
M cell
MLN
PCR
PP
PPR
PBMC
RA
SCFA
TFAN
TGFβ
TLR
Th
TNF
Treg
2´-Fucosyllactose
3-Fucosyllactose
Antigen presenting cell
Bifidobacterium
Cord blood mononuclear cell
Cluster of differentiation
Clostridium
Dendritic cell
Escherichia
Gut associated lymphoid tissue
Germ-free
Human milk oligosaccharides
High Performance Liquid Chromatography
Interferon γ
Immunoglobulin
Interleukin
Isolated lymphoid follicle
Lactobacilli
Lactodifucotetraose
Lacto-N-difucohexaose I
Lacto-N-fucotetraose I
Lacto-N-fucotetraose II
Lacto-N-fucotetraose III
Lacto-N-neotetraose
Lacto-N-tetraose
Lipopolysaccharide
Microbial associated molecular pattern
Microfold cell
Mesenteric lymph node
Polymerase chain reaction
Peyer´s patches
Pathogen recognition receptor
Peripheral blood mononuclear cell
Retinoic acid
Short chain fatty acids
4-Triflouroacetamidoaniline
Transforming growth factor β
Toll-like receptor
T helper cell
Tumor necrosis factor
T regulatory cell
8
INTRODUCTION
INNATE AND ADAPTIVE IMMUNITY
The immune system in mammals is characterised by innate and adaptive immune responses.
The innate immune response is the first defence against pathogens and involves anatomical
and physiological barriers, like the skin and temperature, as well as innate immune cells1.
These cells respond rapidly by phagocytosing pathogens and by secreting substances like
chemokines and cytokines which attract and activate other cells. The adaptive immune system
is slower, reacts to specific antigens and includes immunological memory. Adaptive
immunity is characterised by T and B lymphocytes as well as antibodies produced by the B
cells. There is a constant collaboration between the innate and the adaptive immune system.
Antigen presenting cells (APCs), like macrophages, dendritic cells (DCs) and B cells
recognize microbial associated molecular patterns (MAMPs) via pathogen recognition
receptors (PRRs)2. Common PRRs are Toll like receptors (TLRs) and 10 different TLRs have
been found in man. A putative ligand for TLR2 is bacterial peptidoglycan, whereas TLR4
recognises lipopolysaccharide (LPS) and TLR 9 recognises unmethylated CpG motifs in
bacterial DNA. APCs instruct naïve CD4+ cells to differentiate into either T helper (Th) 1,
Th2 or T regulatory cells (T regs). The different CD4+ T cells are characterised by their
cytokine production with Th1 cells producing mainly IFNγ, Th2 cells secrete IL-4, IL-5 and
IL-13 and different Tregs produce regulatory cytokines like IL-10 and TGFβ.
T and B cell clones have receptors which are specific for diverse antigens1.The B cell presents
the antigen to the activated CD4+ T cell which in turn produces cytokines activating the B
cell. The B cell thereby differentiates into an antibody secreting plasma cell. Apart from being
the antigen receptors on B cells, antibodies can bind antigens facilitating clearance,
complement activation and phagocytoses by cells like macrophages and neutrophils.
9
THE GUT ASSOCIATED IMMUNE SYSTEM
Mucosal epithelia are primary sites for antigen entry 3. Therefore mucosal associated
lymphoid tissue (MALT) is of vast importance for mounting immune responses towards
foreign antigens. There are more lymphocytes in the Gut Associated Lymphoid Tissue
(GALT) than in all other secondary lymphoid tissues combined4.The GALT is an immune
privileged organ with the difficult task to mount immune responses towards antigens
(pathogens) while at the same time being non-responsive to commensal bacteria and food
antigens. The primary barrier of the gut mucosa is the single layer of epithelial cells with the
underlying connective tissue, lamina propia 5. In the lamina propia the GALT has its main
site. The GALT consists of Peyer´s Patches (PP) in the small intestine and of Isolated
Lymphoid Follicles (ILF) in the colon4. Both PP and ILF are aggregates of B-cell follicles
with intervening T-cells. PP and ILF are the inductive sites where activation occurs whereas
the diffuse tissues of the lamina propia are effector sites where high amounts of IgA are
produced6.
There are mainly two types of cells in the gut mucosa which are able to present antigens to
cells in PP and ILF; DCs and Microfold (M) cells. The M cells are integrated in the epithelial
layer and transport live microbes and microbial material from the lumen. DCs, present in the
lamina propia, are able to open tight junctions in the epithelium making it possible for the DC
to send their dendrites into the gut lumen and sample antigens5. The DCs can then present
antigens to lymphocytes in PP and MLNs, but appear to never stray beyond this lymphoid
tissue and thus systemic infection is prevented5.
Secretory IgA (sIgA) is of immense importance for mucosal immunity. It agglutinates
bacteria (and other pathogens) which facilitates the clearance of the bacteria and thereby
prevents invasion of the body. As shown in antibody deficient mice, sIgA produced by GALT
B cells prevent the gut flora from breaching the gut mucosal barrier7. Large amounts of sIgA,
40-60 mg/kg, is produced in the gut lumen every day6. The classical way of activating IgAproducing B cells is T cell dependent and starts in PP where DCs present the antigens to
CD4+Th2 cells. These antigen-primed CD4+Th2 cells then produce IL-4 and TGFβ which
make the B cells undergo µÆ α switching8. IgA+ B cells differentiate further in the
mesenteric lymph nodes before they enter the blood stream6. It appears to be the ability of
10
GALT DCs to produce retinoic acid (RA) (which up regulate gut homing receptors, α4β7 and
CCR9, on the IgA primed B cells) that facilitates the B cell-trafficking to the lamina propia8.
There, the IgA primed B cells finally become IgA secreting plasma cells by the help of IL-5
and IL-6 produced by CD4+Th2 cells8. Lately a T cell independent pathway for the activation
of IgA producing B cells has been postulated8. Work by Mora and colleagues show that the
production of IL-5, IL-6 and RA by DCs in the PP provide a milieu for the B cells to start
producing IgA as well as up regulating gut homing receptors9. Peritoneal B1 cells have also
been shown to produce IgA without T cell help6, 8. It has previously been shown that the
production of commensal specific IgA is independent of T cell activity5.
Fig 1. The structure of the gut associated lympdoid tissue (GALT). Adopted from Kraehenbuhl JP, Corbett
M. Science. 2004; 303(5664):1624-1625. 5
Oral tolerance
The intestinal immune system encounters more antigens than any other part of the body10.
Antigens from pathogens should mount a strong immune response but on the other hand food
and commensal antigens should be tolerated. When not tolerated, diseases like inflammatory
bowel disease, coeliac disease and food allergy could occur. As mentioned above,
11
lymphocytes that are primed in the PP migrate to the MLN for further differentiation, then
enter the bloodstream and further accumulate in the mucosa. As with B lymphocytes, T
lymphocytes up regulate the adhesion molecules CCR9 and α4β7 which facilitates the
migration to the gut mucosa. Both CD4+ and CD8+ T cells inhabit the lamina propia but it is
primarly CD8+ T cells that migrate to the epithelium. Of importance for oral tolerance are
clonal deletion and clonal anergy of antigen-specific CD4+ T cells4. This appears to happen
after high-dose feeding. Many intestinal CD4+ T cells have been postulated to be regulatory T
cells and thus important for maintaining local tolerance towards environmental antigens10.
