Download Licentiate thesis from the Department of Immunology,

Licentiate thesis from the Department of Immunology,
Wenner-Gren Institute, Stockholm University, Sweden
Early gut microbiota in relation to
cytokine responses and allergic disease
Maria A Johansson
Stockholm 2011
SUMMARY
The microbiota, colonizing the mucosal surfaces after birth, perform various functions
important to the host. Postnatal maturation of the immune system, mediated by the
microbiota is crucial, as demonstrated in animal studies. The microbiota has been
implied in various inflammatory conditions, among them allergic disease.
In PAPER I, we investigated the microbiota, early in life, in relation to both parental
allergy and allergic disease at age five in children participating in an allergy cohort.
Bacterial species investigated, were chosen due to their previous associations with
allergy. Interestingly, Lactobacilli colonization differed with parental allergy. A larger
proportion of infants with non-allergic parents harbored Lactobacilli early in life, in
addition to having higher relative amounts and being colonized at more occasions
compared to infants with allergic parents. In relation to allergy, the frequency of
Lactobacilli was higher among the children who were non-allergic at age five, and
similar was observed for Bifidobacterium bifidum. In addition, the non-allergic
children harbored Lactobacilli on several occasions. Further subgrouping of the
children according to both parental allergy and allergic disease, revealed that among
children with allergic parents, a larger proportion of Lactobacilli were the ones
remaining non-allergic were more often colonized with Lactobacilli, thus Lactobacilli
seems to protect against allergy among children with allergic parents. In conclusion,
we demonstrate that parental allergy associates Lactobacilli colonization. We also
conclude that Lactobacilli colonization during infancy was more prevalent among
non-allergics, thus potentially protecting against allergy development.
In PAPER II, we studied whether early colonization influenced the cytokine profile at
two years of age, after in vitro PHA stimulation. Early Lactobacilli colonization was
associated with fewer numbers of cytokine secreting cells, as was persistent
colonization during the first two months of life. A persistent colonization with
Bifidobacteria was also associated with reduced numbers of cytokine producing cells.
On the contrary, S. aureus colonization was associated with increased numbers of
cytokine secreting cells; however this was suppressed if co-colonization with
Lactobacilli occurred. In conclusion, we demonstrate early colonization with different
bacterial species associates with a modulation of cytokine secreting cell numbers.
2
LIST OF PAPERS
This thesis is based on the two original papers listed below, which will be referred to
by their roman numerals in the text.
I.
Johansson MA, Sjögren YM, Persson JO, Nilsson C, SverremarkEkström E.
Early Lactobacilli colonization decreases the risk for allergy at five
years of age despite allergic heredity. Submitted.
II.
Johansson MA, Nilsson C, Sverremark-Ekström E.
The early gut microbiota associates with the number of cytokine
secreting cells at two years of age after in vitro PHA stimulation.
Manuscript.
3
TABLE OF CONTENTS
SUMMARY ......................................................................................................... 2
LIST OF PAPERS............................................................................................... 3
TABLE OF CONTENTS .................................................................................... 4
ABBREVIATIONS ............................................................................................. 5
INTRODUCTION ............................................................................................... 6
THE IMMUNE SYSTEM ................................................................................... 6
INNATE IMMUNITY........................................................................................................... 7
ADAPTIVE IMMUNITY ..................................................................................................... 8
THE GUT ASSOCIATED LYMPHOID TISSUE ............................................................11
ALLERGIC DISEASE.......................................................................................14
THE ALLERGIC REACTION ...........................................................................................14
FACTORS INFLUENCING ALLERGY DEVELOPMENT...........................................16
THE GASTROINTESTINAL MICROBIOTA.................................................18
MICROBIOTA DEVELOPMENT AND COMPOSITION .............................................19
IMMUNE MATURATION AND THE MICROBIOTA..................................................19
EARLY GUT MICROBIOTA AND ALLERGIC DISEASE ..........................................21
PROBIOTIC SUPPLEMENTATION AND ALLERGY..................................................21
PRESENT STUDY .............................................................................................22
OBJECTIVES ......................................................................................................................22
MATERIALS AND METHODS .......................................................................23
STUDY POPULATION......................................................................................................23
CLASSIFICATION OF ALLERGIC DISEASE AND SENSITIZATION.....................24
METHODS...........................................................................................................................24
RESULTS AND DISCUSSION .........................................................................25
PAPER I................................................................................................................................25
PAPER II ..............................................................................................................................28
GENERAL CONCLUSIONS .............................................................................................31
CLINICAL RELEVANCE..................................................................................................32
FUTURE PERSPECTIVES...............................................................................32
BACTERIAL INFLUENCE ON EPITHELIAL CELL RESPONSES............................32
EARLY MICROBIOTA AND REGULATORY IMMUNE FUNCTION LATER IN
CHILDHOOD ......................................................................................................................33
ACKNOWLEDGEMENTS ...............................................................................34
REFERENCES...................................................................................................35
4
ABBREVIATIONS
APC
APRIL
B.
BAFF
C.
CBMC
CD
CSR
DC
ELISpot
GALT
GF
IEC
IFN
Ig
IL
LPS
MHC
MLN
MØ
NK
NLR
PAMP
PBMC
PHA
PP
PRR
SCFA
SPT
TCR
TC
TGF
TH
TLR
TNF
Treg
TSLP
Antigen-presenting cell
A proliferating-inducing ligand
Bifidobacterium
B-cell activating factor
Clostridium
Cord-blood mononuclear cell
Cluster of differentiation
Class switch recombination
Dendritic cell
Enzyme-linked ImmunoSpot
Gut associated lymphoid tissue
Germ free
Intestinal epithelial cell
Interferon
Immunoglobulin
Interleukin
Lipopolysaccharide
Major histocompatibility complex
Mesenteric lymph node
Macrophage
Natural killer
NOD-like receptor
Pathogen-associated molecular pattern
Peripheral blood mononuclear cell
Phytohaemagglutinin
Peyer’s patches
Pattern-recognition receptor
Short chain fatty acid
Skin prick test
T-cell receptor
T cytotoxic
Transforming growth factor
T helper
Toll-like receptor
Tumor necrosis factor
T regulatory
Thymic-stromal lymphopoietin
5
INTRODUCTION
THE IMMUNE SYSTEM
The immune system is crucial for the defense against invading pathogens. It also
faces challenging tasks such as discriminating pathogenic from non-pathogenic
organisms and distinguishing self from non-self, in order to respond in a correct
manner.
We are protected by anatomical, chemical and physical barriers, such as skin, low pH
in the stomach and lytic enzymes which all limit invasion of pathogens. In addition,
the mucosal surfaces are important in reducing pathogenic invasion through the
secretion of antimicrobial peptides. Further, different parts of the immune system
perform various effector functions, all together responsible for an efficient
inflammatory response and clearance of the pathogen. Our immune system can be
divided into two branches, innate and adaptive immunity. The two branches
(discussed separately below) are different in speed and specificity. The innate branch
responds rapidly, however the recognition of pathogens is less specific whereas the
adaptive response is slower to mobilize, but has a high degree of antigenic specificity,
diversity and also results in memory1.
Innate cells are for example monocytes, macrophages (MØ) dendritic cells (DC),
Natural Killer (NK) cells, mast cells and granulocytes like neutrophils, basophils, and
eosinophils. NK cells display cytotoxicity against virus infected- and altered self cells,
whereas other innate cells mainly phagocytose and initiate adaptive responses2. T and
B lymphocytes characterize adaptive immunity, but also NKT cells are included.
