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Doctoral thesis from the Department of Immunology, the Wenner-Gren Institute,
Stockholm University, Sweden
Microbial and maternal influences on allergic sensitization during
childhood: defining a role for monocytes.
Shanie Saghafian Hedengren
Stockholm 2009
Cover illustration: Nils Jarlsbo
All previously published papers were reproduced with permission from the publishers.
Distributed by Stockholm University.
Printed in Sweden by Universitetsservice AB, Stockholm 2009
 Shanie Saghafian Hedengren
ISBN: 978-91-7155-872-5
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SUMMARY
Background: Allergic disorders are inflammatory diseases that develop on the basis of
improper immunological reactions. A decrease in particular childhood infections, or a lower
exposure to microbial components in infancy, has been suggested as contributing factors to
the higher allergy prevalence seen in affluent societies during the last decades. An altered
overall spectrum of infections may set the stage for an imbalanced T-helper type 1 (Th1)/Th2immune profile, which could contribute to the development of allergies. Further, maternal
allergy has been shown to confer a higher risk for allergic sensitization compared to paternal
allergy, suggesting a maternal influence, possibly already in utero, on allergy development.
Toll-like receptors (TLR) 2 and 4, which recognize peptidoglycan (PGN) and
lipopolysaccharide (LPS) respectively, are expressed on e.g. monocytes and have been
implicated in modulating the risk of allergic sensitization. Upon pathogen recognition, TLRsignalling activates intracellular targets, including p38-MAPK, which induce the expression
of genes encoding cytokines and co-stimulatory molecules that regulate adaptive responses.
Aims: The overall aim here was to examine how the developing immune system in young
children is affected by microbial exposure. This was investigated in blood samples from
children participating in a prospective study cohort. Paper I and II addressed how maternal
allergy and allergic disease influence the monocyte’s response to bacterial compounds,
commonly found in the environment. Paper III determined if the timing of primary EpsteinBarr virus (EBV) infection during childhood could be an important factor in modifying the
risk to develop IgE-sensitization and paper IV investigated how innate immunity was affected
by early-life EBV infection by examining monocyte-induced NK-cell cytokine response.
Results: Two-year old infants with allergic mothers have markedly lower p38-MAPK
phosphorylation and IL-6 in response to PGN, a finding that was associated with maternal
allergy but not IgE-sensitization in the child. At 5-years of age, allergic subjects showed a
markedly lower up-regulation of TLR2 on their monocytes in response to PGN. The altered
regulation of TLR2 after PGN challenge was not accompanied by changes in p38-MAPK
activity or inflammatory cytokine production whereas spontaneous IL-6 release was found to
be significantly higher in allergic subjects. Five-year old children who were EBV seropositive
already at the age of 2 were at a significantly lower risk of being chronically IgE-sensitized.
In contrast, acquisition of EBV after 2-years of age was associated with a higher risk of being
IgE-sensitized at the age of 5. Following in vitro stimulation, EBV seropositive 2-year olds
had a significantly lower proportion of IFN-γ+ NK cells that also produced decreased levels of
IFN-γ, when compared to their seronegative counterparts. Also, in vivo IFN-γ levels were
noticeably lower in EBV seropositive infants. EBV infected subjects tended to have lower
proportions of CD14+CD16+ monocytes in a non-stimulated condition. Notably, CD14+CD16+
cells were found to induce NK-cell IFN-γ release more potently than CD14++CD16–
monocytes, which might provide an explanation for the lower innate IFN-γ levels seen in
EBV seropositive infants.
Interpretation: Data from this thesis 1) underscores the importance of early-life microbial
exposure for shaping of proper allergy-protective immune profile 2) highlights EBV as a
potential immunomodulatory candidate and 3) stresses that maternal allergy up to the age of
2, and a defective monocyte TLR2 regulation in pre-school age children with allergic disease
should be considered as factors influencing innate responses. In particular, findings from this
thesis suggest that monocytes could have a role in allergic sensitization as their functionality
appears to be influenced by all the aforementioned aspects.
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SVENSK SAMMANFATTNING
Bakgrund: Mikrobiell exponering tidigt i livet anses viktigt för att vårt immunsystem ska
mogna och balanseras på ett riktigt sätt. Enligt ”hygienhypotesen” leder en ökad
hygienstandard i välfärdsländer till att immunsystemet inte får tillräcklig stimulans via t.ex.
bakterier och virus, vilket leder till obalans och utveckling av sjukdomar som allergier.
Allergi betraktas numera som en folksjukdom. I många fall debuterar den i barndomen och i
Sverige har förekomsten av astma bland barn mer än fördubblats mellan 1970- och 1990-talet.
Att genetik påverkar allergiutveckling är oomtvistat, men på senare år har olika miljöfaktorer
diskuterats alltmer, framför allt i relation till ovan nämnda hygienhypotes. Barn med
allergiska mödrar är mera benägna att själva bli allergiska jämfört med barn som har
allergiska pappor. Resultat från studier tyder på att t.ex. den intrauterina miljön kan påverka
barnets immunsystem och allergiutveckling. Ett flertal olika immunceller uttrycker receptorer
som känner igen mikrober. De mest välstuderade, som också relateras till allergiutveckling, är
TLR2 (detekterar peptidoglykan) och TLR4 (detekterar LPS). Dessa uttrycks till hög grad av
monocyter och deras signalering aktiverar olika intracellulära enheter, t.ex. MAPkinaser, som
initierar produktion av co-stimulatoriska molekyler och cytokiner. Utöver sin centrala roll i
regleringen av adaptiva systemet behövs dessa faktorer även i merparten av naturliga mördar
(NK)-cellers aktivering. Epstein–Barr virus (EBV) är ett mycket vanligt och välkarakteriserat
herpesvirus som normalt ger harmlösa och knappt märkbara infektioner i tidig ålder. Efter
infektion söker sig EBV till immunceller och ligger latent kvar i dessa livet ut. Man har
kunnat visa att EBV-infektion före 2-års ålder associeras med lägre förekomst av tidig IgEsensibilisering. Hur EBV påverkar immunsystemet i tidig ålder, och konsekvensen av en
sådan modulering i relation till allergier, är däremot fortfarande tämligen okänt.
Målsättning & Resultat: Avhandlingens huvudsyfte var att studera hur mikrobiell
stimuli kunde påverka immunsystemet under dess utvecklingsskede. Alla undersökningar
gjordes på perifera blodceller eller plasma från barn som ingår i en prospektiv allergistudie.
Avhandlingens första del undersökte om maternell allergi hade någon påverkan på
avkommans benägenhet att svara på mikrobiell stimuli. I studie 1 undersöktes samma barn vid
födsel och 2-års ålder. Maternell allergi, men inte IgE-sensibilisering, relaterades till ett sämre
peptidoglykaninducerat p38MAPKinas- och IL-6-svar från monocyter hos 2-åringar. I studie
2 sågs ingen maternell effekt på motsvarande svar hos 5-åringar, däremot fanns en defekt i
TLR2-uttryck med avseende på barnens egen allergi. Andra delen av avhandlingen prövade
om tidpunkten för EBV-infektion kunde vara en avgörande faktor för skydd mot IgEsensibilisering samt ifall EBV-seropositiva barns NK-celler och deras interaktion med
monocyter påverkats av EBV-infektionen. Studie 3:s resultat visade att 5-åringar som var
EBV+ redan vid 2 år hade märkbart lägre risk för kronisk IgE-sensibilisering. Däremot var
fler 5-åringar som blev EBV-infekterade efter 2-årsålder IgE-sensibiliserade. Studie 4
jämförde celler från EBV+ och EBV– 2-åringar in vitro. EBV+ barn hade lägre andel IFN-γ+
NK-celler, som även producerade lägre halter av IFN-γ. Denna cytokin fanns dessutom i
mindre koncentration i seropositiva barns plasma. Vidare hade EBV+ barn en lägre andel
CD14+CD16+ monocyter. CD14+CD16+ celler visades kunna inducera högre IFN-γ
produktion hos NK-cellerna, vilket kan förklara det svagare IFN-γ svaret hos EBV+ barn.
Slutsats: Resultaten framhåller att 1) tidig mikrobiell exponering kan vara viktig för
utformningen av en allergiskyddande immunprofil, 2) EBV skulle kunna bidra till en sådan
profil, 3) maternell allergi under tidig barndom samt allergisk sjukdom vid förskoleålder är
faktorer som kan påverka medfödda immunsystemets svar mot mikrober. Sammantaget skulle
monocyterna kunna vara delaktiga i allergisk sensibilisering, då alla föregående aspekter
verkar påverka deras funktion.
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LIST OF PAPERS
This thesis is based on four original articles, listed below, which will be referred to by their
roman numerals in the text:
Paper I
Saghafian-Hedengren S, Holmlund U, Amoudruz P, Nilsson C, Sverremark-Ekström E.