When IL-10 and TGFβ were depleted from lamina propia T cells, they lost their
unresponsiveness towards commensal bacteria indicating that T regulatory cells are involved
in the tolerance towards commensal antigens. The Th3 cells that produce TGFβ have been
isolated from mice MLNs after feeding small doses of antigen for tolerance induction10. Tr1
which produces IL-10 and CD4+ CD25+ T cells could also be important for tolerance
induction in the gut.
Mice undergoing mesenteric lymphadenectomy cannot induce oral tolerance which postulates
a central role for MLNs in oral tolerance induction4. It is not known whether tolerance to
commensals and food antigens is acquired in the same way11. Food antigens can be found in
systemic peripheral lymph organs, however when the MLNs are intact commensals never
stray beyond the MLNs. The commensal flora can also, in contrast to food antigens, signal
through TLRs which might be important for tolerance induction.
DCs are important regarding antigen presentation to T cells. Several different subtypes of
DCs have been identified in the different compartments of the GALT. The most abundant DC
subset in PP produces the anti-inflammatory cytokine IL-10. Furthermore PP DCs induce
antigen specific T cells to produce IL-10 and Th2 cytokines10. Lately some DCs that resemble
the plasmacytoid DC have been identified10. Plasmacytoid DCs have been described to be
tolerogenic in mice10. Interestingly, it has been proposed that DCs only partially mature when
encountering commensal and food antigens leading to T cells with T regulatory properties and
further local IgA production. However, when pathogens are encountered DCs fully mature
with the help of signals from macrophages, mesenchymal cells and epithelial cells. This leads
to Th1 and Th2 activation with inflammatory responses both locally and systemically as well
as local IgA production10. A study by Braat and collegues showed that receptors involved in
Th1 activation, e.g. CD40, are induced more frequently when DCs are matured in the
12
presence of the pathogen Klebsiella pneumoniae than when DCs are matured together with
the commensal Lactobacillus (L.) rhamnosus 12.
The gastrointestinal tract also constitutes the largest reservoir of macrophages in the body
(reviewed in13). These macrophages appear to be blood monocyte-derived but they loose
several receptors and adhesion molecules when they mature into intestinal macrophages.
Therefore they are non-responding to inflammatory stimuli like LPS. Also the intestinal
macrophages do not seem to present antigens to T cells. Instead, this is done by DCs. Still,
intestinal macrophages are good at phagocytosis and scavenging of bacteria.
ALLERGY
Today, atopic allergy is the most common chronic disease among children in the developed
world. In some countries the prevalence is as high as 20-25%14. The symptoms of atopic
allergy are hay fever, allergic rhinitis, gastrointestinal disturbances, asthma and eczema1.
Exposure to common environmental antigens such as plant pollen, animal proteins and house
dust can lead to an allergic response. It is not known why some individuals develop allergic
disorders. Genetic factors seem to be of importance (see below). Some people have a
hereditary predisposition, termed atopy, to the development of allergies. Atopic allergies are
IgE mediated and also called hypersensitivity type 1 or immediate hypersensitivity reactions.
Since the prevalence of atopic allergy has increased during the last decades genetics alone
cannot explain the development of the disorder15. The current view about atopic allergy is that
both genetic and environmental factors seem to interact with each other, leading to the
production of interleukin-4 (IL-4)16. IL-4 is one of the cytokines which play an important role
in developing naive T cells into CD4+ Th2 cells. The Th2 cells secrete IL-4 and IL-5 when
activated by an allergen. This induces the B cells to secrete IgE. Mast cells, eosinophils and
basophils have receptors for IgE1. The immediate allergic response occurs when the allergen
crosslinks IgE Fcε receptors on the mast cells. This leads to degranulation followed by the
release of histamine, heparin and proteases within minutes, all leading to the clinical
symptoms. The clinical symptoms arise from the contraction of intestinal and bronchial
smooth muscles, increased mucus production, vasodilatation and increased vascular
permeability. The eosinophils, the neutrophils and the basophils are responsible for the late
allergic response. This response occurs 6-24 hours after contact with the allergen and the
13
mediator´s, leukotrienes and prostaglandines, effects are more pronounced and long-lasting
than those of histamine.
Figure 2. The allergic reaction. The naïve Th cell is presented to an allergen. In a IL-4 rich mileu the Th cell
will develop into a Th2 cell and produce IL-4, IL-5 and IL-13. When a B cell encounters the allergen it will
produce IgE antibodies in this milieu. The IgE antibodies will attach to Fcε receptors on B cells and eosinophils.
Once the individual encounters an allergen once more this will lead to crosslinking of the Fcε receptors and
release of inflammatory mediators causing the allergic response. Th1 cytokines like IL-12 and IFNγ dampen the
Th2 response and the IgE production from B cells. Adopted from Wills-Karp M, Santeliz J, Karp CL. Nat Rev
Immunol.2001;1(1):69-75.16
Factors influencing allergy development
Parental allergy is a strong risk factor for allergy development17.The allergy status of the
mother is a stronger predictor for allergy development in the child suggesting that the in utero
environment might be of relevance. Polymorphisms in several genes are linked to allergic
disease18. They include e.g. pro- and anti-inflammatory cytokine genes, receptor genes and
HLA alleles. The many genes linked to atopic allergy reflect the complexity of the disease.
The dose, timing and route of allergen exposure seem to influence allergy development. The
foetus can be exposed to allergens already in utero18. It has been shown that maternal
exposure to high levels of birch pollen during pregnancy tends to increase the risk of birch
allergy in the children19. Also, the exposure to high birch pollen levels during the first months
of life significantly increases the risk for allergic asthma and positive skin prick test20,
postulating that the exposure in infancy might be of further importance. In addition, high
exposure to tobacco smoke is associated with childhood asthma and higher IgE production
from cord blood18.
14
The Hygiene hypothesis
In 1989 David Strachan developed the hygiene hypothesis. The base of this hypothesis was
epidemiological studies showing that children with older siblings developed atopic allergy to
a lower extent21. It was postulated that the lower incidence of atopic disease among these
children was due to higher microbial stimulation in these children. Matricardi et al showed
that the seropostivity for food and orofecal pathogens reduced the risk of developing atopic
allergy by 60%22. The association of respiratory infections and allergy is less clear. In one
study no association was seen between seropositivity against respiratory viruses and IgE
sensitization23. However, seropositivity towards the herpesvirus Epstain Barr Virus (EBV)
was negatively associated with IgE sensitization and this was further strengthened if the
subjects simultaneously were seropositive against cytomegalovirus23. Also, a recent study
showed that the presence of few IgG antibodies, against several infectious pathogens,
increases the risk of different atopic disorders24. Nevertheless, several other studies cannot
confirm that childhood infections protect from atopic disease (reviewed in25).