Certain T cells primarily aid B cells in the production of immunoglobulins, while
other T cells exert cytotoxic activity.
In addition to the cells of haematopoietic origin also nonhaematopoietic cells, such as
epithelial cells, are important in immunity as they secrete antimicrobial peptides at the
mucosal surfaces, for instance in the gastrointestinal tract3.
Cooperation between the innate and adaptive branches facilitates the function of the
immune system. For example, innate cells initiate and direct the inflammatory
response, through the presentation of antigens to adaptive T lymphocytes, whereas the
adaptive cells can increase the phagocytic function of the innate cells.
6
Cytokines and chemokines are small soluble peptides produced for communication
between cells or secreted to attract other cells to the site of infection. Cytokines signal
upon binding to their respective cytokine receptor, which result in intracellular
signaling events leading to altered cellular functions. This can include events of both
activating and inhibitory nature, such as the production of other cytokines, an increase
of receptors for other molecules, or the suppression of their own effect by negative
feedback regulation. A particular cytokine may have different effects on different
target cells, depending on its abundance, the presence of cytokine receptors on a given
target cell etc. Further, cytokines display a considerable redundancy, meaning that
many cytokines share similar functions4.
INNATE IMMUNITY
Microbial invasion instantly activates a wide range of innate effector cells and
functions; through recognition either by soluble components or by membrane-bound
receptors. Soluble proteins, for example mannose-binding-lectin or acute phase
proteins like C-reactive protein, activate complement proteins, which facilitate lysis
of the pathogen and mediate opsonization, events that promote the uptake of the
microbe by endocytosis or phagosytosis2. Monocytes, MØs and DCs recognize
conserved microbial motifs, pathogen-associated molecular patterns (PAMPs), on
microorganisms through membrane bound or cytoplasmic pattern-recognition
receptors (PRRs). So far, many different PRRs have been discovered, of which the
Toll-like receptors (TLRs) are the best studied. In humans, the TLR family consists of
10 receptors5. This enables the discrimination of different pathogens, as the
expression is both intra- and extracellular, and the motif recognized varies for the
different TLRs. TLR2 and TLR4 are expressed on the surface of the cells and sense
peptidoglycan (PGN) and lipopolysaccharide (LPS), which are components of gram
positive and gram negative bacteria, respectively. Intracellular expression of TLR3,
TLR7/8 and TLR9 enable recognition of viral RNA/DNA (single or double stranded)
in the cytoplasm. In addition to the TLRs, intracellular Nod-like receptors detect
endogenous danger signals occurring as a consequence of infection or injury.
Ligation of PRRs activates antimicrobial and inflammatory responses through
intracellular signaling cascades with cytokine and chemokine production as a
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consequence. Rapidly secreted cytokines, like tumor-necrosis factor (TNF)-α,
interleukin (IL)-1β and IL-6 are released upon PRR ligation. The cytokines released,
act on both cells and surrounding tissue, thus influencing their behavior. The
recruitment of effector cells to the site of infection is favored by the release of
chemokines, and the increased expression of adhesion molecules on the vascular
epithelium facilitating the extravasation into the tissue. Due to the potency of the
cytokines, regulation of their production is essential as a consequence of excessive
production can result in inflammatory disease or even in septic shock.
Depending on the type of pathogen, the subsequent adaptive response generally
differs; fighting extracellular pathogens requires a humoral response, while clearing
intracellular pathogens demands a cell-mediated response1.
Although discussed separately, the innate and adaptive immune branches are both
required for an effective immune response and cooperation between the two is
necessary. Upon encounter with microbes DCs capture and process the antigen,
before migrating to either the lymph nodes or spleen, where presentation to T cells
occur, thus bridging innate and adaptive immunity. Also, DC secretion of cytokines
supporting inflammation will further direct the following type of adaptive response
initiated.
ADAPTIVE IMMUNITY
Whereas components of innate immunity are present prior to the onset of infection,
recruitment of adaptive effector functions require days.
Characterizing adaptive immunity is diversity, antigenic specificity, self versus
nonself recognition and generation of antigen-specific long lasting memory. The
primary adaptive response may need approximately one week, whereas a secondary
response upon rechallenge with the same antigen, only requires a couple of days.
Adaptive immune recognition relies on antigen receptors expressed on B and T
lymphocytes. Rearrangement and assembly of the germ line genes by recombinationactivating gene (RAG)- enzyme enable a diverse repertoire of receptors specific for
an antigen. In response to a certain antigen clonal expansion of the specific population
of lymphocytes occurs, further increasing the specificity through additional
8
mechanisms such as non-template nucleotide addition, gene conversion and also in
the case of B lymphocytes, somatic hypermutation.
The initiation of the adaptive response requires the cooperation of antigen-presenting
cells (APC) scanning the periphery for pathogens, phagocytosing and processing
proteins before migrating to the lymph nodes or spleen where interaction with
adaptive cells occur.
T lymphocytes originate from bone marrow, but mature in the thymus. Two major
subgroups of T cells are the T helper (TH) and T cytotoxic (TC) cells, divided based on
their expression of surface glycoproteins: cluster of differentiation (CD) 4 and CD8,
respectively. T cell receptors (TCR) recognize antigens only in the context of major
histocompatibility complex (MHC) class I or class II molecules expressed on antigen
APCs. TC cells are important in clearing infected and transformed cells in the body,
while the function of TH cells is mainly cytokine secretion, important for both
humoral and cell-mediated immunity through the activation of other effector cell
types such as B cells, TC cells and MØs. Both CD4 and CD8 T cells require IL-2 for
their activation and differentiation.
Depending on the co-stimulatory signals and cytokine environment, TH cells can
differentiate into distinct subsets of T helper cells important in the defense against
different pathogens. TH1 cells, important in the defense of intracellular bacteria,
produce IFN-γ and activate macrophages and other effector functions whereas TH2
cells, characterized by IL-4, IL5 and IL-13 production are important in the defense
against helminth infections. Further, an IL-17 producing subset of T-helper cells is
considered important in the defense against extracellular bacteria and fungi.
The different subsets of TH cells counteract each other by negative feedback
mechanisms; IFN-γ inhibits the development of TH2 cells and IL-4 counteracts the
TH1 type of immunity. Each of the different TH lineages are controlled by individual
transcription factors; T-bet regulates TH1 whereas GATA-binding protein 3 (GATA3)
and retinoic-acid-receptor-related orphan receptor γt (RORγt) control TH2 and TH17
respectively.
B lymphocytes both derive from and mature in the bone marrow. The main function
of B cells is to secrete antigen specific immunoglobulins (Ig)/antibodies. Activation
of naïve B cells can be both T-cell dependent and T-cell independent resulting in
differentiation into antibody secreting plasma cells and memory cells. Activation of B
9
cells result in class switch recombination (CSR). The different isotypes of Ig
subclasses that exist are IgM, IgD, IgG, IgE and IgA. Effector functions of the
antibodies are mainly promoting opsonization thus phagocytosis of microbes,
activating complement proteins or mediating antibody-dependent cell mediated
cytotoxicity thus directing cytotoxic activity against the infected cells. In humans, a
TH1 response induces IgG1, IgG3 isotypes, whereas a TH2 type of response mediates
IgE and IgG4 production.