Maternal allergy influences p38-mitogen-activated protein kinase activity upon microbial
challenge in CD14+ monocytes from 2-year-old children Clin. Exp. Allergy. 2008; 38: 449-57.
Paper II
Amoudruz P, Holmlund U, Saghafian-Hedengren S, Nilsson C, Sverremark-Ekström E.
Impaired Toll-like receptor 2 signalling in monocytes from 5-year-old allergic children. Clin.
Exp. Imunol. 2009; 155: 387-94
Paper III
Saghafian-Hedengren S, Sverremark-Ekström E, Linde A, Lilja G, Nilsson C. Early-life
EBV infection protects against persistent IgE-sensitization. Submitted.
Paper IV
Saghafian-Hedengren S‡, Sundström Y‡, Sohlberg E, Nilsson C, Linde A, Troye-Blomberg
M, Berg L§, Sverremark-Ekström E§. Herpesvirus seropositivity in childhood associates with
decreased monocyte-induced NK cell IFN-gamma production. J. Immunol. 2009; 15: 2511-7.
‡§
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These authors contributed equally to the work.
Genius is 1% inspiration, 99% perspiration.
Thomas A. Edison (Harper's Monthly, 1932)
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TABLE OF CONTENTS
SUMMARY
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SVENSK SAMMANFATTNING
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LIST OF PAPERS
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ABBREVIATIONS
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FOREWORD AND GENERAL AIM OF THE THESIS
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BACKGROUND
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INTRODUCTION TO THE IMMUNE SYSTEM
COMPONENTS OF THE IMMUNE SYSTEM
Monocytes and macrophages
Dendritic cells
Granulocytes
Mast cells
NK cells
B lymphocytes
T lymphocytes
INNATE RECOGNITION OF MICROBES
TOLL-LIKE RECEPTORS
TLR ELICITED SIGNALLING
p38-mitogen activated protein kinases
CYTOKINES AND CHEMOKINES
TNF, IL-1β, IL-8 and IL-12
IL-10
IL-6
IFN-γ
Regulation of helper T-cell responses
MONOCYTES AND THEIR INTERPLAY WITH NK CELLS DURING INFECTION
MONOCYTES
Phenotype
Migratory behaviour
Function
MONOCYTE INDUCED NK-CELL ACTIVATION DURING INFECTION
ALLERGY
MECHANISMS BEHIND IGE-MEDIATED ALLERGY
Risk factors
EPIDEMIOLOGICAL EVIDENCE BEHIND THE “HYGIENE HYPOTHESIS”
Molecular mechanisms?
EFFECT OF ALLERGIC HEREDITY AND HERPESVIRUSES ON EARLY-LIFE INNATE RESPONSES
Maternal allergy
Epstein-Barr virus
IS THERE A ROLE FOR MONOCYTES IN THE DEVELOPMENT OF ALLERGIES?
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PRESENT STUDY
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OBJECTIVES
SUBJECTS
STUDY COHORT , EVALUATION OF IGE-SENSITIZATION AND ALLERGIC DISEASE
DETERMINATION OF EBV AND CMV SEROSTATUS AT 2- AND 5-YEARS OF AGE
Earlier cohort findings
METHODS
PHOSPHO-SPECIFIC FLOW CYTOMETRY
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RESULTS AND DISCUSSION
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PAPER I & II
PAPER III & IV
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GENERAL CONCLUSIONS
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FUTURE PERSPECTIVES AND PRELIMINARY RESULTS
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STUDY I & II
STUDY III & IV
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ACKNOWLEDGEMENTS
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REFERENCES
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ABBREVIATIONS
AEDS: atopic eczema/dermatitis syndrome
APC: antigen-presenting cell
BCR: B-cell receptor
CBA: cytometric bead array
CBMCs: cord-blood mononuclear cells
CMV: Cytomegalovirus
EBV: Epstein-Barr virus
GeoMFI: geometrical mean fluorescence intensity
Ig: Immunoglobulin
IL: interleukin
LPS: lipopolysaccharide
mDC: monocyte-derived dendritic cell
MHC: major histocompatibility complex
NK cell: natural killer cell
NOD: nucleotide oligomerization domain
PAMPs: pathogen-associated molecular patterns
PBMCs: peripheral blood mononuclear cells
PGN: peptidoglycan
PHA: phytohaemagglutinin
PRRs: pattern recognition receptors
p38-MAPK: p38-mitogen activated protein kinase
RA: rheumatoid arthritis
SEB: Staphylococcus aureus exotoxin B
SPT: skin prick test
sgp130: soluble glycoprotein 130
sIL6R: soluble IL-6 receptor
TCR: T-cell receptor
Th1: T-helper type 1
Th2: T-helper type 2
TLR: toll-like receptor
Tregs: regulatory T-cells
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FOREWORD AND GENERAL AIM OF THE THESIS
Allergic disorders are inflammatory diseases that develop on the basis of improper
immunological reactions. The prevalence of allergic disorders among the world’s affluent
population has steadily increased during the past few decades and the reason behind this,
which is most pronounced among children, is still unknown. It has been hypothesized that
changes in exposure to environmental factors may be responsible for the observed ‘allergy
epidemics’. Among the suggested environmental influences are bacterial and viral exposures
during childhood. During the last years, great effort has been made to understand how
interactions between the immune system and microorganisms may alter the tendency to
become allergic. The overall purpose of this thesis was to further investigate this aspect to
better understand the impact of microbial exposure in relation to allergy development during
childhood.
BACKGROUND
INTRODUCTION TO THE IMMUNE SYSTEM
The condition of being immune, immunity, is protection from unwanted biological invasion
that causes infection or disease. The immune system in vertebrates is composed of two highly
interrelated components; innate and adaptive, which are mutually required for the resolution
of most infections. The concept of innate immunity refers to the first line of defence that
operates to limit infection in the early stages after pathogen exposure. The ability to rapidly
recognize and limit infectious challenge is mediated by pre-existing molecular and cellular
mechanisms, with specificity against broad classes of molecules that distinguish common and
frequently encountered pathogens. There are different anatomical levels where the
components of the innate immune system protect the host from pathogen invasion. For
instance, the initial barriers such as the skin- and mucosal membranes and the acidity of the
stomach prevent microbes from entering the host’s body. If these barriers are trespassed, an
inflammatory response is elicited where cells and anti-microbial proteins, contribute to the
elimination of invaders1,2.
Eventually the innate system alerts acquired (or adaptive) immunity, which
displays a high degree of specificity and diversity. The adaptive immune cells possess the
capacity to mount memory. As a result of memory, the adaptive immune cells act faster and
are more powerful in neutralizing pathogens upon a second encounter. Ultimately, the
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immune response must be resolved, which requires sequential and extremely well coordinated
endogenous mechanisms that result in the conversion of insulted tissue back to its pre-injured
state3. Indeed, the inflammatory process is like a double-edged sword; in its absence
infections and injuries cannot heal which would lead to loss of organ function and
compromised host survival. On the other hand, inflammation that runs unchecked can also set
the stage for a variety of diseases such as atherosclerosis and rheumatoid arthritis (RA).
Components of the immune system
The immune system comprises both cellular and soluble members. Examples of the latter are
anti-microbial peptides, the complement system, cytokines and chemokines. In the following
pages, there will be a brief introduction to cells of the immune system. In the next section,
cytokines and chemokines that are relevant to my studies will be described; remaining soluble
factors will not be presented. Since the main cells in focus in this thesis are the monocytes,
there will be a more detailed description about monocytes and their cross talk with natural
killer (NK) cells in a separate chapter. NKT cells will not be brought up in this thesis.
Innate cells
Monocytes and macrophages
Monocytes and macrophages are vital for the regulation of immune responses and the
development of inflammation. They originate from common progenitors in the bone marrow
and are members of the mononuclear phagocytic system. During haematopoiesis, ganulocytemonocyte progenitors differentiate and give rise to blood monocytes, which account for 510% of circulating leukocytes. Upon entering peripheral sites, monocytes differentiate into
tissue macrophages as well as other specialized cells including dendritic cells (DCs). Proinflammatory, metabolic and immune stimuli increase the recruitment of monocytes to
peripheral sites where they contribute to host defence, tissue remodelling and repair4.
Macrophages are dispersed throughout the body and comprise a heterogeneous
population with a central role in pathogen eradication and in maintaining tissue homeostasis.
Upon ingestion, pathogens become trapped in the macrophage phagosomes that further fuse
with lysosomes to form phagolysosomes. These contain enzymes and reactive oxygen- and
nitrogen species, which digest the pathogen. Macrophages also express receptors that bind
antibodies, which are antigen-specific molecules (secreted by B cells) that bind to target cells.