There is a lower prevalence of atopic allergy among children growing up on farm26. In this
environment children are more exposed to microbial products like endotoxin and bacterial
DNA27. Consequently, it might be the microbial products and not the infections in per se that
prevent allergic disease. Children exposed to higher levels of endotoxin are less frequently
allergic28. Interestingly, it was shown that mice with a deficient TLR4 receptor respond with
high IgE responses and anaphylactic shock when a food allergen coupled to an adjuvant was
administered 29. However, if the mice simultaneously were given CpG oligodeoxynucleotides
(mimicking bacterial DNA) no such responses were seen.
There appears to be an imbalance between Th1 and Th2 cytokines in individuals developing
atopic disorders (reviewed in30). The immune system of the neonate is somewhat Th2 skewed,
partly to achieve a successful pregnancy. The Th2 cytokine IL-4 is important for IgE
production in B cells. It is believed that a lower load of microbial products early in life leads
to a lower activation of TLRs on e.g. innate cells and subsequently to a poorer development of
Th1 cells. These cells secrete pro-inflammatory cytokines i.e. IFNγ which are able to down
regulate Th2 cytokines. Lower amount of Th1 cytokines therefore creates a milieu for allergic
sensitisation. It has been shown in vitro that substances mimicking microbial products switch
the allergen-specific response from Th2 to Th1 by increasing the production of Th1
15
cytokines31. Studies from our group further indicate an early immaturity of anti-microbial
immune responses by monocytes in children with allergic mothers, an impairment which
seems to persist during the first 2 years of life32,33.
Peripheral blood mononuclear cells (PBMC) from already allergic subjects appear to produce
high amounts of IL-4, IL-5, IL-9 and IL-13 after stimulation with both allergen and
polyclonal activators34, 35. However, the amount of Th1 cytokines produced by PBMC from
allergic subjects appears to differ depending on the stimuli. Polyclonal activators appear to
induce a lower production of Th1 cytokines by PBMC from allergic subjects than PBMC
from non-allergic individuals. Allergens might instead induce increased Th1 cytokine
responses34. Allergic subjects also appears to have an enhanced expression of the Th2
transcription factor GATA-3 and instead a decreased production of T-bet which is involved in
Th1 differentiation30.
There might be an imbalance in cytokine production in children developing allergic disease
already prenatally. One study showed a significantly lower proportion of IL-12 producing
cord blood mononuclear cells (CBMC) following allergen stimulation in children who
became IgE-sensitized at two years of age when compared to CMBC from non-sensitized
children36. However, others have shown that allergen stimulated Th1 cytokine production
from CBMC, does not correlate with sensitization at two years of age37. This study instead
postulates a role for the postnatal priming of T cells since sensitisation correlates with
significantly higher amounts of Th2 cytokines produced by PBMC from 6 month year old
infants.
T regulatory cells appear to dampen both Th1 and Th2 responses (reviewed in30). Cytokines
secreted by Tregs are anti–inflammatory cytokines like IL-10 and TGFβ. Treg cells producing
IL-10 have been shown to switch antibody production away from IgE towards IgG4. It has
been postulated that factors leading to poorer development of Treg cells, like insufficient
microbial stimulation, are involved in allergy development.
16
THE COMMENSAL GUT FLORA
The gut harbours around 800 different species of bacteria38. These species belong to nine
different phyla of bacteria with Firmicutes and Bacteroides being the most predominant. The
total amount of bacteria is estimated to weigh approximately one kg and outnumbers the
number of cells in the human body by a factor of ten39,
40
. The bacteria are differently
distributed along the gastrointestinal tract, with few species in the acidic stomach. The
bacterial density increases in the small intestine and the colon harbours the highest number of
bacteria, 1012 colony forming units (CFU)/ml38. Both anaerobic and aerobic genera of bacteria
harbour the gut 38, 40however the majority is strict anaerobes41. Bifidobacterium, Clostridium,
Bacteroides, Lactobacilli and Eubacterium are among the most common anaerobic genera of
bacteria40, 41 in the gut. Streptococcus, Enterococcus and Escherichia (E.), together with other
Enterobacter, are common aerobic genera of bacteria.
A polysaccharide rich mucus gel layer is overlying the gut epithelium. This mucus layer
provides carbohydrate motifs that enable microbes to attach to the mucus and form biofilms42.
The biofilm promotes several of the functions exerted by the microflora42. It also makes the
microbes resist the forces of gut peristalsis4. The gut microbiota is important regarding several
metabolic functions like synthesis of vitamins e.g. vitamin K and biotin. Furthermore, the
commensal flora ferments non-digestible carbohydrates (see below). Apart from being
important for immune system development (see below) the commensal flora also protects the
host from pathogens in additional ways. Lactic acid producing bacteria e.g. Lactobacilli and
Bifidobacteria lower the pH in the gut and create a milieu less friendly for pathogens. The
commensal flora also competes with pathogens for nutrients and receptors. It has been
postulated that the commensal flora can induce the production of antimicrobial peptides4. The
antimicrobial peptides shape the composition of the gut flora in the small intestine43. These
cationic peptides are produced by paneth cells in the crypts of the small intestinal mucosa and
disrupt the membrane of microbes. Dependent on different cationic activity different
antimicrobic peptides have bactericidal activity against diverse bacteria.
17
Fig. 3 Gut microbial ecology. Adopted from O'Hara AM, Shanahan F. EMBORep.2006;7(7):688-693.40
Factors influencing the composition of the gut flora
The composition of the intestinal flora is rather stable in healthy individuals44 but can be
altered by several factors like drugs, diet, stress, disease and aging. Mitsouka has studied the
gut flora in relation to age and show that Bifdobacteria, E. coli and Streptococcus are most
numerous in infancy (see below for further discussion on gut flora in infancy)44. Bacteroides
and Eubacterium appear thereafter and the numbers of E. coli and Streptococcus decreases. In
elderly people Bifidobacteria numbers are decreased, however other bacteria like Lactobacilli,
Clostridia, E. coli and Streptococcus increase in numbers. The consumption of various
antibiotics alters the gut flora45, 46. Numbers of Bifidobacterium and Bacteroides have been
shown to decrease after antibiotic consumption45. Clostridium (Cl.) difficile is regarded as a
pathogen but can persist in the lumen without causing disease. However, when the existing
community is changed e.g. by antibiotics it can give rise to pseudomembranousus colitis38.
The host genotype also appears to influence the diversity of the gut flora38.
Differing
genotypes could give rise to differing attachment sites for microbes. Also the major
histocompatobility complex genotype has been reported to influence the murine fecal
microbiota. Interestingly, sIgA has been proposed to be a mediator of bacterial selection. sIgA
does not only prevent microbes from increasing in numbers but might also be able to anchor
microbes to enterocytes47. This was demonstrated with a subset of sIgA that was able to
anchor cultured human fecal bacteria and E. coli to an enteroyte-like cell line. Microbes that
are responsible for the de-glycosylation on enterocytes could be creating binding sites for
sIgA which makes this anchoring possible42. The possibility that there is a host selection for
specific bacteria indicates cooperation between the host and the bacteria. The term
18
commensal, which means that one part benefits while the other part is unaffected, is therefore
misleading and one would rather refer to the gut bacteria as mutualistic48.