The importance of the immune system for host health and survival is evident in the
case of different immune deficiencies, which result in more or less unsuccessful
protection of the host. As important it is to mount an immune response when required,
it is equally essential that suppression of the inflammatory response occur, as failure
can be detrimental to the host. A dys-regulated immune response may lead to
exaggerated inflammation and tissue damage, and immune responses towards self or
harmless antigens may cause autoimmune and allergic disease, respectively.
The CD4+ regulatory T (Treg) cells are important in active suppression of the immune
response. There are several types of Tregs distinguished by different expression of
surface molecules and secreted cytokines. Natural (n) Tregs developing in the thymus
are CD4+CD25+ cells expressing high levels of the Foxp3 transcription factor. These
cells secrete the anti-inflammatory cytokines IL-10, transforming growth factor
(TGF)-β and express inhibitory the molecule CTLA-4. The nTregs are considered
responsible for systemic homeostasis and prevention of autoimmune disease6.
Induction of regulatory cells also can occur in the periphery, for example in the
gastrointestinal tract. The induced Tr1 and Th3 cells are characterized by secretion of
IL-10 and TGF-β, respectively. These regulatory cytokines inhibit cytokine secretion
by activated T cells and suppress expression of co-stimulatory molecules on APCs7.
10
THE GUT ASSOCIATED LYMPHOID TISSUE
The mucosal surfaces lining the respiratory and digestive tracts are the largest areas in
the body with a constant contact with antigens, and are also the main routes for
pathogen entry. Moreover the gastrointestinal tract is in close contact with the
commensal microbiota (discussed below) inhabiting the intestine; only one single
layer of epithelial cells separates the lumen from the underlying immune structures.
The gut-associated lymphoid tissue (GALT) is not only important for our immune
defense, but it is also a site for oral tolerance induction to harmless antigens.
The intestinal immune system, GALT can be divided into effector sites and organized
tissues. The effector sites contain lymphocytes present within the epithelium and
throughout lamina propria. The organized tissues, Peyer´s patches (PP), mesenteric
lymph nodes (MLN) and the smaller isolated lymphoid follicles, responsible for the
induction of an immune response, are present throughout the wall of the small and
large intestines8.
Peyer´s patches are aggregates of lymphocytes, with large B-cell follicles and
intervening T cell areas situated below the subepithelial dome (SED), an area
immediately under the follicle associated epithelium. The SED is infiltrated by
macrophages, DCs, T and B cells, which distinguishes it from the epithelium covering
the villous mucosa. The presence of Microfold (M) cells is also characterizing the
SED. The M cells are specialized in uptake of antigens and in delivering them to
APCs. In addition, DCs themselves can capture luminal antigens through the
extension of transepithelial protrusions before migrating to the PPs and MLNs where
priming of naive lymphocytes occurs (Figure 1).
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Figure 1. Left Anatomy of the intestinal immune system, showing the epithelium separating lumen
from the underlying immune structures. Dendritic cells sample and process antigens before interaction
with naïve T cells occurs and initiation of adaptive responses take place. Both T cell dependent and
independent pathways leading to induction of class switch recombination to IgA can appear. The poly
Immunoglobulin Receptor facilitates transport of sIgA to the lumen. Right Depending on the cytokines
produced, by both epithelial and dendritic cells, and on the co-stimulatory interactions that occur, the
following adaptive response to an antigen differs. Adapted from Cerutti Immunity 20089
In addition to the conventional lymphocytes, there are several other innate-like
lymphocytes, like natural-killer T (NKT) cells and gammadelta (γδ) T cells present in
high numbers in the intestine. These cell types have a reduced diversity of their
antigen receptors, resulting in a more restricted recognition towards predefined
antigens compared to the conventional lymphocytes. NKT cells interact with nonclassical MHC molecules also known as CD1d, presenting mainly bacterial lipids and
additional data suggest that γδ T cells also recognize phospholipid antigens. Although
the function of these cells is less studied, γδ T cells are implied in killing infected,
activated and transformed cells among other functions10.
Additionally, intestinal epithelial cells (IECs) play a central role in homeostasis and
immunity in the gastrointestine. IECs express both classical MHC and non-classical
CD1 molecules enabling antigen presentation. However, the expression of positive
and negative co-stimulatory molecules on epithelial cells is under debate3. Moreover,
in response to various stimuli epithelial cells produce cytokines, with the potential to
12
skew subsequent immune responses. Among others, IECs produce thymic stromal
lymphopoietin (TSLP) and possibly also IL-10, TGF-β and retinoic acid, all
promoting tolerogenic or TH2 responses (Figure 1 Right)9.
The induction of immunosuppressive mechanisms to food- and commensal antigens
in the gut, often referred to as oral tolerance, and intestinal homeostasis is crucial, as
failure in these mechanisms can result in hypersensitivity reactions or inflammation in
the gut11. Several subsets of DCs are important for both immune responses and
tolerance induction in the gut as they direct adaptive responses (Figure 1 Right)9. The
regulatory cytokines IL-10 and TGF-β induce functionally suppressive Tregs.
Retinoic acid conditions tolerogenic DCs that induce Tregs cells but also a low
expression of co-stimulatory molecules CD80/86 can result in T cell anergy7. Retinoic
acid is also important as a factor influencing the expression of CC-chemokine
receptor 9 (CCR9) and integrin α4β7, and therefore regulating the homing of B- and T
cells back to the mucosa thus mediating homeostasis. DCs also secrete B-cell
stimulating factors including a proliferating-inducing ligand (APRIL) and B-cell
activating factor (BAFF) thus stimulating T-cell independent CSR into IgA12. Other
cytokines of importance in the CSR to IgA are IL-10 and TGF-β, which also induce
IgA in T cell dependent manner9.
Secretory IgA (sIgA) is the major antibody isotype produced at the mucosa. In
humans, both IgA1 and IgA2 exist; the latter being more resistant to degradation by
bacterial proteases in the lumen. Together with antimicrobial peptides, IgA provides
instant immune protection. Poly immunoglobulin receptors (pIgRs), expressed on the
basolateral surface of mucosal epithelial cells, bind dimeric IgA connected by a Jchain and facilitate the transport of IgA to the intestinal lumen. Cleavage of the pIgR
results in a secretory component responsible for mucophilic properties of sIgA.
Functionally, sIgA neutralize toxins and mediate immune exclusion without evoking
an inflammatory response. Moreover, sIgA promote a mutualistic relationship with
the commensal microbiota by facilitating the formation of a biofilm favoring growth
of non-pathogenic bacteria, thus limiting the growth of pathogenic organisms13.
13
ALLERGIC DISEASE
Failure in tolerance induction to innocuous antigens (allergens) may result in
hypersensitivity reactions. An immediate, type-I, hypersensitivity reaction is
characterized by a TH2 response favoring IL-4, IL-5, IL-9 and IL-13 and IgE
production (IgE-mediated allergy)14. A genetic predisposition to produce IgE
antibodies (atopy), is important for the development of allergic diseases of this kind;
however the increase in allergic diseases observed in Westernized countries the last
decades are not likely to depend only on predisposition, but also environmental
factors seem important15. An allergic phenotype is often initiated early in childhood
with symptoms that are skin or food-related, which switch into respiratory related
symptoms later during childhood, a phenomenon known as the “atopic march”16.