This directs the otherwise non-specific phagocytes towards specific antigen binding. Based on
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their anatomical location macrophages exert specialized functions. For example, alveolar
macrophages express high levels of receptors that are needed for eradication of inhaled
microorganisms and particles from the environment, while intestinal macrophages display
phenotypic characteristics related to high phagocytic and anti-microbial activity but
diminished release of inflammatory cytokines4.
Their phenotypic and functional heterogeneity also reflects their differential
activation modes, which are divided into three major classes5. Classically activated
macrophages are products of the cell-mediated response. They arise in response to microbes
and interferon (IFN)-γ and exhibit increased killing of intracellular pathogens, have high
levels of co-stimulatory molecules, release inflammatory cytokines and mediators and
promote extracellular matrix breakdown. Classically activated macrophages have been shown
to be mediators of immunopathology in several autoimmune diseases. Alternatively activated
macrophages contribute to homeostatic processes in the host. They develop in the presence of
interleukin (IL)-4 or IL-13, but can also arise in response to helminths. Alternatively activated
macrophages express scavenger receptors, mannose receptors and secrete molecules that
contribute to the resolution of inflammation and wound healing due to their antiinflammatory, fibrotic and angiogenic activities. These cells have, however, also been
implicated in the pathophysiology of allergic asthma5,6. Regulatory macrophages arise in
response to a number of stimuli including immune complexes, prostaglandins,
glucocorticoids, apoptotic cells and the regulatory cytokine IL-10. A consistent characteristic
of regulatory macrophages is their production of marked levels of IL-10 along with
diminished amounts of IL-127, while they do not contribute to extracellular matrix restoration
and can express high levels of co-stimulatory molecules5. Further, a number of bacterial, viral
and parasitic pathogens can induce the development of regulatory macrophages to interfere
with phagocytosis5.
Dendritic cells
DCs are professional antigen-presenting cells (APCs) with a predominant role in priming and
directing adaptive immune responses, as well as in the induction of self-tolerance8. They are
present in small amounts but are distributed throughout the body, particularly at sites of
pathogen encounter such as the skin and the mucosal surfaces. They can also be found in the
circulation. Apart from being superior in the priming of naïve T cells, DCs possess the ability
to migrate to other locations where optimal interactions with adaptive cells can occur, to
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present antigens they have captured in the periphery. This is a feature that appears to
distinguish DCs from other professional APCs9.
DCs comprise two main categories: conventional and plasmacytoid (p). The
conventional DCs can be further divided into two variants based on the paths they follow to
access lymphoid tissues during steady state conditions10. Migratory DCs are tissue-derived
and travel via afferent lymphatics to reach local draining lymph nodes while the remaining
constitute resident or blood-derived DCs. Monocyte-derived (m) DC is the most common DC
type used in studies on human and mouse DC biology. It is a distinct DC type that develops
during inflammatory processes and microbial stimuli, but whether it corresponds to any of the
lymphoid-tissue DCs during steady state is unclear8,10. pDCs were identified during the past
decade and today intensive research is performed for understanding of their functions in
immunity. They are round-shaped in resting stage, circulate in blood and lymphoid tissues
and have a critical role in recognition of, and defence against, viruses. However, the role of
pDCs in antigen presentation and T-cell priming is unclear as they display low amount of costimulatory and antigen-presenting receptors and have insufficient capacity to capture and
process antigens11.
Granulocytes
Granulocytes denote neutrophils, eosinophils and basophils on the basis of cellular
morphology and staining characteristics of their cytoplasm. Neutrophils are the most abundant
and account for 50-70% of the circulating white blood cells in humans. They are specialized
in the phagocytosis and digestion of pathogens and are generally the first to arrive to sites of
infection or tissue injury. They home to these sites upon receiving chemotactic signals such as
inflammatory mediators and pathogen- or host-derived molecules. Following phagocytosis,
neutrophils release various cytokines and chemokines that contribute to the inflammatory
response. Once phagocytosis and digestion of microbes is complete, neutrophils undergo
apoptosis, which is followed by their clearance by mononuclear phagocytes at the site of
inflammation. Indeed, neutrophil apoptosis is not only essential for maintaining immune
system homeostasis but also required for facilitating the ultimate resolution of inflammation12.
Eosinophils, which represent 1-3% of peripheral blood leukocytes, have minor
phagocytic activity compared to neutrophils. They have historically been believed to play a
role in defence against parasitic infections. Under steady state conditions the majority of
eosinophils home to the gastrointestinal tract. They are also present in the thymus, mammary
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gland and uterus. Their arrival to sites of inflammation is mediated by a number of factors,
particularly cytokines that are associated with allergic reactions. Of note, eosinophils are
strongly associated with the pathogenesis of allergic disorders, as in allergic asthma13.
Basophils are non-phagocytic granulocytes that account for less than 1% of
circulating blood leukocytes. Like mast cells (described below), basophils express the high
affinity immunoglobulin (Ig)-E receptor (FcεRI) on their surface. The physiological role of
basophils in immunity remains poorly defined, as this granulocyte population has been largely
overlooked. However, recent evidence from studies in mice suggests that they could provide
an innate source of the cytokine IL-4 in response to allergic inflammation and parasitic
helminth infections14. In addition, it has been shown that isolated basophils from mouse
spleen, liver or bone marrow support the development of T-helper type (Th) 2-cytokine
responses via IL-4-dependent mechanisms15.
Mast cells
Mast-cell precursors are formed in the bone marrow and do not differentiate into mast cells
until they leave the circulation and enter the tissues. Mast cells are abundant in a wide range
of tissue sites where the body comes in contact with the external environment, including the
skin, lung, conjunctiva, nasal mucosa as well as the mucosal epithelial tissue of the
respiratory and digestive tracts16,17. As already mentioned, mast cells express FcεRI, through
which they act as key effectors in IgE-related immune responses such as allergic
hypersensitivity reactions and parasite infections18. Mast cells can also contribute to host
defence against bacteria as they have the capacity to recognize microbial products and aid
microbial clearance by recruiting neutrophils to the site of infection19.
NK cells
NK cells were originally classified as a population of large granular lymphocytes that can kill
target cells in a spontaneous manner without prior sensitization. They contribute to host
defence through effective cytolysis of virus-infected and tumour cells. Further, NK cells are
an important source of cytokines and chemokines that contribute to inflammation and
polarization of T-cell responses20. In contrast to B- and T-lymphocytes, NK cells employ a set
of germ-line encoded activating and inhibitory receptors for their killing of altered cells,
which appears to result from the net outcome of simultaneous activation and inhibition
signals. Today it is known that direct NK cell activation is triggered by aberrant expression of
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major histocompatibility complex (MHC) class I or non-MHC ligands for inhibitory
receptors, and/or the up-regulation of stress-induced molecules that are ligands for NK-cell
activating receptors21,22. Interestingly, none of the activating receptors, except CD16
(FcγRIII), are able to elicit effector functions by themselves in freshly isolated human NK
cells23. Rather, it appears that engagement of different pair-wise combinations initiates
cytolysis and cytokine production23. It is appreciated that the positive and negative signals
transmitted by activating and inhibitory receptors are balanced under baseline conditions for
maintenance of tolerance24.
NK cells comprise roughly 15% of circulating human lymphocytes and are
widely distributed throughout the body25,26. Observations in mice have localized NK cells to
several compartments including the lung, liver, spleen, bone marrow, lymph nodes and
thymus
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. In addition, NK cell numbers are greatly increased in the mucosal lining of the
uterus during pregnancy of humans and mice; approximately 70% of the infiltrating
leukocytes are NK cells28,29. Today NK cells are regarded as a heterogeneous population with
functional differences and varying subset distribution between distinct anatomical sites30. The
two major subsets include CD56bright CD16– NK cells, which have been suggested to display
low cytolytic activity but high cytokine production, and CD56dim CD16+ cells that
predominate in peripheral blood and inflamed tissues and display lower cytokine release but
high cytotoxic activity25,30,31. Of note, characterization of human secondary lymphatic tissues
has revealed that up to 75% of lymph node- and tonsil NK cells constitute the
CD56brightCD16– subset, which produce high levels of IFN-γ26 – an NK-cell signature cytokine
with antiviral, immunoregulatory, and anti-tumour properties32. In addition to IFN-γ
production, NK cells have the capacity to produce a variety of factors including
immunosuppressive and angiogenetic cytokines25, which indicates that they could have a role
in dampening immune responses20 as well as in tissue remodelling28,29. Interestingly, they
have also been suggested to control overstated reactions by directly targeting activated
immune cells during the course of an immune response33.
Indeed, direct NK-cell activation against certain pathogens, which induce
ligands for NK-cell cytotoxicity receptors34 or downregulate MHC class I ligands for NK-cell
inhibitory receptors35, has been demonstrated. In the context of many infectious challenges
however, a large body of experimental data points to the inability of isolated NK cells to
become activated36 – a condition that is reversed through signals provided by for instance
monocytes37,38, DCs38,39 and macrophages40,41.