Infant gut flora
The foetus is sterile in utero but the colonisation starts immediately after birth49. It has been
shown that the type of delivery influences the gut flora. Neonates born with caesarean section
often have less Bifidobacteria and Bacteroides45 and are colonised later with E.coli,
Lactobacilli, Bacteroides and Bifidobacterium (B.) species50, 51. It can take up to over one
month before infants born with caesarean section have similar levels of these bacteria
compared to those of vaginally delivered infants51. A long vaginal delivery appears to
increase the chance of finding live bacteria in the mouth and stomach of newborn infants49.
Neonates born with caesarean section are initially mainly exposed to bacteria in the hospital
environment and of nursing staff. Studies have been performed to investigate infant gut flora
in developing countries compared to developed countries. A higher prevalence of Ethiopian
and Estonian infants harbour Lactobacilli than Swedish infants52, 53. Adlerbeth et al showed
that Pakistani infants were colonised earlier than Swedish infants and also harboured a more
diverse enterobacterial flora54. The hygienic procedures in developed countries might be
responsible for this difference which also might have unfavourable consequences (see below).
The early colonisers E. coli and Streptococcus followed by Bifidobacteria and Bacteroides
appear to be influenced by the diet. In breast-fed infants higher numbers of Bifidobacteria can
be detected55 whereas Clostridia have been found in higher numbers in formula-fed infants49.
Streptococcus, Bacteroides and enterobacteria might also be higher in formula-fed infants49.
Microflora associated characteristics also appear to differ between breast-fed and formula-fed
infants56. During weaning the gut flora of previously breast-fed infants assumes the phenotype
of formula-fed infants and when only solid food is given the gut flora resemble the adult gut
flora.
Bacterial fermentation, prebiotics and probiotics
Several attempts have been made to affect the composition of the gut flora. As mentioned
above the diet appears to influence the gut flora. Non-digested carbohydrates like dietary
19
fibers, resistant starch and oligosaccharides are main fermentable dietary substrates57. The end
products produced following this fermentation are mainly short chained fatty acids (SCFA)
and several gases58. Bifidobacteria and Lactobacilli are producers of lactate and therefore
often called lactic acid producing bacteria44. Bifidobacteria also produce acetate. Acetate and
lactate are common fermentation products in breast-fed infants58. Genes involved in
poly/oligosaccharide metabolism have been shown to account for 8% of the genome of B.
longum however Bacteroides thetaiotaomicron have even more genes responsible for
polysaccharide breakdown59. The diversity of these genes is important for the substrate
utilization of different bacteria.
Prebiotics are defined as “non-digestible food ingredients that beneficially affects the host by
selectively stimulating one or a limited number of bacterial species already resident in the
human colon”57. Prebiotics such as inulin and fructo-oligosaccharides have been shown to
function as substrates for Bifidobacteria and Lactobacilli and thereby increasing their growth,
but investigations on how the whole gut microbiota is affected by prebiotics are lacking.
Nevertheless prebiotics have been shown to improve gut health by increasing faecal weight
and thereby increase gut peristalsis counteracting constipation60.
Probiotic bacteria are live orally administered bacteria with potential health benefits61.
Mainly, species of Bifidobacteria and Lactobacilli have been used as probiotics. Probiotics
have been postulated to have many effects but are so far only concluded to have effects on
lactose intolerance (since they increase the production of lactase) and some types of
diarrhoea. Still, there is no conclusive evidence that probiotics actually colonise the gut thus
further research is needed62.
Gut flora and the immune system
To investigate the role of the commensal flora germ-free (GF) animals have been used. These
animals have alterations in their intestinal morphology and thicker muscle cell wall compared
to animals reared conventionally40, 63. The large intestine of these animals accumulates mucus
due to the lack of mucus-degrading enzymes and they therefore have a larger caecum. The
GALT of GF animals is poorly developed11 and these animals are more susceptible to
infections40. Also other peripheral lymphoid organs in GF mice contain structural defects4.
20
The CD4+CD25+ T cells of GF mice elicits lower suppressor activity than CD4+CD25+ T
cells from conventional mice64. Neonatal and GF mice have very few IgA+ B cells6. However,
after colonisation increased populations of IgA+ B cells can be detected. Also increases in
IgA, both total and commensal specific, can be seen after colonisation63. Several species like
sheep, cattle, rabbits and pigs are dependent on the GALT for the final somatic diversification
of the antibody genes. In rabbits it is only in the presence of intestinal microbiota that the Blymphocytes proliferate and produce diverse antibodies65. It has been shown that the biofilm
formation of commensal bacteria is important for the maturation of B cells in the GALT of
rabbits65.
Several studies have investigated the role of the commensal flora regarding intestinal
homestasis and oral tolerance66,
67
. Following gut injury, mice with a disturbed gut flora
produce less cytokines compared to mice with a normal gut flora66. Also, in the same study it
was shown that mice deficient in MyD88, an adaptor molecule essential for TLR-mediated
induction of inflammatory cytokines, produce significantly less cytokines than wild type mice
after gut injury. This postulates a role for the commensal flora in intestinal homeostasis and
demonstrates that the activation of TLRs is important for the recognition of the commensal
flora. Germ-free mice have also been shown to produce low amounts of IFNγ and IgG2a after
tolerogenic doses of ovalbumin (OVA) followed by a systemic challenge with OVA67.
Instead, these mice produce high amounts of IgE, IgG1 and IL-4. When Bifidobacterium
infantis was introduced to GF mice the IFNγ and IgG2a response was restored in neonatal
mice but not in adult mice67. This postulates a role for the commensal flora in early tolerance
induction. Interestingly, also administration of LPS to germ-free mice has been shown to
restore the induction of oral tolerance4. The proportion of CD4+ T cells in GF mice appears to
be affected systemically64, 68. However, a polysaccharide from Bacteroides fragilis was able
to restore the proportion of CD4+ T cells in the spleen of GF mice68. GF mice are also
somewhat Th2 skewed and this polysaccharide is able to drive the T cell development
towards a Th1 phenotype.
Gut flora and allergy
The gut flora could have changed according to increased hygiene conditions. There appears to
be an early colonisation with “skin bacteria” like Staphyloccous today compared to decades
ago69. Also, differences in gut flora between children from affluent countries compared to
21
developing countries postulates a role for increased hygiene as a modulator of the gut flora5254
. Differences in gut flora have also been shown between children raised with an antroposofic
life style with more fermented food and less use of antibiotics46. Antroposophic children less
often develop allergies compared to children raised during conventional conditions.
In the late 1990: s studies postulated a role for the gut flora in relation to allergy development.
Children who were allergic at two years of age were less often colonised with Bifidobacteria
and Lactobacilli whereas they had higher counts of coliforms and Staphylococcus aureus70.