Today, 30 % of the population in developed countries is considered to be atopic of
which approximately 10 % have allergic symptoms17.
THE ALLERGIC REACTION
Normally, humoral immunity with production of IgE antibodies is important in the
defense against extracellular parasites such as helminth infections. However, specific
IgE antibodies directed against harmless antigens instead mediate allergic disease.
TH2 cell differentiation is favored by IL-4, which is also one of the major effector
cytokines that together with IL-13 induce IgE class switch in B cells. Where the initial
IL-4 comes from is not clear, however NKT cells have been implied in the early
production of IL-418. Further, TSLP produced by epithelial cells, can activate DCs to
prime naïve CD4 cells to TH2 type3. Once produced, specific IgE binds to high
affinity receptors (FcεRI) constitutively expressed at high levels on mast cells and
basophils, and at lower levels on eosinophils, a process termed sensitization. Upon
reencounter with the specific allergen, crosslinking of the IgE antibodies confers the
release of granules containing preformed mediators like histamine, proteases and
chemotactic factors responsible for the clinical manifestation of the allergic response
(Figure 2).
14
Figure 2. Allergic mechanisms An APC process and present antigen in the context of MHC II to a
naïve T cell, which then differentiates into a TH2 cell producing IL-4 and IL-13, promoting CSR to
allergen specific IgE antibody production. The produced IgE binds to high affinity receptors on mast
cells, thus sensitizing the individual. Upon reencounter with the specific allergen, crosslinking of IgE
results in release of soluble mediators like histamine responsible for the symptoms of allergic disease.
During the chronic course of allergic inflammation, also other TH effector mediators are involved.
Holgate & Polosa Nat Rev Immunol 200819.
Histamine acts on both tissues, resulting in vasodilation and smooth muscle
contraction, as well as on effector cells including eosinophils, T cells and neutrophils.
Secondary mediators synthesized by breakdown of phospholipids or after target cell
activation, include leukotrienes, prostaglandins and a variety of cytokines and
chemokines. While biological effects of histamine are instant, the secondary
mediators account for longer lasting ones including continuation of the airway
constriction, mucus production, smooth muscle contraction and vascular permeability.
Cytokines and chemokines produced during an allergic reaction account for both local
and systemic effects through the recruitment of eosinophils. Moreover, the long-term
effects during chronic allergic inflammation, tissue destruction and remodeling also
involve elements of other TH subsets19.
15
FACTORS INFLUENCING ALLERGY DEVELOPMENT
Both genetic and environmental factors are associated with an increased or decreased
risk for development of allergic disease, highlighting the complexity in the etiology of
allergy. Genes of interest, in relation to allergic disease are many, however, they can
be classified into groups of genes depending on their functions. The first group is
genes involved in sensing the environment and microorganisms, such as PRRs.
Indeed, polymorphisms in TLR4 as well as the co-receptor CD14 have been
associated with atopy and asthma20. The second group of genes of interest are genes
involved in barrier function. Several genes important in epithelial barrier integrity
have been associated with allergic disease. Among others, the Filaggrin gene has
been associated with increased risk for both atopic dermatitis and sensitization. The
third group of genes involved in allergic disease is the one engaged in inflammation.
More specifically, genes that are involved in regulation of TH responses and effector
functions, such as STAT6, IL4RA, and IL13 have also been implicated. The fourth
group of genes considered, are the genes implicated in modulating the tissue
responses during chronic inflammation, for instance ADAM33 and PDE4D expressed
in smooth muscle and fibroblasts. It should be noticed that some genes can belong to
more than one group of genes, and some are highly involved in the sensing of
environment. This suggests that the risk for allergic disease can be modulated with for
example microbial exposure21.
As for environmental factors associated with an altered risk for allergic disease, these
include parental smoking, air pollution but also by allergen exposure22. However, the
main environmental exposures discussed during the last decades are microbial
exposures. Westernized countries have experienced a profound increase in incidence
and prevalence of allergic disease coinciding with for example improvements in
hygienic standards and antibiotic usage and in urban standards of living. Late in the
80s, the “hygiene hypothesis” was coined based on epidemiological studies showing
that larger sib-ship size and early day care attendance decreased the risk for allergy23.
A decreased microbial exposure, early in life, has been proposed to explain the
increase in allergy disease15. Since then, many studies have continued exploring the
relationship between early exposure of microorganisms and development of allergic
disease. A landmark study proved, in 2001, that farm environment substantially
reduced the risk of becoming allergic24. Also, level of exposure to endotoxin was
16
shown to be inversely associated with allergic disease25,26. Since then many studies on
preferentially commensal bacterial exposures in relation to allergic disease have been
performed (discussed below).
Not only exposure to bacteria has been investigated, but also viral infections have
been implicated in the development of allergy. Children suffering from many upper
respiratory infections, early in life, are at higher risk of developing respiratory
conditions such as wheezing and asthma. More specifically, the respiratory syncytical
virus has been associated with an increase in asthma development27. Further, Hepatitis
A seropositivity has been inversely associated with atopic disease in some studies28
but not in others29. Additionally, our group has shown that children seropositive for
Epstein Barr Virus before two years of age have a reduced risk for persistent IgE
sensitization, whereas seroconversion after two years of age was associated with
increased risk for sensitization at five30. This indicates that both exposure, but also
that the timing of exposure may be of importance and that there may be a “window of
opportunity” early in life during when- a modulation of the immune system may
occur.
Fighting helminth infections require a humoral response, accompanied by the
production of IgE antibodies. Interestingly, helminth infections and allergic symptoms
do not coincide despite the occurrence of antigen specific IgE antibodies in helminthinfected individuals. The possible mechanism behind the allergy protection is that,
although high levels of IgE, also the regulatory cytokines IL-10 and TGF-β are
increased31. Indeed, long-term anti-helminth treatment associates with increased skin
prick test reactivity32.
Additional support that exposures to microorganisms modulate the risk of allergic
disease is that receiving antibiotics during infancy associates with higher risk for
allergy33-35, a risk also observed for children born with caesarean delivery36.
17
THE GASTROINTESTINAL MICROBIOTA
Co-evolution of man and microbes has created a mutualistic relationship between the
host and the microbiota that inhabits skin and mucosal surfaces. Approximately 5001000 different species of bacteria are present in the gastrointestinal tract at
concentrations ranging from 102-3 cells/gram in the proximal ileum and jejunum to
107-8 in the distal ileum and highest density with 1011 in ascending colon. Two major
phyla of bacteria, Bacteroidetes and Firmicutes, account for approximately 60-90% of
all species37. The microbiota performs various metabolic functions crucial to host
fitness such as synthesis of vitamins and bile salt metabolism in addition to
production of short chain fatty acids (SCFA)38. Produced by fermentation of
indigestible carbohydrates, SCFAs serve as an energy source for the epithelium and
host in addition to influencing intestinal homeostasis39. Moreover, the microbiota has
a large impact on the immune system in the host38. Not only does it compete with and
exclude pathogens but it induces the maturation of the host immune system after birth
(as discussed below)40. Recognition of peptidoglycan from the microbiota has been
shown to enhance systemic innate immunity41. In addition, our microbiota promotes
angiogenesis and epithelial cell homeostasis42. Due to its energy harvesting
properties, the microbiota has been associated with obesity43,44 and overweight45 and
clearly dysbiosis is associated with inflammatory bowel disease46.