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Adaptive cells
The sensors of adaptive immunity are the B-cell receptors (BCRs) and the T-cell receptors
(TCRs, only αβ T-cells will be considered here). B- and T-cells have the capacity to
recognize a diverse range of substances and distinguish between minor differences among
these. The high level of specificity and diversity results from rearrangements of BCR or TCR
genes from their germline configuration, generating a random repertoire of antigen-binding
receptors42. Antigen recognition triggers the activation, proliferation and differentiation of
adaptive cells. This clonal selection accounts for the majority of the fundamental properties of
the adaptive immune system. Since the adaptive immunity selectively identifies and
remembers antigens it has encountered over time, it will continuously develop and mature
upon its meetings with foreign substances. Consequently, adaptive immune responses are not
uniform among all members of the same species; but result from the host’s reactions against
specific antigenic challenges43.
B lymphocytes
B lymphocytes contribute to humoral immunity where the effectors are antigen-specific
antibodies, also called immunoglobulins. The Ig exists both as a membrane-bound receptor
(the BCR) and in a soluble form secreted by plasma cells. The surface Ig is involved in
processes that are fundamental to B-cell activation, differentiation and apoptosis, whereas the
secreted antibody is able to directly neutralize antigens and induce additional effector
mechanisms that contribute to antigen clearance. In humans and mice, B cells arise from
lymphoid progenitors and develop to become functionally mature in the bone marrow. Naïve
antigen inexperienced B cells are IgM+ and IgD+. They circulate in the blood and lymph and
are found in secondary lymphatic organs, especially in the spleen and lymph nodes. It is
appreciated that B cells can detect and mount responses against small soluble antigens as well
as immobilized particulate antigens on the membrane of follicular DCs and macrophages44.
Activation and differentiation of naïve B cells are antigen-driven and those that do not
encounter antigen have a short life span and will die by apoptosis within weeks. For most
antigens, B cells need assistance from T cells for their antibody response, which is referred to
as thymus-dependent responses.
Even if the precise factors that determine activated B-cell fate remain uncertain,
they appear to differentiate along two pathways. In secondary lymphoid organs, they can
differentiate to form extra-follicular plasmablasts that are important for a rapid release of
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antibodies and contribution to early protective responses. B cells also go through germinalcentre reactions, during which three important events take place; affinity maturation, classswitching and differentiation to plasma and memory-B cells. The process of affinity
maturation results from repeated rounds of point mutations in the antigen-binding domains of
Igs and the selective expansion of the clones that progressively increase their antibodyaffinity against the initially encountered antigen45. Class-switch is a mechanism that retains
the antigen-specificity of antibodies while changing their isotype, for instance from IgM to
IgG. Isotype switching promotes changes in biological function of antibodies to the most
suited for elimination of the encountered invader. The memory cells formed after a primary
response comprise recirculating memory-B cells and stationary plasma cells, which can be
long-lived46. A recall response has a shorter lag period, reaches a higher magnitude and lasts
longer with increased antigen-affinity as well as other isotypes than IgM.
T lymphocytes
Progenitor-T cells arise in the bone marrow and migrate to the thymus where they further
differentiate and generate mature and functionally diverse CD3+ helper (CD4+) and cytotoxic
(CD8+) T cells47. Unlike B cells, which can recognize free antigen, T cells recognize peptide
antigens only in the context of MHC molecules. Helper-T cells are vital for the immune
system as they secrete cytokines that act co-stimulatory or regulatory on immune responses.
They are restricted to MHC class II molecules that are found on APCs, and in their naïve
form, CD4+ cells do not become fully activated unless they receive co-stimulatory signals via
professional APCs. Dependent on the nature of signals that naïve helper-T cells receive
during their activation, they can differentiate into diverse effector subsets; T-helper type 1
(Th1), Th2 and Th17 that produce different sets of cytokines tailored for the most favourable
response against different encountered substances48,49.
Th1 cells have a crucial role in cell-mediated immunity. They promote the
cytotoxic effector functions of CTLs, NK cells and macrophages and support the B-cell
production of IgG1 in humans and IgG2a in mice, which are isotypes that promote antibodydependent cell-mediated cytotoxicity (ADCC)50. Th2 cells, on the other hand, support
humoral (extracellular) immunity, which has a prominent role in defence against helminthic
infections. The Th2-promoted immunity is mediated through B-cell synthesis of IgG4 and IgE
in humans and IgG1 and IgE in mice50,51, which are antibody classes that activate mast cells
and eosinophils and support eosinophil-mediated attack on helminths. Recently, a new lineage
18
of IL-17-producing helper-T cells (Th17) has been characterized, which seems to play a role
in the pathogenesis of organ-specific autoimmune diseases52 and in allergic inflammation53. In
addition to its pro-inflammatory role, IL-17 has been observed to be important in protection
against extracellular bacteria and against fungi54.
Cytotoxic-T lymphocytes (CTLs) are MHC class I restricted and implicated in
the killing of virus infected- and tumour cells, as well as in transplant rejection. The precise
mechanism of activation of naïve cytotoxic-T cells to effector CTLs is not entirely clear. It
appears to involve peptide presentation by helper-T cell ‘licensed’-APCs along with costimulation55. Also, IL-2 is likely to be involved since it is a principal cytokine needed for
proliferation and differentiation of activated precursor CTLs to effector cells55. As for the B
cells, memory-T cells may persist long after an infection has resolved and can quickly expand
to large numbers of effectors upon re-exposure to their cognate antigen.
There is another population of T cells called regulatory-T cells (Tregs, formerly
known as suppressor-T cells), which have been shown to suppress autoimmune responses via
inhibition of self-reactive T-cells56. Today, Tregs are regarded essential in mediating peripheral
tolerance, preventing several autoimmune diseases, dampening chronic inflammatory
conditions and inhibiting responses against microbial infections56. Currently it is believed that
the Treg-immune suppression on effector-T cells functions through the following mechanisms;
suppression by inhibitory cytokines including IL-10 and transforming growth factor (TGF)-β,
suppression by cytolysis of target cells, suppression by metabolic disruption of activated cells
and suppression via DC targeting57,58. Two major subsets of CD4+ Tregs include the naturally
occurring and adaptive. Naturally occurring Tregs constitute 5-10% of all Th-cells, are
CD4+CD25+FoxP3+ and thymus-derived. Their suppression is antigen specific and requires
cell-to-cell contact in vitro56. Adaptive or induced Tregs include type-1 regulatory (Tr1) and
Th3 cells, which can be generated through immunosuppressive cytokines and orally delivered
antigens, respectively56. Their suppressive action is believed to occur mainly through IL-10
and TGF-β56,58,59.
INNATE RECOGNITION OF MICROBES
It was long considered that innate immunity was a non-specific system that had its principal
role in capturing and presenting antigens to adaptive cells. Yet, there were observations that
challenged this view and suggested additional roles for innate immunity. Experimental
findings demonstrated that MHC-TCR ligation and APC-derived co-stimulation had to be
19
delivered by the same cell to generate clonal expansion of helper-T cells60. It was also shown
that a variety of pathogen-derived substances potently induced the expression of costimulatory molecules on innate cells61. These findings lead to the postulation that innate cells
detect infectious agents through pathogen-specific receptors whose signals trigger costimulation and integrate innate and adaptive responses62,63. Progressively the innate receptors
were suggested to recognize common structural patterns among large groups of
microorganisms, as the receptors were found to be germ-line encoded. The patternrecognition receptors (PRRs) known then, like the serum protein mannose-binding lectin, did
not induce co-stimulation and did not fulfil a candidate role in integrating innate and adaptive
responses. The conceptual breakthrough came with the identification of Toll-like receptors
(TLRs), which are undoubtedly the best-characterized PRRs to date61-64.
TLR signalling has been subjected to intense research65, 66, and today it is known
that pathogen-associated molecular pattern (PAMP) recognition by a given TLR elicits
distinct signalling pathways that result in the activation of various sets of genes encoding
cytokines, chemokines, co-stimulatory molecules and anti-microbial compounds65-67. This
indicates that the activation of innate responses has a great impact on the initiation and
development of adaptive functions68,69. Furthermore, other classes of PRRs including the
cytosolic NOD-like receptors (NLRs), NALP proteins and RIG-like helicases have recently
been identified (Table 1)66,70. It should also be emphasized that the definition PAMP can be
somewhat misleading, as PRRs also recognize non-pathogenic microbial AMPs.
Nonetheless, innate immunity is also capable to respond to endogenous
molecules that under normal circumstances do not easily come in contact with PRRs. Host
cells release these so called alarmins due to abnormal cell death, infection, tissue damage and
certain pathological conditions71,72. For instance, heat-shock proteins, high mobility group
box 1 and self nucleic acids, which are normally confined to restricted intracellular
compartments, can during stress conditions translocate and elicit immune responses via
interactions with TLRs71,73. To date, the recognition of alarmins by PRRs is strongly
correlated with the pathogenesis of autoimmune diseases72,73.