Also, species of Bifidobacteria appear to differ between allergic and non-allergic children.
Colonisation with B. adolescentis, was more common in children with atopic allergies as
compared to non-allergic children who were colonised with B. infantis, B. bifidum and B.
breve to a higher extent71. However, Murray et al did not see any differences, regarding
species of Bifidobacteria, when they compared the faecal microbiota between sensitized
wheezy and non-sensitised non-wheezy children72. Yet, they found that children with eczema
had lower amounts of Bifidobacteria. These studies indicate that there might be a difference in
the gut flora between allergic and non-allergic children.
Nevertheless, these studies do not explain whether it is potential differences in the infant gut
flora that drives development of atopic disease or if atopic disease might influence the gut
flora. Therefore it is also relevant to study the infant gut flora and relate to subsequent allergy
development. Such prospective studies have mainly been focusing on genera of bacteria and
show interesting but not quite concurrent results. One study showed that children who had
become allergic at the age of two years were less often colonised with enterococci and
Bifidobacteria but more frequently colonised with Staphylococcus aureus and had higher
counts of Clostridia already as infants39. Kalliomäki et al did also show that children who had
become allergic at 12 months of age had higher counts of Clostridia at three weeks of age73.
No other genera of bacteria appeared to differ but the fatty acid profile of bacteria were
different between the two groups suggesting that children who develop allergic disease have a
different gut flora composition. A large study by Adlerbeth et al showed that increased total
IgE in serum at 18 months correlated with later colonisation by Lactobacilli species50. Lately,
the KOALA study has shown alterations in Clostridium difficle and E. coli colonisation at 1
month in children who develop allergy during their first 2 years74. However, no alterations in
total Bifidobacteria74 or at Bifidobacterium species level75 were seen between infants who
developed allergy compared to infants who did not. Evaluation of allergic outcome was
22
performed early in all of these studies (at 12, 18 or 24 months). Therefore, later development
of allergic disease is not considered.
IMMUNOLOGY OF HUMAN MILK
Human milk is the ideal nutrition for infants. It contains optimal proportions of the
macronutrients; carbohydrates, lipids and proteins, as well as micronutrients such as vitamins
and minerals. Apart from containing nutrients important for the growth of the infant, human
milk also contains several immunomodulatory components76. The immune system of the
infant is far from developed at birth and therefore human milk IgA, lactoferrin,
oligosaccharides, leukocytes, cytokines and lysozyme play a substantial role in protecting the
infant from pathogens77. The production of Ig isotypes is impaired at birth and IgG present in
the circulation is mainly of maternal origin78. Maternal IgG acquired via the placenta is
mainly catabolised two months after birth making human milk sIgA even more important
against mucosal infections at this time79. Since the lactating mammary glands belong to the
mucosal immune system, the human milk antibodies reflect the antigens the MALT of the
mother has been stimulated with79. Therefore, the sIgA in human milk protects the infant from
the antigens the mother has been exposed to. The infants own production of sIgA is rare
during the first ten days after birth indicating the importance of microbial stimulation of the
GALT for sIgA production79. Thereafter, numbers of sIgA producing plasma cells increases
and peak around 12 month after birth.
Several different leukocytes are present in human milk (reviewed in77). They are most
predominant in colostrum decreasing during the course of lactation. Macrophages and
neutrophils are most common but also lymphocytes, mainly T cells, are present. The human
milk macrophages express activation markers and appear to activate the infant’s lymphocytes,
while the role of the neutrophils in human milk is not known.
Both anti- inflammatory (e.g. IL-10, TGFβ) and pro-inflammatory (e.g. IL-1β, IL-6, IL-8,
TNFα) cytokines are present in human milk (76, 77). They mainly arise from production in the
mammary gland, although the leukocytes in human milk contribute to the production of
cytokines77. The physiological role of these cytokines is not completely understood since it is
not known to which extent the cytokines survive the passage through the stomach. Still the
23
cytokines in human milk could be of importance for the development of the immune system
in infants (see below).
Lactoferrin is a major protein in human milk80. It chelates free iron leading to increased
absorption of iron for the infant as well as making this nutrient unavailable for bacteria.
Another antimicrobial peptide in human milk is lysozyme which is able to break bonds in
peptidoglycan, a component in the cell wall of bacteria.
Exosomes have also been found in human milk81. These human milk exosomes have been
found to increase the number of Foxp3+CD4+CD25+ T regulatory cells and also to regulate
the production of several pro-inflammatory cytokines.
Breast feeding in relation to allergy development
It has been estimated that 13% of the world deaths of children below five could be prevented
if the World Health Organisation’s breast feeding guidelines (exclusive breastfeeding during
the first 6 months) were followed76. However, the role of breastfeeding in allergy prevention
is controversial. One meta-analysis, regarding exclusive breastfeeding for the first 3 months,
showed a decreased risk of breastfeeding on development of allergic rhinitis82. The effect was
however not that pronounced for children with atopic heredity. Kull et al showed that
exclusive breastfeeding for 4 months or more decreased the risk of developing eczema and
asthma83, 84 However, another large Swedish study showed that breastfeeding did not protect
children from atopic dermatitis during their first year85. Therefore, it is debated whether the
different immunomodulatory components in human milk differ between mothers. Indeed,
some studies show that low levels of sIgA is associated with increased risk of cow’s milk
allergy in infants, however not all studies confirm this86. Levels of antigens like casein, egg
proteins and peanut proteins have been detected in human milk though it is not clear if they
have any role in allergy development86. The composition of polyunsaturated fatty acids in
milk might differ between allergic and non-allergic mothers and could also be related to
allergy outcome in children87. The levels of cytokines might vary between allergic and nonallergic mothers with significantly higher IL-4 concentrations in the milk from allergic
mothers88. A recent study from our group indicates that mothers with higher exposure to
24
infectious, before the age of ten, have increased levels of TGFβ1, IL-2 and IL-8 in their breast
milk89.
Human milk oligosaccharides
Human milk oligosaccharides (HMOs) are the third most abundant solid component in human
milk after lactose and lipids90. Colostrum contains roughly 20g HMOs/L while the
concentration in mature milk is lower, 12-14g/L
91
. HMOs are complex carbohydrate
structures (3-10 monosaccharide units) that are synthesised in the mammary gland by
glycosyl- and fucosyltransferases92. HMOs contain lactose in the reducing end91, 93. Lactose
consists of the two monomers D-glucose and D-galactose. Other monomers of HMOs are Nacetylglucosamine, L-fucose and sialic acid. Different enzymes combine these monomers into
manifold structures. Approximately 130 different HMOs have been characterised so far94. Of
these are roughly 90 % neutral and the rest acidic. It is the secretor status and the Lewis blood
group of the mother that determines the pattern of oligosaccharides in the breast milk.