With the increasing evidence that the microbiota is crucial to the host, the methods to
analyze it are of great importance. The primary samples used to investigate the
microbiota are fecal samples. The main methods to analyze the microbiota are both
culture dependent and culture independent. Culturing bacteria is time consuming and
importantly all species of bacteria are not cultivable. Recent molecular methods use
16SrRNA genes for the investigation of the microbiota. These genes contain
conserved and variable regions enabling the design of primers and probes for the
detection of specific bacteria. With primers and probes, fluorescent in situ
hybridization and Quantitative Real time PCR detect and quantify known bacterial
species, groups or genus. The drawback, though with these molecular methods is the
inability to distinguish between dead or alive bacteria37.
18
MICROBIOTA DEVELOPMENT AND COMPOSITION
The fetus develops in a sterile in utero environment, however during birth
colonization of the skin and mucosal surfaces is initiated. The first colonizers of the
gastrointestinal tract are facultative anaerobes consuming oxygen, thus preparing for
more anaerobic bacteria to begin their colonization. Prominent bacteria postnatally
are Proteobacteria like Escherichia coli and Bifidobacterium (B.) species (spp)
belonging to Actinobacteria before Bacteroidetes and Firmicutes begin to establish47.
Colonization is a dynamic process and bacterial composition is influenced by various
factors. Caesarean sections largely impact the composition of the microbiota, as the
first bacteria these newborns encounter are skin- related species of bacteria, instead of
vaginal and fecal species48,49. Further, country of birth and level of affluence seem to
have impact on the colonization in addition to prematurity, antibiotic use and infant
feeding50. Breast milk contains up to 109 microbes per liter, primarily Staphylococcus
spp, Streptococcus spp in addition to Lactobacillus and Bifidobacterium spp.51,52.
Further, breast milk content of oligosaccharides is high53, supporting growth of the
microbiota. Life style factors, like family size and home environment have been less
studied, however children having older siblings seem to possess a more mature
microbiota at the age of 12 months compared to children without siblings54,55. In
addition, host genetics have also been implied in determining the composition of the
colonizing bacteria56,57.
IMMUNE MATURATION AND THE MICROBIOTA
Although all components of the immune system are present at birth, the neonatal
immune system needs education to become fully functional. In the womb, the fetus
develops in a protected environment with a low exposure to foreign antigens. Once
born, neonates rely to a larger extent on the innate branch of immunity for their
defense neonates are susceptible to infections, mainly due to their antigen
inexperience and immature adaptive immunity58. Maternal IgG transferred across the
placenta during the last trimester, and sIgA in the breast milk will add to the
protection of the newborn. Further, breast milk contains immunoprotective factors
such as lactoferrin and lysozyme and soluble TLR2 that potentially inhibit signaling
via membrane bound receptor59. Additionally, in mice, the milk contains antigen-IgG
immune complexes that potently induce oral tolerance in newborn pups60.
19
Exposure to microbial components may, through the activation of innate immunity
accelerate the maturation of adaptive immunity. Most of the knowledge about the
influence of the microbiota on the maturation of the immune system originates from
animal studies, reviewed in40. Breeding animals under germ free (GF) conditions have
facilitated a model of investigating the impact of not only the whole microbiota but
also the species-specific contribution to the maturation of the immune system.
Generally, immune structures like PPs and MLNs are underdeveloped in GF animals;
both numbers and cellularity are reduced, highlighting the microbiota as inducer of
these immune inductive sites. Both innate and adaptive compartments are affected.
Locally in the intestine, DC numbers are reduced, as is the number of MØs61 however
restoration can be accomplished after administration of bacterial species to the GF
animals62. Also, the microbiota seems important in the expansion of NKT cells63.
Moreover, the number of IgA producing plasma cells is lower in GF animals
correlating with a reduced amount of IgA. Also systemically, plasma cell numbers are
reduced
together
with
fewer
germinal
centers
and
Ig
levels.
Again,
conventionalization of the GF animals restores these reduced numbers/levels64.
Interestingly, the number of IgE positive B cells in PPs is increased65 together with
serum IgE levels in GF animals compared to conventional and antibiotic treated
animals, respectively66. In addition TH and Tregs are also induced by the microbiota6770
.
In humans, much less is known about the function of the microbiota in the induction
of the immune system. However, early colonization with Bacteroides fragilis or
Bifidobacteria and Lactobacilli has been associated with an increase in IgA/M
secreting cells71 and higher levels of secretory IgA in saliva72, respectively. Moreover,
only peripheral blood mononuclear cells (PBMC), from infants colonized with
Bacteroides fragilis early in life, produced IFN-γ spontaneously as compared to the
children not colonized. Also, the level of Bacteroides fragilis colonization was
inversely correlated with the IL-6 and CCL-4 response after LPS stimulation
indicating
an
immunomodulatory function72. Furthermore,
Lactobacilli
and
Bifidobacteria colonization has been associated with lower cytokine responses
following in vitro allergen stimulation and also the level of Bifidobacterium
colonization was associated with increased Foxp3 expression73.
20
EARLY GUT MICROBIOTA AND ALLERGIC DISEASE
As the microbiota influences the maturation of immune system and the exposure to
microbial products reduces the risk for allergy, a link between the intestinal
microbiota and development of allergic disease is investigated. Comparison of
allergic and non-allergic children revealed differences in the composition of the
microbiota. Björkstén et al.74 was the first to show this in a study investigating the
microbiota in children from Estonia and Sweden, countries with low and high
prevalence of allergic diseases, respectively. Since then, several other studies have
continued exploring the early microbiota and its association to future allergic disease.
Although there is considerable inconsistency between the results from the different
studies, a pattern can be distinguished. Some species of bacteria seem to be more
prevalent among children not becoming allergic whereas some bacterial species are
more commonly detected in children later turning allergic. Generally, Clostridium
(C.) difficile75,76 and Staphylococcus (S.) aureus77 have been associated with allergy
development whereas the opposite association has been reported regarding several
different species of Bifidobacteria and Lactobacilli54,55,78. Indeed, the microbiota
seems to be less diverse early in life in children later having atopic eczema79.
PROBIOTIC SUPPLEMENTATION AND ALLERGY
As a reduced microbial exposure is shown to influence development of allergic
diseases, supplementation of probiotic bacteria to prevent allergy has been performed.
The World Health Organisation defines probiotics as “live microorganisms which
when administered in adequate amounts confer a health benefit on the host”.
Different administration strategies have been used. In some studies, the probiotic has
been administered to the pregnant mother and the offspring postnatally, whereas in
others, either the mother or the infant only have received the supplementation,
respectively. So far, the outcome of probiotic supplementation in prevention of
allergy has been inconsistent. Some studies show a reduced risk for allergic
phenotypes with probiotics80-82, whereas others report no or even higher risk for
allergic outcomes compared to the control groups83,84.
21
PRESENT STUDY
OBJECTIVES
Generally, the aim of this work was to investigate the early gut microbiota in relation
to allergic heredity, allergic disease at five years of age and cytokine responses at 24
months of age.
Our specific aims were to:
•
Investigate if parental allergy influences the early gut microbiota and to
examine whether the early microbiota associates with allergic disease at five
years of age (PAPER I).