20
Table 1. Pattern-recognition receptors and their ligands. Int. Immunol. 2009; 21: 318. Reprinted with permission from the
authors Kawai T. & Akira S., and Oxford University Press, © 2009 by The Japanese Society for Immunology.
Toll-like receptors
Currently, 13 members of TLRs have been characterized in the mammalian species. TLR1 to
TLR9 are conserved between humans and mice whereas TLR10 is non-functional in mice and
TLR11-TLR13 are lost in the human genome66. Each TLR can recognize molecules that have
a common pattern among classes of pathogens (Table 1). For instance, lipopolysaccharide
(LPS, also known as endotoxin) is a cell-wall component of Gram-negative bacteria and the
PRR for LPS, TLR4, detects basically any Gram-negative bacterial infection. TLR2 detects a
wide variety of microbial compounds including zymosan in fungi and peptidoglycan (PGN),
which is a component abundant in the outer membrane of Gram-positive bacteria. The ligand
specificity of TLR2 is dependent on heterodimeric interactions with other TLRs including
TLR1 or TLR6. TLR5 has been shown to sense flagellin from both Gram-positive and Gramnegative bacteria. Intracellular TLRs detect PAMPs in the lumen of intracellular vesicles and
include TLR3; which recognizes double-stranded viral RNA; TLR7 and TLR8 that are
implicated in the detection of single-stranded virus RNA and TLR9; which detects CpG
motifs from bacterial and viral DNA as well as hemozoin from malaria parasites 66,68,74.
21
TLRs are predominantly expressed by innate immune cells; including
professional phagocytes, DCs, macrophages and monocytes75-78. Human neutrophils express
mRNA for all the TLRs except TLR377. The highest densities of TLR2 and TLR4, which are
the two best-characterized TLRs today, are found on monocytes75-77. Monocytes also express
high mRNA levels for TLR1, 5, 8 and lower levels of TLR676,79. Blood DCs express TLR1, 2,
3, 5, 6, 8 and 1079 whereas mDCs also express TLR4 prior to activation76. pDCs express high
amounts of TLR7 and 9 and to a much lower extent TLR1, 6 and 1079. One study found that
human NK cells express TLR1-8 and TLR10 mRNA and are able to directly respond to TLR2
and TLR5 agonists80. Adaptive cells also express TLRs, although to a lesser extent. Human B
cells are generally agreed to express moderate to high levels of TLR1 and TLR6-1081. There
is also evidence for intracellular T-cell expression of TLR1, 2, 3, 4, 5 and 982 and extracellular
TLR2 and TLR4 expression on activated human T cells83. Triggering of human Treg TLR584
and TLR885 increases or dampens their suppressive function, respectively. On the whole
however, it appears that TLR signalling in T cells acts as co-stimulatory upon responses that
are triggered by TCR rather that inducing direct cellular activities82,83.
TLR elicited signalling
TLRs are transmembrane glycoproteins with leucine-rich-repeat motifs in their extracellular
ligand-binding domains. Their cytoplasmic signalling domain resembles that of the IL-1
receptor (IL-1R) and is known as Toll/IL-R1 homology (TIR) domain. A principal signalling
pathway common to all TLRs triggers the activation of the myeloid differentiation primaryresponse protein 88 (MyD88) adaptor-molecule as the primary downstream target. This
initiates sequential activation events that involve the activation of the nuclear factor-κB (NFκB) and the mitogen-activated protein kinases (MAPKs, Figure 1).
Upon recognition of ligands, TLRs dimerize and undergo conformational
changes that trigger the association of downstream adaptor molecules to the TIR-domains
starting with MyD88 followed by the recruitment of IL-1R-associated kinases 4 (IRAK-4)
and IRAK-1. For MyD88-dependent TLR2 and TLR4 signalling an additional adaptor
molecule termed TIR-associated protein (TIRAP)/MyD88-adaptor-like (MAL) is required for
the association of MyD88 to the cytoplasmic TIR-domains. The binding of IRAK-4 to
MyD88
assists
IRAK-4-mediated
phosphorylation
of
IRAK-1,
which
leads
to
autophosphorylation of the N-terminus of IRAK-1 that creates a binding site for another
adaptor protein called tumour-necrosis-factor-receptor-associated factor 6 (TRAF6). The
22
IRAK-1-TRAF6 composite (the latter is an ubiquitin protein ligase) associates with other
ubiquitin-conjugating enzymes, dissociates from the receptor and activates a complex of
proteins consisting of the transforming-growth-factor-β-activated kinase 1 (TAK1) and the
adaptors TAK1-binding protein 1 (TAB1), TAB2 and TAB3.
TAK1, which belongs the MAPK kinase kinase (MAPKKK) family,
phosphorylates a subunit of the inhibitor of NF-κB (IκB) kinase (IKK) complex and MAPK
kinase 6 (MKK6). Activated IKK in turn phosphorylates the IκBs that bind to and prevent the
translocation of NF-κB to the nucleus, which leads to the rapid polyubiquitylation and
degradation of IκBs. The activated MKK6 simultaneously catalyses downstream members of
the MAPK cascade, resulting in the activation of a family of transcription factors referred to
as activating protein-1 (AP-1), which like the freed NF-κB are critical for the induction of
genes for co-stimulatory molecules and pro-inflammatory cytokines (Figure 1, all reviewed
in65,74,86,87). The pattern of gene expression can to some extent differ between the TLR-elicited
pathways. For instance, TLR3, TLR7 and TLR9 but not TLR2 signalling mediates the
production of type I interferons (IFNs)88, which are crucial in the induction of antiviral
conditions in cells.
Figure 1. TLR-induced signalling. Reprinted from Seminars in Immunology, Vol. 21, Kawai T. & Akira S.
TLR-signaling: 24-32, © 2007 with permission from Elsevier Ltd.
23
p38-mitogen activated protein kinases
MAPKs are a group of protein kinases that are highly conserved among eukaryotic species.
They play an essential role in cellular processes such as stress responses, cell proliferation,
differentiation, apoptosis and immune defence89-91. Indeed, pathogens have evolved a number
of mechanisms to manipulate MAPK functions and circumvent the immune system92, which
highlights a critical role for MAPKs in anti-pathogenic responses. There are three welldefined families of MAPKs in mammals. The extracellular signal-regulated protein kinases
(ERKs) respond to growth factors and mitogens and function mainly in regulating cell
proliferation. The c-Jun NH2-terminal kinases (JNK) and p38-MAPKs appear to have
partially overlapping functions regarding their activation upon environmental perturbations
and inflammatory cytokine stimulation90,93,94. The functional role of JNK in innate immune
cells is poorly understood since there has been a lack of specific JNK-inhibitors until recent
years95. MAPK pathways result from cascades of sequential activation events that are
triggered with the phosphorylation of MKKs by MAPKKKs. Activation of MAPKs by MKKs
is mediated by conserved protein kinase cascades (Figure 2) and occurs through the dual
phosphorylation of threonine and tyrosine at tripeptide motifs of threonine-X-tyrosine90.
Phosphorylation of MAPK threonine and tyrosine residues results in conformational changes
that increase accessibility to, and catalysis of, substrates. Of note, MAPK pathways
themselves can be modulated by several mechanisms, including the dissociation of signalling
complexes and deactivation of pathway intermediates. The latter can occur through
deposphorylation of kinases by MAPK phosphatases, which have been suggested to play a
central role in the regulation of immune responses96.
The p38 family, from now on referred to as p38-MAPK, includes five members;
α, β, γ, δ and ERK697. p38α is the best characterized and the principal isoform in the majority
of inflammatory cells including monocytes89,94,97. As large body of evidence suggests that
p38-MAPK activity is critical for inflammatory responses, p38-MAPK is currently implicated
as a potential target for therapeutics in a number of inflammatory disorders98-100. The
importance of p38-MAPK during innate responses has been demonstrated by several means.
For instance, macrophages from mice knocked-out for Mkk3, which preferentially
phosphorylates p38-MAPK, display reduced p38-MAPK activity and a defective IL-12p70
release after LPS challenge101. Similar defective responses have been verified in humans
where the use of p38-MAPK inhibitor SB203580 blocks LPS mediated IL-12p70 production
in mDCs102. Notably, suppression of the p38-MAPK pathway inhibits the upregulation of costimulatory molecules on mDCs and their allostimulatory capacity103. Other MAPK members
24
can also affect co-stimulatory molecule expression and cytokine release by APCs67,90,95, but
inhibition of p38-MAPK seems to confer the most pronounced effects67. It appears that p38MAPK, ERK and JNK regulate APC-maturation pathways differentially; while p38-MAPK
seems crucial in achieving T-cell activation, inhibition of ERK and JNK partially affects DC
maturation and does not seem to be sufficient for dampening DC’s allostimulatory activity95.