Therefore, at least four different HMO patterns exist94. Approximately 77% of Caucasians are
secretors meaning they have the enzyme α1-2 fucosyltransferase93. This enzyme is needed for
the formation of 2-fucosyllatose (2-FL) and lacto-N-fucopentaose I (LNFP I). Non-secretors
therefore lack these HMOs. Other common HMOs are (Lacto-N-tetraose (LNT) and Lacto-Nneo-tetraose (LNnT) which form core structures important for longer HMOs like LNFP I, II,
III and Lacto-N-difuco-hexaose I (LNDFH I). 2-FL together with 3-Fucosyllactose (3-FL)
and Lactodifucotetraose (LDFT) are shorter oligosaccharides.
The HMOs have been shown to be resistant towards digestive enzymes and therefore reaches
the intestines intact95. The high concentrations of HMOs combined with the fact that they are
resistant to digestion have raised the question of the function of these glycans. As yet the
biological function of HMOs is not fully understood94. However several studies postulate two
important functions of these oligosaccharides80, 94. The HMOs are assumed to be the major
components responsible for the differences seen between breast fed and formula fed infants
(see above)94. HMOs function as the first prebiotics since they are fermented by bacteria like
B. infantis96. The N-acetyl-glucosamine containing oligosaccharides have been shown to be
essential for the growth of a subspecies of B. bifidum62. Also Coppa and collegues have
25
shown that high concentrations of oligosaccharides in milk correlate with a higher diversity of
Bifidobacterium species92.
Interestingly, HMOs also appear to act as anti-adhesion agents inhibiting pathogens from
binding to gut epithelial surfaces31,
90
. As mentioned above the HMOs are synthesised by
glycosyl- and fucosyltranferases. These enzymes are also responsible for the formation of the
glycans present on different cell types. Therefore HMOs resemble glycans on human cells.
Pathogens use these glycans to adhere to epithelial cells. When free HMOs are present in the
gut lumen the pathogens bind to these and therefore HMOs decrease the infection load of
orofeacal pathogens. Indeed, HMOs have been shown to protect infants from infectious
diarrhoea97. High concentrations of α1-2 linked HMOs have been shown to be most
protective.
PRESENT STUDY
AIMS
Several studies postulate a role for the infant gut flora in relation to the development of the
immune system and subsequent allergy development. Also, the role of breast feeding in
allergy prevention is debated. The overall aim of this thesis was therefore to investigate the
infant gut flora and the consumption of human milk oligosaccharides, which stimulate species
of the gut flora, and relate to allergy development in children.
The specific aims of each study were to:
Study I: Investigate whether the colostrum from allergic and non-allergic mothers differ in
composition regarding neutral oligosaccharides and if there is a difference in the consumption
of colostrum oligosaccharides between neonates who at the age of 18 months had developed
allergic disease compared to neonates who had not developed allergy at 18 months.
Study II: Study the presence and amounts of four Bifidobacterium species, 2 groups of
Lactobacilli, Cl. difficile and Bacteroides fragilis in infant faecal samples collected at 1 week,
1 month and 2 months of age and relate to the development of allergic disease in these
children at five years of age.
26
MATERIAL AND METHODS
The methods of the studies are further discussed in each paper. The different study
populations in each study are described below.
Study population study I
Mothers attending the Antenatal Health Care Centres in Linköping August 1993 to March
1996 and in the beginning of 2004 were invited to participate in a prospective study of the
development of atopic symptoms in relation to environmental factors and maternal immunity.
The children were born between January 1994 to July 1997 and June to October 2004. All the
children were delivered at term and they had an uncomplicated perinatal period. Mothers who
breast-fed their babies for less than 3 months were excluded.
Clinical examinations and skin prick tests (SPT) against fresh hen’s egg, milk and extracts
from cat and peanut (ALK, Denmark) were carried out at 6, 12 and 18 months of age, and
whenever allergic symptoms were suspected. At these appointments the parents also
completed a questionnaire regarding clinical symptoms, nutrition and allergen exposure to
pets of their babies.
The diagnosis of atopy in the parents was based on a convincing clinical history of bronchial
asthma, allergic rhinoconjunctivitis, atopic eczema and food allergy. The mothers were
classified as allergic if they showed clinical symptoms of allergic disease and had circulating
IgE antibodies against a panel of common allergens, measured with allergy screen test (Magic
Lite™, ALK, Hørsholm, Denmark) or Phadiatop (Pharmacia CAP System RAST FEIA,
Pharmacia Diagnostics AB, Uppsala, Sweden). Mothers not reporting clinical symptoms of
allergy and having a negative allergy screen test or Phadiatop were classified as non-allergic.
Children were classified as non-allergic when they did not show any symptoms of allergy and
had a negative SPT. Children were classified as allergic if they showed clinical symptoms of
allergic disease.
The SPT was considered to be positive if the mean diameter of the wheal reaction was ≥3
mm. Atopic eczema was defined as pruritic chronic or chronically relapsing dermatitis with
typical morphology and distribution. Food allergy was defined as a positive skin prick test,
combined with a positive clinical history of immediate skin and/or gastrointestinal reactions
or atopic symptoms upon exposure to a certain food and clinical remission of atopic
27
symptoms on an exclusion diet. Breast-feeding was defined as exclusive when all cows milk
formula, except for extensively hydrolysed formula (i.e. Nutramigen™, Bristol Meyers), were
avoided.
From the original study population, we randomly selected 20 women (11 allergic and 9 nonallergic) and 20 children. Eleven of these children were non-allergic and nine showed
symptoms of atopic eczema, food allergy or both (table 1).
The local Ethical Committee at the University Hospital, Linköping, Sweden, approved the
study.
Study population study II
This study population has been described in further detail by Voor et al98. Pregnant women
and their families attending maternity clinics in Linköping, Sweden were invited to participate
in a prospective study of the development of atopic symptoms in relation to environmental
factors. The children were born during March 1996 to October 1999. All the children were
delivered at term and they had an uncomplicated perinatal period.
A clinical examination of the babies was made at 3, 6, 12, 24 and 60 months of age. At these
occasions questionnaires were completed regarding symptoms of allergy, use of antibiotics
and incidence of infections. Atopic eczema was defined as pruritic chronic or chronically
relapsing dermatitis with typical morphology and distribution99. Asthma was defined as three
or more episodes of bronchial obstruction during the last 12-month period, of which at least
one should be verified by a physician. Allergic rhinitis/conjunctivitis was defined as rhinitis
and/or conjunctivitis following at least twice in one hour after allergen exposure and not in
relation to infection. Urticaria was defined as allergic if it appeared at least twice in one hour
after allergen exposure.
Also SPT were performed at the follow-ups. These included standardized allergen extracts
with cow´s milk- and egg-, cat-, dog-, timothy- and birch allergen (ALK, Horsholm,
Denmark). The SPT was considered to be positive if the mean diameter of the wheal reaction
was ≥3 mm.
Children regarded as allergic had shown symptoms of allergic disease during their first five
years as well as positive SPT. Non-allergic children had not shown any symptoms of allergic
disease nor a positive SPT during their first five years. Inclusion in this study was based on
availability of faecal samples and allergic status during the first 60 months. Thirty seven
28
children were included in total. Fourteen of these children had developed allergy during their
first five years while 23 had not. None of the children included in this study had received oral
antibiotics during their first 3 months. All children, except two in each group, were
exclusively breastfed for at least 3 months. Two children in the non-allergic group were
delivered with caesarean section. The rest of the children were delivered vaginally.