•
Explore if the cytokine profile, in terms of numbers of IL-4, IL-10, IL-12 and
IFN-γ secreting cells, at 24 months of age relates to the early-life gut
microbiota (PAPER II).
22
MATERIALS AND METHODS
STUDY POPULATION
A total of 58 (PAPER I) and 30 (PAPER II) children, participating in a prospective
allergy cohort (n=281), were included here. The cohort has previously been described
in detail elsewhere85. Briefly, parents-to-be were invited to participate, only if selfreported allergic disease was confirmed with positive or negative skin prick test
results. The children were followed from birth and were clinically examined by the
same pediatrician (C.N) at 6, 12, 18, 24 and 60 months of age. Skin prick test (SPT)
were performed against food and inhalant allergens according to the manufacturer’s
instruction (ALK, Copenhagen, Denmark). The SPT included food allergens: egg
white (Soluprick weight to volume ratio 1/1000), cod (Soluprick 1/20), peanut
(Soluprick 1/20) cow’s milk (3% fat, standard milk), and soybean protein (Soja Semp;
Semper AB, Stockholm, Sweden). SPTs were also performed for inhalant allergens:
cat, dog, Dermatophagoides farinae, birch and timothy (Soluprick 10 Histamine
Equivalent Prick test). Histamine chloride (10 mg/mL) and allergen diluent served as
positive and negative control, respectively. The SPT was considered positive if the
wheal diameter was ≥3mm after 15 minutes. Also both the mothers and fathers
underwent SPT to furred pets and pollens common for Sweden.
Serological analysis of specific IgE, to the same allergens tested with SPT, was
performed using ImmunoCAP (Phadia AB, Uppsala, Sweden). Samples with
allergen-specific levels of ≥35 kU/L were considered positive.
All families gave their informed consent to participate and ethical permission was
granted from the Human Ethics Committee at Huddinge University Hospital,
Stockholm, Sweden.
23
CLASSIFICATION OF ALLERGIC DISEASE AND SENSITIZATION
In PAPER I, children was classified as allergic if at least 1 SPT was positive and/or if
specific IgE to at least 1 of the selected allergens was positive, together with allergic
symptoms at five years of age, according to the World Allergy Organisation
nomenclature86.
In PAPER II, children were classified as sensitized if at least 1 SPT was positive
and/or if specific IgE to at least 1 of the selected allergens was positive.
METHODS
Description of the methods used, are found in detail in PAPER I and II, respectively.
Briefly, DNA was purified from faeces, using Qiagen DNA Stool Mini Kit. For
detection of the different bacteria, species-specific primer pairs were applied together
with Real Time PCR using SYBR green chemistry (PAPER I and II).
In PAPER I, B. adolescentis, B. bifidum, B. breve, Clostridium (C.) difficile, and S.
aureus were analyzed. Also, three species of Lactobacilli, L. casei, L. paracasei, L.
rhamnosus were detected with one primer pair, and will be referred to as
“Lactobacilli” from now on. In PAPER II, all but C. difficile were analyzed, as it was
only detected in few individuals in PAPER I.
In PAPER II, mononuclear cells were isolated from cord blood or peripheral blood,
sampled at 24 months of age, by Ficoll-Paque gradient centrifugation, before being
frozen in liquid nitrogen until analysis. Cells were thawed, washed and thereafter
cultivated with or without phytohaemagglutinin (PHA) for 4 hours before being
plated for additional 44 hours. IL-4, IL-10, IL-12 and IFN-γ secreting cells were
determined using ELISpot technique. Numbers of cytokine secreting cells are
expressed as cells/105 cells and are the mean of three wells after subtraction of
medium control (PAPER II).
24
RESULTS AND DISCUSSION
PAPER I
The microbiota has, in many studies, been associated with allergic diseases during
childhood. There are, however, contradictory results between studies and several
factors account for the inconsistency between the studies performed. Often, the
outcome of allergy is considered between 18-24 months of life and the outcome is
often eczema and/or wheezing. Both eczema and asthma are common during
childhood, but is not necessarily related to IgE-mediated allergic symptoms.
Additionally, the microbiota is dynamic early in life, thus sampling at only one
occasion may generate incomparable results between studies. Furthermore, the early
microbiota is influenced by many factors such as mode of delivery, feeding, and
siblings etc. Also, recent studies show that host genetics, and the immune system
influence the colonization57,87 therefore we included infants with allergic and nonallergic parents, respectively. All infants included were born term, vaginally
delivered, breast fed for a minimum of three months and none received antibiotics
during this period. All these factors have been associated to influence the microbiota
composition and/or alter the risk for allergic disease. Further, we evaluated allergy,
both SPT and/or specific IgE and allergic symptoms at five years of age, when
diagnosis is more secure.
Indeed, in this study, we show that colonization associates with parental allergy.
Lactobacilli colonization, early in life, is more prevalent among infants with nonallergic parents compared to infants with allergic parents. Further the infants with
non-allergic parents were also colonized with Lactobacilli on more occasions and the
relative amounts of Lactobacilli were higher among infants with non-allergic parents,
which was also true for B. bifidum at one week of age. None of the other species
investigated differed depending on parental allergy. This is the first time that the early
microbiota has been related to allergic heredity. In previous studies investigating the
microbiota, a vast majority of the parents are allergic, making it impossible to study
the impact of heredity.
In relation to allergic disease, we found that Lactobacilli and B. bifidum were more
often detected in early fecal samples from the children who remained non-allergic at
the age of five compared to the allergic children. Also, the relative amounts of
25
Lactobacilli were higher in the non-allergics compared to the allergic children. The
pattern of B. breve colonization, in relation to allergic disease, was similar to the one
observed for Lactobacilli and B. bifidum, however not statistically significant. No
differences in B. adolescentis frequencies or amounts were observed between allergic
and non-allergic children.
Early B. bifidum colonization has previously been associated with higher levels of
IgA in saliva72. IgA has been associated with protection against clinical symptoms in
sensitized infants, through blockage of allergen and IgE interaction26. Interestingly,
high levels of fecal IgA associates with reduced risk for sensitization88.
A large number of infants was colonized with S. aureus, as has previously been
reported89,90. Contrary to Lactobacilli and B. bifidum, S. aureus seems to be more
prevalent among children allergic at age five, however this was not statistically
significant. Similar result has previously been obtained77, but again others show
opposite association91.
C. difficile has previously been associated with allergic disease75-77. In our study, C.
difficile colonization was rare during the first two months of life and not related to
allergic disease. All infants in our study were vaginally delivered and breast-fed the
first three months, probably explaining the few number of infants harboring C.
difficile, as it has been shown that C. difficile is more common in caesarean delivered
and formula-fed infants54.
Our results confirm and extend previous findings from our group55 and others75,77,78;
all reporting that Lactobacilli and Bifidobacteria as more prevalent colonizers, early
in life, in non-allergic children. Thus, there seem to be differences in the microbiota
prior to the development of allergic disease.
As both parental allergy and allergy development are associated with the early
Lactobacilli colonization pattern, we wanted to investigate colonization in relation to
both these factors. Thus, we grouped the children according to both parental allergy
and allergic disease at age five. Interestingly, among the children with allergic
parents, infants colonized with Lactobacilli were less likely to be allergic at age five,
suggesting that Lactobacilli may protect against allergic disease in children with
allergic parents. Whether allergic children born to non-allergic parents instead lack
Lactobacilli early in life, is not possible to elucidate from our study, as too few
children among the infants with non-allergic parents were allergic at age five to
perform any statistical analysis. Even though the early-life colonization differed, at
26
twelve months of age however, there were no differences in relation to allergic
disease, indicating that it is the early microbiota that is of importance for the future
development of allergic diseases.