Figure 2. MAPKinase signalling. Annu. Rev. Immunol. 2002. 20: 56. Reprinted with permission from the authors Dong C.,
Davis R. & Flavell R. and Annual Review of Immunology, Volume 20 © 2002 by Annual Reviews, www.annualreviews.org.
Cytokines and chemokines
The development of successful immune responses relies on complex interactions between
different immune cells, which are partially mediated through a group of soluble proteins
collectively referred to as cytokines. The term IL (interleukin), describes cytokines that are
secreted by, and can act on, leukocytes. Monokines denote cytokines that originate from
monocytes and macrophages. Chemokines belong to a family of small polypeptides, which
regulate leukocyte trafficking by affecting their migratory behaviour. This is accomplished by
the ability of chemokines to selectively control the adhesion, chemotaxis and activation of
leukocyte populations and subsets. Here, I will focus on presenting cytokines and chemokines
that were investigated in this thesis and briefly discuss cytokines that direct the development
of Th1- and Th2-responses.
TNF, IL-1β , IL-8 and IL-12
TNF, also known as TNF-α, is primarily produced by activated monocytes, macrophages and
T cells. It can however be released by other immune cells including NK cells, B lymphocytes,
mast cells, neutrophils and eosinophils104,105. The levels of circulating TNF are usually very
low in healthy subjects but increase during, and correlate with the severity of, inflammatory
and infectious conditions105. TNF mediates its activity by launching downstream pathways in
25
a similar manner as TLR-ligands13. Many of the TNF-induced pro-inflammatory effects has
been explained in the context of endothelial and leukocyte interactions. TNF mediates the
expression of various adhesion molecules by vascular endothelium, which in combination
with chemokines results in the recruitment of different leukocytes to the site of infection. The
physiological effects of TNF include the induction of acute-phase responses, neutrophil
activation and promotion of apoptosis in various cell types. However, this essential cytokine
has detrimental effects when it is inappropriately or excessively produced, and is currently
implicated in the pathophysiology of several inflammatory conditions including RA and
asthma104,105.
IL-1β is a well-characterized member of the expanding IL-1 cytokine family
and originates from various cell types including monocytes, macrophages, DCs, and epithelial
cells. Among the mediators initiating IL-1β synthesis are PAMPs and pro-inflammatory
cytokines including TNF and type I IFNs106. IL-1 is a potent inducer of fever, and acts in
synergy with TNF to provoke acute-phase as well as inflammatory responses. The signalling
pathways mediated by IL-1 are alike those initiated by PAMPs65, which consequently lead to
up-regulation of chemokine and inflammatory-cytokine genes. IL-1 has pathophysiological
roles in several inflammatory disorders including RA and neonatal-onset multisystem
inflammatory disease where treatment of the latter with IL-1R antagonist (A) has shown
promising results107. IL-1 also seems to be involved in the differentiation of Th17-cells in
humans108, which might further support its pathophysiological role in autoimmune disorders.
IL-8, or CXCL8, is a chemokine that regulates leukocyte trafficking during the
acute phase of inflammation109. Factors such as TLR agonists and TNF110 can induce the
production of IL-8 by a range of cells for example monocytes, granulocytes, lymphocytes,
fibroblasts and endothelial cells. The principal function of IL-8 is to attract neutrophils to the
site of inflammation. IL-8 intensifies the oxidative burst in pre-activated neutrophils and acts
as a growth factor in an autocrine manner in tumour cells such as malign melanoma and in
liver and colon carcinomas111. For that reason the influx of neutrophils and their reactive
oxygen species have been speculated to explain the observed association between
inflammation and carcinogenesis110.
IL-12, also called IL-12p70, is a heterodimeric cytokine that consists of the two
subunits p40 and p35. The former subunit is found in large excess and is shared with another
member of the IL-12 cytokine family called IL-2332,108. The production of IL-12 can be
triggered by various stimuli including pathogens, cytokines and co-stimulatory molecules on
lymphocytes. It is predominantly produced by monocytes, macrophages, DCs and neutrophils,
26
but to a lower extent also by B cells32,112. IL-12 is a potent innate cytokine that directs naïve
T-cell differentiation towards Th1 cells and supports the production of Th1-induced antibody
classes32. IL-12 is a crucial for a number of T-cell activities; it induces their proliferation,
enhances cytotoxicity, and stimulates the production of cytokines particularly IFN-γ. During
the initial phase of infection, IL-12 participates in a positive activation loop by inducing IFNγ production that in turn primes phagocytes for further IL-12 release. This feedback loop is
necessary for enhancing innate functions including the bactericidal activity, antigenpresentation and co-stimulation by APCs32,108,112. Interestingly, it has become increasingly
appreciated that some of the suggested pathophysiological roles of IL-12 in chronic
inflammation are rather attributed to the presence of IL-23, an inaccuracy thought to be
primarily caused by the usage of reagents and mouse models that target only p40108, 113.
IL-10
IL-10, earlier described as cytokine synthesis inhibitory factor, is a multifunctional cytokine
with a main function in the limitation and resolution of inflammatory responses. A variety of
cells synthesize IL-10; monocytes, macrophages, DCs, and the majority of T-cell subsets
appear to be important producers114, but also mast cells, eosinophils, B cells, keratinocytes,
epithelial and tumour cells can release this cytokine115,116. Monocytes can release IL-10 upon
stimulation with LPS and PGN, with the latter inducing the highest levels (SaghafianHedengren, unpublished data). The main biological function of IL-10 seems to be conducted
on APCs. IL-10 inhibits production of a range of monocyte-produced cytokines, including
TNF, IL-1, IL-6, IL-12 and chemokines including IL-8, MIP-1α and MIP-1β. It also
suppresses the expression of antigen-presentation and co-stimulatory molecules, which results
in the attenuation of T-cell activation. In addition, IL-10 increases the expression of
molecules that antagonize the activity of pro-inflammatory mediators, for example IL-1RA
and soluble (s) TNFR. It can also have direct inhibitory effects on T cells by dampening their
production of cytokines like IL-2, IL-5 and TNF. In contrast, IL-10 can enhance certain
cellular activities, for instance it increases B-cell survival, proliferation, and antibody
production. IL-10 might also be an essential immunoregulator in the intestinal tract as IL-10
knockout mice develop inflammatory bowel disease115,117.
Interestingly, a number of viruses including Epstein-Barr virus (EBV) and
cytomegalovirus (CMV) produce human IL-10 homologues; which highlights the importance
of IL-10-associated regulatory functions. The EBV homologue is approximately 85%
identical with human IL-10, which explains why the majority of available anti-human IL-10
27
antibodies cross-react with this homologue115,118. There is evidence suggesting a negative role
for IL-10 in infectious diseases such as Listeria and Leshmania, as enhanced pathogenspecific immune responses have been demonstrated after IL-10 inhibition (all reviewed
in115,117,119). In addition, elevated numbers of IL-10-secreting monocytes with the functional
capacity to propagate allergic responses have been found in the circulation of subjects with
atopic disease120.
IL-6
IL-6 has a chief role in the induction of acute phase responses121. However, it is a
multifunctional cytokine that can influence a range of immune functions including the
induction of plasma-cell differentiation and T-cell growth and development122. Further, IL-6
together
with
macrophage
colony-stimulating
factor
(M-CSF)
favours
monocyte
differentiation from DCs to macrophages123, which is essential for the molecular control of
APC development and pathogen clearance121. The most important sources of IL-6 are
monocytes and macrophages, but other cells such as DCs, endothelial cells and B cells can
also release this cytokine. The various effects of IL-6 are mediated by signalling through a
receptor complex consisting of a glycoprotein (gp) 130 homodimer together with either its
transmembrane (IL-6R), or its naturally occurring soluble receptor (sIL-6R)124. The
expression of gp130, which is the signal-transducing component, is ubiquitous while IL-6R
expression is mainly restricted to hepatocytes and subsets of leukocytes including neutrophils,
monocytes and lymphocytes125,126. gp130 signalling leads to the activation of signal
transducer and activator of transcription (STAT)-3, which translocates to the nucleus and
alters gene expression122. IL-6 unresponsive cells can be rendered responsive to IL-6 through
sIL-6R, a mechanism referred to as IL-6-trans-signalling127. In addition, gp130 can also be
found in a soluble form, the presence of which selectively antagonizes IL-6-trans-signalling
by inhibiting transmission of signals by membrane-bound gp130128.
Is IL-6 an inflammatory cytokine or not? The answer to this question primarily
depends on the stage of the ongoing immune response. During the initial phase, IL-6 is a
potent inducer of acute-phase proteins, which in turn induce pro-inflammatory
responses125,129. Later on, IL-6 contributes to the resolution of acute inflammation by inducing
mechanisms that initiate a shift from innate to adaptive responses. For instance, IL-6
suppresses the level of pro-inflammatory cytokines130, stimulates the production of IL-1RA129
and rescues T cells from apoptosis131. IL-6-trans-signalling induces neutrophil apoptosis and
28
directs mononuclear cell recruitment by for instance enhancing monocyte chemotactic
protein-1 secretion and CD62ligand (L) expression132,133.