The study was approved by the Regional Ethics Committee for Human Research at Linköping
University. The parents of all children gave their informed consent in writing.
RESULTS AND DISCUSSION
Study I
Some components in human milk appear to differ between allergic and non-allergic mothers87,
88
. Oligosaccharides are the third most abundant solid component in human milk90. In order to
investigate whether maternal allergy influences the content of these, the concentration of the
neutral oligosaccharides LNDFH I, LNFP I, LNFP II, LNFP III, LNT, LNnT, 3-FL, 2´-FL
and LDFT were analysed in colostrum samples from allergic (n=11) and non-allergic (n=9)
women. The colostrum samples were collected at the same time of the day and at the same
time in the lactation period, 2-4 days post partum. In accordance with previous studies97, 100,
101
, 2´-FL and LNFP I were the most abundant neutral oligosaccharides. There was no
difference in the amount of neutral oligosaccharides in the colostrum from allergic compared
to non-allergic mothers. This suggests that the amount of neutral oligosaccharides in human
milk is not influenced by the allergy status of the mother.
The variation in oligosaccharide concentration in milk from different women was immense.
This variation is in accordance with previous studies and varies according to the secretor
status, the ABO blood group type, and the Lewis blood group type102. It is the same enzymes,
glycosyltransferases including fucosyltransferases, which are necessary for the production of
breast milk oligosaccharides that are involved in the production of the cell surface glycolipids
that determine blood group type in erythrocytes102. Also, these enzymes are responsible for
the production of cell surface glycoconjugates, which can bind particles and pathogens.
Consequently, blood group phenotype and secretor status have been associated with certain
29
diseases i.e. urinary tract infection and asthma102,
103
. Additionally, high amounts of α1,2-
linked fucosylated oligosaccharides appears to protect the infant from several types of
diarrhoea97. Consequently, the variation in HMOs could be of medical importance.
The oligosaccharides in human milk have been shown to be resistant toward digestive
enzymes and therefore reach the intestines intact95. This in combination with the fact that they
occur in high concentrations postulate a significant role for the HMOs in the intestine. They
might function as the first prebiotics by increasing the growth certain commensal gut
bacteria94,
96
. Also, as mentioned above, HMOs might prevent the binding of certain
pathogens to the mucosa and thus lowering the infection rate by these pathogens90, 95. These
two functions postulate a role for the oligosaccharides in early immunity making it relevant to
study oligosaccharide consumption by infants in relation to allergy development in these
children. Our results indicate that children who had developed allergic symptoms at the age of
18 months tended to have consumed colostrum with higher concentrations of neutral
oligosaccharides in total, although the difference did not reach statistical significance
(p=0.12). This tendency could however not be attributed to any specific neutral
oligosaccharide. The fact that oligosaccharides in human milk act as receptor homologues97,
102
and thus prevent pathogens from binding to the cells in the mucosa implies that a high
amount of neutral oligosaccharides protects against orofecal pathogens. Interestingly, it has
been shown that infections with food and orofecal pathogens were able to reduce the risk of
atopy with 60%22. Accordingly, the ability of human milk oligosaccharides to bind pathogens
and serve as anti-infective agents might influence the development of the immune system
more than their ability to stimulate gut microbiota-immune interactions.
Study II
The commensal gut flora appears to be important for oral tolerance induction and gut
homeostasis66,
67
. Previous studies have shown differences in the infant gut flora between
children who develop allergic disease compared to children who do not39, 73, 74. However, this
has not been confirmed to such an extent by others50. By using Real-time PCR we
investigated the presence and amounts of four different Bifidobacterium species, two groups
of Lactobacilli, Cl. difficile and Bacteroides fragilis in faecal samples collected from infants 1
week, 1 month and 2 months old. This was related to the allergy outcome in these children up
30
to five years of age. Interestingly, infants who harboured Lactobacilli group I (L. rhamnosus,
L. paracasei, L. casei) at 1 week of age significantly less frequently developed allergic
disease than infants who did not harbour these bacteria at the same time (p=0.004). Also, the
presence of these bacteria at several time points was more common among non-allergic
children suggesting that a persistent colonisation with these species of Lactobacilli might be
of importance. Children that developed allergic disease also significantly less commonly
harboured B. adolescentis as one month old infants (p=0.008). In comparision, B. adolescentis
has been shown to be more common among children with an already established allergic
disease compared to non-allergic children71.
Mainly genera of bacteria in infant faecal samples have been studied in relationship to the
development of allergy in children previously39, 73, 74. This might explain why some studies
show an association with certain gut bacteria and allergy development while other studies do
not show this difference. We showed differences at species level namely that Lactobacilli
group I (L. rhamnosus, L. paracasei, L. casei) and B. adolescentis were less common in
faeces of allergic infants at one week and one month respectively. Indeed different species of
these bacteria might elicit differing cytokine patterns. Several species of Bifidobacteria, e.g.
B. bifidum and B. infantis, have been shown to be good inducers of IL-10 production by
macrophages104. B. adolescentis and B.longum did instead appear to induce TNF, IL-6 and IL12 production. Diverse Bifidobacterium species have also been shown to induce activation
markers (CD83, CD86) on DCs generated from PBMCs. Regarding the DCs it was only B.
bifidum, B.longum and B. pseudocatenulatum that induced IL-10 production105. A balanced
Bifidobacterium flora might therefore induce appropriate cytokine responses. Lactobacillus
rhamnosus stimulated DCs have been shown to induce hyporesponsive T cells106. The study
by Smiths et al show that some lactobacilli species (L. reuteri, L. casei) bind to the PRR DCSIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) on DCs
leading to priming of IL-10 producing Tregs107. Taken together these studies postulate that
different commensal bacteria are responsible for various cytokine responses. A diverse gut
flora might therefore be of importance for a balance in cytokine production which could be
important for the development of a fully functional immune system.
It is important to consider that we do not gain full insight into the entire gut microbiota when
analysing bacteria from faecal samples. Different bacteria might adhere at various sites within
the diverse compartments of the gut. These bacteria might therefore exert different functions
31
throughout the gut. Also, aerobe bacteria might appear to exist in higher numbers when faeces
are studied instead of biopsies from the more anaerobe gut108. Another useful tool, for
studying differences in the gut flora between two populations, is to use microflora-associated
characteristics. This method measures the function of the microbes since metabolic products,
like short chained fatty acids, are studied. Böttcher et al have investigated gut flora associated
characteristics and indeed found differences between already allergic and non-allergic
children109.
Clinical relevance
By studying the infant gut flora in relation to allergy development we might gain insight into
which bacteria that might be of importance in allergy prevention and thus appropriate
probiotic candidates. Studies have investigated the role of giving probiotics from birth (or
prenataly to pregnant mothers) and subsequent development of allergic disease110,
111
.