Whether the differences in Lactobacilli colonization predominantly depend on
genetics or environmental factors is still an open question. As there is a difference in
the early Lactobacilli colonization pattern within the group of children with allergic
heredity depending on if the child develops allergy or not, this suggests that
Lactobacilli colonization is influenced by environmental factors, unless other genes,
not involved in allergic phenotype determine this colonization. We have not
investigated the maternal vaginal flora or their breast-milk thus we cannot exclude
that the children are differently exposed. Brest milk from allergic mothers has
previously been reported to contain fewer counts of Bifidobacteria compared to nonallergic individuals51.
We should acknowledge that we only have investigated a few number of species, and
thus no complete picture of the microbiota is presented here. However, the species
were chosen for their previous association with allergic disease and real-time PCR
was used, as it is a very sensitive method for detection of specific species.
Interestingly, probiotic supplementation has in some studies reduced clinical
outcomes like eczema and/or IgE sensitization81,82 whereas others have reported no
beneficial or even adverse effect on outcomes following probiotic treatment83,84,92.
Also, in animal models administration of L. rhamnosus HN001 has had beneficial
effects, protecting against allergen-induced allergic disease93.
Several factors account for these controversies. The strategies for probiotic
supplementation have been different; administration of probiotics to the pregnant
woman with following administration also to the offspring, administration of
probiotics to the mother, but not to the newborn, and further supplementation only to
the newborn. Moreover, the bacterial species administered have varied, thus
comparisons between studies are not feasible. In addition, the measured outcome is
different between studies, as is the age of the allergy diagnosis. Interestingly,
administration of Lactobacillus spp. to already allergic individuals shows promising
immunomodulatory effects. L. casei Shirota given to seasonal rhinitis patients
conferred a reduction of antigen-specific IgE in favor of IgG and also a reduction
antigen-induced cytokine levels94. Moreover, administration of L. gasseri to allergic
27
children reduced their clinical symptoms of allergy and suppressed their production of
pro-inflammatory cytokines from PBMCs compared to placebo controls95.
Indeed, several bacterial species have immunomodulatory effects and can potentially
be used for prevention of allergic disease, although more knowledge about the
mechanisms of interaction with and influence on the immune system is needed.
PAPER II
From animal studies, it has become increasingly clear that the microbiota induces
maturation of the immune system among many other crucial functions. For obvious
reasons less is known in the human situation, however alterations in the microbiota
have been associated with inflammatory conditions, like autoimmune disease96 and
IBD46. Also, in PAPER I we show that differences in the microbiota associates with
allergic disease later in childhood. In mice, several bacterial species have specific
functions on the immune system; segmented filamentous bacteria induce maturation
of CD4+ IL-17 and IL-22 producing cells68, whereas Bacteroides fragilis mediate
conversion to Foxp3+ IL-10 producing cells69. Whether this is the situation also in
humans is not thoroughly investigated. Thus, in this study, we investigated early
colonization in relation to numbers of cytokine secreting cells after in vitro PHA
stimulation, at 24 months of age. IL-4 and IFN-γ secreting cells were examined as
they are important for humoral- and cell-mediated immunity, respectively. Also, the
number of cells producing the regulatory cytokine IL-10 was investigated, as was
numbers of cells secreting IL-12 due to their potency to direct cell-mediated immune
responses. A dysregulated immune response, like hyperreactivity, may originate from
failure in tolerance induction early in life, a process in which a balanced microbiota is
believed to be of importance. Indeed, elevated stimulated cytokine production
associates with allergic symptoms in infancy/childhood97-99 illustrating that allergy
associates with an exaggerated immune responsiveness, at least in vitro. Despite that
no association with sensitization was observed increased ratios of IL-4, IL-5 and IL10 over IFN-γ have been associated with an increased risk wheezing and several
episodes of wheezing100.
In this study, we show that early colonization with different species associates with a
modulation of cytokine secreting cell numbers. Infant colonization with Lactobacilli
associated with fewer numbers of cytokine secreting cells, after PHA stimulation,
28
compared to non-colonized infants, significant for IL-12 and tendencies for IL-4, IL10 and IFN-γ, respectively. Additionally, persistent colonization with Lactobacilli
during the first two months of life associated with significantly fewer IL-4 and IL-12
producing cells, compared to infants only having Lactobacilli on no or one occasion.
Administration of Lactobacillus species generally seems to have suppressive effects
on cytokine responses94,95,101 and could potentially modulate the risk for allergy.
Intranasally administered Lactobacillus to mice resulted in a diminished expression of
several pro-inflammatory cytokines, via a TLR-independent pathway102 In line with
our results, colonization with Lactobacilli and Bifidobacteria has previously been
reported to associate with lower cytokine responses following allergen stimulation73.
This is interesting in relation to our previous findings that, in two unrelated cohorts,
early Lactobacilli colonization is more common in children who are non-allergic at
five years of age55, (PAPER I). Our findings are supported by the results from a large
cohort investigating more than 600 infants78 supporting that Lactobacilli early in life
associates with a decreased risk for allergic disease possibly through the influence on
the immune system.
Colonization with Bifidobacterium at any early time point investigated, did not alter
the numbers of cytokine-secreting cells. However similarly to the Lactobacilli,
persistent colonization with B. bifidum associated with significantly fewer IL-4 and
IL-12 secreting cells compared to infants harboring B. bifidum at no or one occasion.
Also colonization with B. breve at several occasions tended to associate with reduced
numbers of IL-10 producing cells at 24 months. This suggests that bacterial species
within the genus might have distinct effects on and influence the immune system
differently. Low counts of Bifidobacteria have previously been associated with
allergy55,75,77 and we show that B. bifidum is more often detected in infants remaining
non-allergic at five years of age (PAPER I). Early colonization with Bifidobacteria
species is associated with higher levels of secretory IgA in saliva72 Interestingly, high
levels of secretory IgA26 and fecal IgA88 have been associated with reduced risk for
developing allergic symptoms and sensitization, at two years of age, respectively.
Minimization of Bifidobacteria, in neonatal rats, increased the levels of IL-4 in
plasma and postponed the maturation of PPs DCs103 hence revealing their importance
in the maturation of the immune structures.
S. aureus, is a common species in Swedish infants89,104 and also in our studies
(PAPER I and II), a majority of the infants harbor S. aureus. Early colonization with
29
S. aureus associated with significantly increased numbers of IL-4-, IL-10- and IL-12secreting cells at 24 months, after PHA stimulation. Also, colonization at several
occasions with S. aureus associated with more IL-10-secreting cells. In PAPER I, S.
aureus tended to be more frequent among children developing allergic disease, an
association previously reported also by others77,105. S. aureus has been shown to
induce the inflammatory cytokine IL-17, after in vitro stimulation of peripheral blood
cells from neonates and infants90.