There is a strong correlation between IL-6 levels and the progression of
inflammatory diseases such as Crohn’s disease134 and RA135. This might be related to IL-6mediated effects on cell survival, as mice that develop lymphocyte-dependent autoimmune
arthritis have an exaggerated gp130-induced STAT3 activity that is correlated with the
resistance of T cells to apoptosis136,137. It is also possible that the pathophysiological role of
IL-6 is mediated by its involvement in the generation of Th17-cells138, as these cells are
believed to sustain chronic inflammation53,54. IL-6 released after stimulation of DCs with TLR
agonists is able to block T-cell suppression by Tregs139. On one hand, this is necessary for an
optimal response against pathogens but on the other hand it is harmful in the context of
chronic inflammation. Similarly, staphylococcal exotoxin B (SEB) stimulation of nasal polyp
explant cultures results in reduced Treg numbers and function, which can be partly reversed by
inhibition of IL-6140. Besides the direct pathophysiological effects of IL-6, a dysregulation via
IL-6-trans-signalling caused by localized excess of sIL-6R in human allergic asthma141 and
autoimmune disorders125 has been proposed. Another recent study in mice demonstrated that
IL-6-trans-signalling could inhibit the differentiation of naïve T-cells to Tregs142 whereas
direct IL-6 signalling accounted for a partial suppression142,143.
IFN-γ
IFN-γ, the only type II IFN, is a potent macrophage activator and a critical cytokine for innate
and adaptive immunity against infections with viruses and intracellular bacteria. IFN-γ is also
essential in tumour control. The primary sources of innate IFN-γ are NK and NKT cells, while
adaptive IFN-γ derives from CD8+ and Th1 effectors144. The production of IFN-γ is not
directly induced by TLRs, although many innate IFN-γ responses require factors derived from
pathogen-activated accessory cells36. The significance of IFN-γ in immunity relies on its
effectiveness to directly inhibit pathogen replication, and most importantly on its stimulatory
and modulatory effects on the immune system.
Indeed, IFN-γ signalling is fundamental in directing Th1-cell differentiation144.
IFN-γ also enhances the expression of genes that control cellular MHC class I expression,
MHC class II expression on APCs, phagocytosis and microbicidial activity in macrophages
and intracellular enzymes that inhibit viral replication145. Interestingly, experimental data
demonstrate that IFN-γ can also influence APC development by switching monocyte
29
differentiation from DCs to macrophages146, highlighting the correlation between
monocyte/macrophage activity and IFN-γ.
Regulation of helper T-cell responses
It is believed that naïve CD4+ T-cells can have four distinct effector fates that are determined
by the nature of signals they receive upon their initial contact with antigens: Th1, Th2, Th17
and induced Tregs. Here, there will be a brief description about the regulation of the three first
subsets.
During the mid 80’s two distinct CD4+ T-cell populations (Th1 and Th2 cells)
were identified, which were characterized by their diverse pattern of cytokine
production147,148. Moreover, Th1 and Th2 responses appear to be mutually regulated; the
products from each subset mediate their own maintenance and prevent the differentiation of
naïve-T cells to the opposite subset147-149. The process of CD4 T-cell lineage differentiation is
strongly influenced by the nature of the infecting pathogen and the cytokine environment
emanating from the innate cells in response to pathogens144,149. As a rule IL-12 and IFN-γ
have a key role in driving Th1 responses150, while IL-4 is regarded to be essential in antigenspecific Th2 polarization and maintenance151. IL-6 has been shown to confer Th2 bias
indirectly by inducing IL-4 and IL-5 production, as well as by suppressing IFN-γ mediated
signalling in T cells152,153. Paradoxically, the absence of IL-6 does not affect in vivo Th2
differentiation154. Of note, the fact that many of the above-mentioned cytokines can be
produced by innate cells stresses that type 1 and 2 responses are not simply restricted to
differentiated helper-T cells.
The novel subset of cytokine-producing Th17 cells is distinct from Th1 and Th2
effectors. Th17-cell function is partially mediated by their release of IL-17 (IL-17A and IL17F), which evokes inflammation149. In mice, commitment to the Th17 lineage depends on
TGF-β and IL-654. However, the mechanism of human Th17-differentiation is not entirely
clear but experimental data strongly support an important role for IL-1β and IL-23 while the
influence of TGF-β and IL-6 is still a matter of controversy155-157.
30
MONOCYTES AND THEIR INTERPLAY WITH NK CELLS DURING INFECTION
Monocytes
Phenotype
Monocytes have previously been regarded as a homogenous population of cells.
Accumulating evidence during the past few decades have lead to the agreement on the
existence of monocytic subsets, which are roughly divided into two major populations in
humans and mice158. The two subsets are classified based on their differential expression
degrees of CD14, a component of the receptor complex for LPS recognition, and CD16, the
low affinity Fc-receptor for IgG. The majority (90-95%) of monocytes are CD14++CD16- and
the remaining (5-10%) are CD14+CD16+. The CD14++CD16- cells are referred to as classical
monocytes whereas CD14+CD16+ cells, which also express higher levels of MHC class II and
co-stimulatory molecules, are called non-classical or pro-inflammatory monocytes4,159.
Studies focusing on TLR expression in human monocytes have revealed that they express
high mRNA levels of TLR1, 2, 4, 5 and 8, with TLR2 and TLR4 levels exceeding all the
others, and low levels of TLR6 and no TLR375,76,79,160.
The knowledge about surface expression of TLRs and their differential levels on
human monocytic subsets is limited and somewhat discordant. For instance, an earlier study
failed to detect surface TLR4 on neither subset but found CD14+CD16+ cells to display higher
levels of TLR2161. In contrast, others detected surface TLR2 and TLR4 on all monocytes, but
found the CD14+CD16+ subset to express only higher TLR4162. This discrepancy could
partially depend on differences in experimental setup, as TLR expression can fluctuate during
the course of cell culturing162 (Amoudruz & al, unpublished data). Moreover, the pattern of
TLR2 and TLR4 regulation on the two subsets appears to differ following stimulation162.
Migratory behaviour
The two subsets also differ with respect to adhesion molecule and chemokine receptor (CCR)
expression. CD14++CD16− monocytes express CCR2 and CD62L but not CCR5, while
CD14+CD16+ cells express CCR5 but lack surface CCR2 and CD62L4,158,159. Both subsets
express the fractalkine receptor, CX3CR1, however CD14+CD16+ monocytes express
characteristically higher levels158,163. Interestingly, it has been speculated that the
CD14++CD16− monocytes and CD14+CD16+ subsets not only possess different migratory
activities but may also face varying susceptibility to different infectious agents for example to
HIV, which uses CX3CR1 as one of its co-receptors164.
31
Function
In addition to the expression of diverse phenotypic markers and chemokine receptors,
CD14++CD16– and CD14+CD16+ subsets are believed to possess different physiological
functions. For instance, in a trans-endothelial in vitro setup where human monocytes were
able to migrate across the endothelial barrier and differentiate, the CD14+CD16+ subset was
shown more likely to differentiate into DCs165. This is interesting, since they might represent
a potential subset that can affect T-cell priming and responses in vivo. However, the mouse
population corresponding to human CD14+CD16+ does not show any significant migratory
capacity to sites of inflammation in vivo163. Besides their increased expansion-capacity after
TLR stimulation, CD14+CD16+ monocytes produce larger amounts of TNF upon challenge
with TLR2 and TLR4 agonists and lower levels of the regulatory cytokine IL-10 after LPS
challenge compared to classical monocytes161,162,166. Yet, when it comes to the allostimulatory
capacity of the subsets, activated DCs originating from human CD16+ monocytes seem to
preferentially promote Th2 responses in vitro167,168. Further, murine CD14+CD16+ equivalents
have been suggested to replenish resident myeloid cells in non-inflamed tissues163.
Circulating CD16+ monocytes have been shown to expand during several
inflammatory conditions such as atherosclerosis169, RA170, allergy171 and sepsis162,172 and
therefore these monocytes have been designated as inflammatory. This designation could
however be somewhat contradictory, given that the migration of the CD16+ subset to
inflammatory sites is not substantial in vivo, compared with CCR2+ (classical) monocytes163.
Monocyte induced NK-cell activation during infection
At current, a new appreciation is beginning to emerge that professional APCs play an
important intermediary role in the activation of NK cells during infection. In this respect,
soluble mediators and contact-dependent mechanism have been pointed out as crucial36.
Below, a number of monocyte-related factors will be depicted.