Interestingly a strain of L. rhamnosus, given to pregnant mothers and their infants, reduced
the frequency of eczema in the children indicating that this species might be important in
eczema prevention110. However, total IgE or positive skin prick test did not differ between
the probiotic or the placebo treated group. A more recent double-blind randomised placebocontrolled trial investigated the role of Lactobacillus reuteri administration to mothers
prenataly and to their infants for their first 12 months111. The prevalence of eczema outcome
and asthma did not differ between the two groups however the probiotic group had
significantly lower proportion of IgE associated eczema. Studies showing no association with
probiotics have also been completed112. Today there is therefore still not enough data to
recommend probiotics in the prevention of allergic disease113.
Importantly, since new research is constantly emerging new insights into the complexity of
the gut flora the correct way might not be to administer one strain of probiotic bacteria but to
increase the diversity of the gut flora by several means like promoting breast feeding, vaginal
delivery and even prebiotics. Indeed one double-blind randomised placebo-controlled trial
study where several probiotics and prebiotic galacto-oligosaccharides were administered
showed promising results regarding eczema and atopic eczema outcome at two years of age,
however the cumulative incidence of atopic disease was not decreased114. Our study regarding
consumption of colostrum oligosaccharides did not show any effect on allergy development at
32
18 months of age. However this pilot study was relatively small and measured the allergy
outcome at a relatively early age. Additional studies might be of importance for studying the
impact of HMOs on the gut flora and subsequent allergy development. Human milk has also
been shown to modulate the TLR responses which are of importance in the recognition of
microbes. Additional substances in human milk might also stimulate species of the gut flora91.
Therefore, it might be of relevance to promote breastfeeding also for its ability to stimulate
gut flora immune interactions.
33
CONCLUDING REMARKS
•
The content of neutral oligosaccharides in colostrum does not depend on maternal
allergy, nor does consumption of colostrum with high amounts of oligosaccharides
protect children against allergy development.
•
Infants who harbour Lactobacilli group I (L. rhamnosus, L. paracasei, L. casei) at 1
week of age and B adolescentis at 1 month of age significantly less frequently develop
allergic disease compared to infants who do not harbour these bacteria at the same
time. This might imply the importance of a diverse gut flora early in life for the
development of a fully functional immune system.
ONGOING AND FUTURE PROJECTS
STIMULATION OF CORD BLOOD MONONUCLEAR CELLS AND PERIPHERAL
BLOOD MONONUCLEAR CELLS WITH COMMENSAL BACTERIAL DNA.
The intracellular pathogen recognition receptor TLR 9 is expressed in B cells and pDCs115.
Following TLR 9 activation with e.g. bacterial DNA and synthetic CpG oligodinucleotides,
mimicking bacterial DNA, these cells produce cytokines like IL-6, IL-10 and type 1
interferons. This further activates other cells like NK cells, monocytes and neutrophils
resulting mainly in production of Th1 cytokines115. CpG oligodinucleotides have been shown
to directly inhibit IgE and IgG1 synthesis in B cells116. Some trials, investigating the use of
synthetic CpG oligodinucleotides as allergy vaccine, have been performed with promising
results117. The unmethylated DNA from different bacterial species appears to lead to different
cytokine responses largely depending on the different frequencies of CG dinucleotide
content118. We have investigated the presence and amount of bacterial DNA in faeces from
infants developing and not developing allergies and indeed found differences. We are
therefore interested in studying the cytokine response following activation of PBMC and
CBMC with DNA from different commensal bacterial species. We have previously shown
that PBMC and CBMC produce IL-6 and IL-10, after incubation for 24 hours, when
stimulated with at least 5µg bacterial DNA/ml. DNA from different bacterial species appears
to induce production of different amounts of cytokines, however an increase in concentration
might be needed to further investigate this. We will therefore continue this work and use
higher concentrations of DNA from different species, incubate for several time points and
34
measure IL-2, 4, 6, 10, TNF and IFNγ with cytometric bead array. We also have the
possibility to measure the cytokine production with Luminex to detect additional cytokines.
We will evaluate whether bacterial DNA from various commensal species induces varying
cytokine responses and whether PBMC and CBMC respond differently towards the stimuli.
Human intestinal epithelia express TLR9 mRNA119. However, whether intestinal epithelia
respond to CpG oligodinucleotides is not quite clear and might differ between primary
epithelia and cell lines119. Therefore we will also stimulate epithelial cell lines, foetal and
adult, with commensal bacterial DNA. Syntetic CpG oligodinucleotides will be used as
positive reference control.
HUMAN MILK OLIGOSACCHARIDES IN RELATION TO INFANT GUT FLORA
AND ALLERGY DEVELOPMENT IN CHILDREN AT FIVE YEARS OF AGE.
Our study regarding consumption of colostrum oligosaccharides did not show any effect on
allergy development up to 18 months of age. However this pilot study was relatively small
and measured the allergy outcome at a relatively early age. In our second study we
investigated the early infant gut flora in relation to allergy development up to five years of age
and found differences at species level. Since human milk is the only food during the first two
months it might influence the early gut flora. Interestingly, also breast milk consumed by the
children in study II has been collected. Therefore, using methods described in paper I, we will
measure the neutral oligosaccharide content in colostrum and mature milk consumed by these
children. These results will then be related to the early gut flora and to the allergy
development up to five years of age in these children.
35
ACKNOWLEDGEMENTS
I am most grateful to my supervisor Eva Sverremark Ekström for being the best supervisor a
PhD student could ever have. I would like to thank her for giving me the opportunity to do a
PhD, for believing in me and for pushing me in the right direction.
I would also especially like to thank Bengt “Muck” Björkstén for inspiring me and for all
interesting discussions. I am also grateful to him for introducing us to the Linköping staff.
Another person without whom this work would not have been possible is Frank “Zeke”
Lindh. Thank you for bringing me to Uppsala and Isosep and teaching me the procedure of
HMO analysis. Thanks also for the fun we had there and for keeping the “godisskål” filled!
I am also very grateful to Karel Duchén and Maria C Jenmalm at Linköping University
Hospital for supplying me with samples and for interesting discussions. Your previous work
has also been an inspiration.
My room mates Yvonne, Nora and Petra should also be acknowledged. Thank you for letting
me ask “stupid” questions and for being great friends! I would also like to thank Shanie, Anna
and Lisa for fun lunches and the nice times outside work.
I would like to thank all present and former staff at the Immunology department.
Thank you, Anette, Thomas, Ulrika, Björn, Pär and Håkan at Isosep for nice lunches, fun
parties and all the gossip!
Mina underbara vänner, tack! För att ni aldrig tröttnar på att prata. För allt galet och alla
skratt.
Jag är väldigt tacksam mot min familj. Tack mamma och pappa för att ni alltid har trott på
mig och för att ni gett mig så många möjligheter! Moster Maggan för att du som familjens
första doktor gett mig fotspår att vandra i. Lillebror Sven och kusin Hanna för att ni stått ut
med eran bossiga storasyster.
Ett stort tack till Jacob som försöker förstå det jag håller på med. Du finns alltid där och
lyssnar och du betyder så väldigt mycket för mig!
36
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