As the majority of infants are colonized with S. aureus early in life, we speculate that
it is due to the lack of other bacterial species, potentially Lactobacillus, which may
cause an inappropriate immune stimulation. Due to the opposite pattern of
colonization of Lactobacilli and S. aureus, both in relation to allergic disease (PAPER
I) and in relation to cytokine- secreting cell numbers, we went on investigating cocolonization. Indeed, early co-colonization of Lactobacilli and S. aureus suppressed
the numbers of IL-4, IL-10, IL-12 and IFN-γ secreting cells compared to colonization
with S. aureus alone, indicating that the simultaneous presence of Lactobacilli early in
life can modulate an S. aureus induced effect on the immune system. The mechanism
of how Lactobacilli exert their function needs further investigation, however it has
recently been reported that lactic acid, degrade gram-positive lipoteichoic acid and
reduce pathogen-induced cellular cytotoxicity106. Also, SCFA, produced by the
microbiota is demonstrated to be crucial for the resolution of inflammatory responses
in the intestinal tract39.
We further investigated numbers of cytokine secreting cells in cord blood as it has
recently been shown that host genetics and the immune system of the host might
influence the colonization56. Here, we did not observe any association between cord
blood responses and subsequent colonization with the species investigated, suggesting
that cytokine responses at birth do not significantly influence the colonization. Cord
blood responses have previously been associated with future development of allergic
disease. Low numbers of IL-12-secreting cells in cord blood have been associated
with increased risk for sensitization at 24 months of age107 whereas high levels of IL5 and IL-12 in PHA-stimulated cord blood, associates with IgE sensitization108. In
relation to IgE sensitization, no associations between cord blood- or PBMC cytokine
secreting cells were observed neither did the numbers of cord blood cytokine
secreting cells associate with cytokine secreting cell numbers at 24 months.
30
For the reasons previously discussed, probiotic supplementation, has been more or
less successful in the prevention of allergic diseases, while its administration to
already allergic individuals has mediated immunomodulatory effects on both
cytokines and antibody isotypes produced and to a certain degree reduced clinical
symptoms94,95,101.
The mechanisms how different species interact with and influence the immune system
need further investigation, but clearly dysbiosis in the early microbiota may indeed
modulate the risk of developing inflammatory disease like allergy later in life.
GENERAL CONCLUSIONS
In this work, we investigated the microbiota in relation to both allergic disease at age
five and numbers of cytokine-secreting cells at 24 months, after PHA stimulation. We
show that different bacterial species positively and negatively associate with allergic
disease at five years of age, which is also mirrored in relation to numbers of cytokineproducing cells at 24 months of age. More specifically, colonization with Lactobacilli
and Bifidobacteria early in life associates with being non-allergic at age five. These
species of bacteria also modulate the numbers of cytokine-secreting cells, lowering
cytokine responses after PHA stimulation, which may be favorable in relation to
allergic disease. On the contrary, S. aureus colonization in infancy, seemed to be
more frequent among allergic children, and associated with higher numbers of
cytokine-secreting cells. Interestingly, the S. aureus induced numbers of cytokinesecreting cells, was reduced if co-colonization with Lactobacilli was present,
suggesting that some species of bacteria influences the effect of other species. Thus,
the presence of
“unfavorable” bacteria does not matter, as long as “beneficial”
bacteria are counteracting their adverse effect on the immune system. Although some
species of bacteria have been ascribed specific functions in animal models, the
maturation of the immune system does probably not only rely on single species of
bacteria, but rather a balanced diverse microbiota providing proper signals.
31
CLINICAL RELEVANCE
Dysbiosis in the microbiota is implied in a variety of inflammatory conditions, thus
investigating the early microbiota in relation to the maturation of the immune system
will contribute to increasing knowledge about both etiology and potentially
prevention of these immune mediated diseases.
Indeed supplementation of probiotics has potent immunostimulatory effects, in both
animals and humans. In animals, Lactobacillus species protected against virusinduced inflammation102, but also influenced non-infectious conditions like body fat
storage109 and protected against development of allergen-induced allergic disease93. In
humans, beneficial effects of probiotics have been observed during both infectious
and non-infectious conditions as recently reviewed110. Also, supplementation of
probiotics, to already allergic individuals has to a certain degree reduced their clinical
symptoms and modulated their immune responsiveness94,95,101.
Probiotic administration, for the prevention of allergic disease, has been more or less
successful, stressing the need for further investigation of how, mechanistically,
different species of bacteria influence the immune system. However, due to the
immunostimulatory potential of bacterial species, more thorough investigations of
their influence on the immature newborn immune system need to be considered as its
modulation may have both beneficial and adverse effects.
FUTURE PERSPECTIVES
BACTERIAL INFLUENCE ON EPITHELIAL CELL RESPONSES
any questions remain to be answered regarding the interaction of the microbiota with
the intestinal epithelium and the immune system. Previous in vitro studies exploring
bacterial interactions with immune cells have to a large degree ignored the epithelium.
The intestinal epithelium is far from a passive border separating the immune structure
from the lumen, but consists of cell types secreting antimicrobial peptides,
transporting sIgA in addition to secreting cytokines among many functions.
With respect to the findings in both PAPER I and II, we aim to further study how
different bacterial species interact with and influence intestinal epithelial cells in in
vitro as well as in animal models. We aim to continue exploring whether the
32
interactions depend on soluble factors produced by the bacterial species or contact
dependent interactions. Unpublished results, from our group and others111, indicate
that soluble factors influence cytokine/chemokine production from epithelial cells.
Also, breast milk has been reported to modulate the bacterial induced cytokine release
by epithelial cells112,113 thus the modulatory effect of breast milk will be further
investigated.
EARLY MICROBIOTA AND REGULATORY IMMUNE FUNCTION
LATER IN CHILDHOOD
We demonstrate that the different species of bacteria modulate the cytokine profile at
24 months of age (PAPER II), which potentially has implications for future
development of, for instance, allergic disease, which our group (PAPER I) and others
have observed.
Whether early colonization has a pronounced effect on the immune system later in
childhood is not known. As a follow up, of the children in PAPER I and II, has been
performed we have the possibility to relate the early colonization to immune
phenotype and function at age five. As important it is to mount an immune response,
in case of infection, it is equally crucial that negative regulation of the response
occurs, as failure in this can lead to chronic inflammation. In light of the findings of
others that allergic individuals have a hyper-activated immune response and
suboptimal Treg responses, it would be interesting to evaluate how early colonization
relates to the phenotype and function of Tregs after both microbial and allergen
stimulations. Does the microbiota early in life associate with an alteration in
phenotype and function of Tregs?
As a sidetrack, we will also explore how, the early gut microbiota relates to body
mass index throughout childhood, as it has been shown that S. aureus associates with
overweight45 and other studies have seen differences in lean and obese individuals114.
33
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to Eva Sverremark-Ekström for being
an outstanding supervisor, for always being present and giving me the support that I
need!
To my co-supervisors, Klavs Berzins and Caroline Nilsson, for your support.
To seniors, Carmen Fernandez, Eva Severinsson and especially Marita TroyeBlomberg for thorough reading of my thesis.
Ylva Sjögren-Bolin, thanks for your friendship and for introducing me into the
project.
Former and present students at the department, Gelana, Maggan and Anna-Leena,
thank you for being friendly to me.
Shanie Saghafian-Hedengren and Ebba Sohlberg for the discussions about science,
immunology, life in general and always being present.
To my Family, for your support and patience with me through the ups and downs
For your never-ending love, support and belief in me, Micke, I love you!
34
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