Blocking IL-12 in vitro markedly reduces IFN-γ secretion by human NK cells in
response to Gram-positive bacteria such as Staphylococcus aureus and Lactobacillus
johnsonii37 as well as Plasmodium falciparum173. IL-15 is a monokine that is crucial for the
development, proliferation and cytotoxicity of NK cells174,175. Another monokine, IL-18, is
necessary for optimal NK-cell IFN-γ responses176 and killing177. However, in contrast to IL18 knockout mice, IL-12 knockouts succumb to infectious challenge with viruses176. Further,
the release of NK-cell cytokines seems to be controlled by pair-wise combinations of IL-12
32
with different monokines. A combination of IL-12 and IL-15 induces higher IL-10 and TNF
production by NK cells in comparison to IL-12 and IL-18. In contrast, higher IFN-γ levels are
obtained by a combination of IL-12 and IL-18 and not by IL-12 with IL-15178.
Several co-stimulatory molecules involved in T-cell activation have also been
found to contribute to NK-cell activity. For instance, inhibition of CD80 or CD86
significantly reduces the capacity of L. johnsonii to stimulate IFN-γ production from human
NK-cell and monocyte cultures37. There are also indications that signals delivered by CD40
on APCs might be important in helminthic infections; blockage of CD40L in mouse
macrophage and NK cell co-cultures was found to decrease IFN-γ levels40. Nonetheless,
human NK-cell activation by LPS-matured DCs requires the formation of an immunological
synapse for effective delivery of IL-12179. In addition, secreted IL-15 is trans-presented by
accessory cells via their IL-15Rα to NK cells in a cell-contact-dependent manner180. Thus, it
is possible that contact-dependent mechanisms of monocyte induced NK-cell activities could
in part be a further attribute of cytokine delivery36.
ALLERGY
The prevalence of allergies has steadily increased during the past decades and seems to be
associated with a modern lifestyle. Currently the view is that “certain environmental factors,
to which children are exposed during early-life, interact with the specific genotype of a child
and influence the developing immune system in a way that either predisposes to or protects
from development of allergic disease”181. It is also well established that the development of
allergic disorders is related to a Th2-skewed profile. Th2 cells activate complex immune
reactions that trigger the release of mediators and the recruitment of inflammatory cells,
which in turn elicit inflammatory responses that lead to clinical manifestations of allergic
disease182,183.
Allergens are common and otherwise harmless environmental antigens that
typically elicit allergic responses in susceptible individuals182. Why some individuals mount a
specific IgE response upon their initial exposure to allergens is still not fully understood. In
accordance with the World Allergy Organization184 allergy is defined as a “hypersensitivity
reaction initiated by specific immunologic mechanisms” mediated either by antibodies or
cells. In a majority of patients with allergic symptoms from mucosal membranes in the
airways and gastrointestinal tract, the antibody belongs to the IgE subclass, and these patients
are said to have an IgE-mediated allergy. IgE-sensitization on the other hand describes the
33
presence of allergen-specific IgE in blood or skin irrespective of clinical symptoms. Atopy
describes a personal and/or familial tendency to produce IgE antibodies in response to
ubiquitous environmental antigens and this term should not be used until an IgE-sensitization
has been documented by the presence of specific IgE in serum or by a positive skin prick test
(SPT).
Mechanisms behind IgE-mediated allergy
When APCs present allergens to naïve T cells in a Th2 promoting environment, the T cells
start to produce Th2 cytokines such as IL-4, IL-5, IL-9 and IL-13, which drives the
differentiation and class-switching of IgE+ B cells. These IgE antibodies circulate and bind
FcεRI on mast cells and at this stage a person is classified as IgE-sensitized. Following reexposure to the same allergen, the cross-linking of the bound IgE antibodies on the sensitized
cells causes their degranulation of pharmacologically active mediators such as histamine,
leukotrienes, chemokines and cytokines (Figure 3). These mediators collectively trigger
allergic symptoms including vascular-permeability, smooth-muscle contraction and mucus
secretion. This phase is known as the early allergic response. The late allergic response
emanates when mediators of the acute reaction stimulate the synthesis of secondary
mediators, which direct the recruitment of other inflammatory cells, particularly neutrophils
and eosinophils, to the site of allergen exposure. These cells will contribute to sustained
swelling and epithelial disruption182,183,185,186.
Figure 3. Allergic mechanism.
34
Risk factors
A familial history of allergy is a strong predictor for the development of allergic disease and
several regions of the genome have been suggested to relate to an increased risk of acquiring
allergic phenotypes181. For instance, chromosome 5q has been linked to asthma and allergy
susceptibility as multiple cytokine genes including IL-4 and IL-13 that are important for the
regulation of IgE are found within this region187. Notwithstanding the importance of genetic
factors, the relatively short timeframe during which allergic disorders have increased would
indicate that environmental changes are likely to contribute to the pathogenesis of allergies.
Currently, it is appreciated that gene-environment factors acting during early-life are
fundamental events for the development of allergic disease. The environmental risk factors
include air pollution, tobacco smoke, pet ownership, allergen exposure (such as house dust
mite) and maternal factors188,189. Also respiratory tract infections caused mainly by respiratory
syncytical virus (RSV) have been suggested as risk factors for development of childhood
wheezing and asthma189,190. The majority of children become infected with RSV during
postnatal years where mild RSV bronchiolitis is a self-limiting condition. A minor percentage
of children become hospitalized, and it is among the severe RSV bronchiolitis cases that RSV
has been pointed out as a risk factor for development of allergic asthma during
adolescence191. In addition, early-life exposure to microbes and/or microbial products has
been related with modification of the risk for developing allergic disease192,193, findings that
have initiated numerous investigations regarding the underlying mechanisms behind such
modulatory action181,194. The following pages will focus on two factors: maternal and
microbial, in relation to the development of allergic responses during childhood.
Epidemiological evidence behind the “hygiene hypothesis”
During 1989 the so-called “hygiene hypothesis” was developed to explain the notion that
eczema and hay fever were less prevalent among children from larger families than among
children from families with one child195. It was presumed that exposure to infectious agents
increased by having siblings and then further speculated that modern hygiene practices and
health care had lead to lower exposure to microbial agents. This would therefore result in an
imbalanced immune system that in turn predisposes individuals to the development of allergic
diseases.
Major support for the hygiene hypothesis came from studies comparing the
prevalence of allergic disorders among children growing up in rural or urban environments.
35
Indeed, there is a strong association between growing up on a farm and a reduced risk of
developing allergic disease196,197. It has also been shown that the most protective effect of the
farming environment is related to frequent contact with livestock198,199. Interestingly, it
appears as early-life or even prenatal exposure to farming environment confers the strongest
protection, as the development of bronchial asthma is significantly lower in children whose
mothers continued working in stables during pregnancy200,201. Farm milk consumption is
another feature of farming life-style that has been strongly correlated with protection against
development of allergic disorders202,203.
Which organisms may contribute to the protection then? Shirakawa & al.
demonstrated a strong inverse relation between delayed hypersensitivity to Mycobacterium
tuberculosis and atopy among Japanese school children204. Further, higher tuberculosis
notification rates have been associated with an absolute decrease in the prevalence of
symptoms related with asthma205. A study in Guinea-Bissau demonstrated a positive relation
between childhood measles and protection against IgE-sensitization in comparison to
vaccinated subjects206. However, the role of measles infection in development of atopy in
affluent societies is contradictory190. Hepatitis A seropositivity has been suggested to be a
marker of poor hygiene practices and demonstrated to inversely correlate with the prevalence
of atopic disease207. TIM-1 is expressed by activated T cells upon Th2-cell differentiation and
the receptor for hepatitis A208,209. Interestingly, having an allelic variant of TIM-1 gene, which
affects viral entry into cells, at the same time as being seropositive for hepatitis A, is
associated with protection from atopic disease209. This may offer mechanistic support for the
hygiene hypothesis since it provides a possible link between infection and susceptibility to
atopy.
Janson & al did not support a protective role for hepatitis A infection in their
study including 1249 adults from Iceland, Sweden and Estonia210. Their findings rather
indicated an accumulative protective effect of ≥ 3 microbial infections against allergic disease
and degree of IgE-sensitization210. Also, repeated virus infections with the herpes type
varicella, zoster, febrilis, labialis, exanthema subitum and somatitis within the first 3 years of
life have been related to protection against asthma at the age of 7211. The role of EBV in
development of atopic sensitization and disease has proved controversial however. A couple
of studies show an inverse relationship between seropositivity and allergic sensitization in
infants below 2.5-years of age212,
213
,
while others who investigated 4-year old children214
failed to demonstrate any relation. Laske & al found no relation between seropositivity for
herpes group viruses EBV, CMV and varicella zoster at 1-years of age and incidence of atopic
36
disorder at the age of 7215. It should be pointed out that in this study only 1% (n=4) of the
children were EBV seropositive, which is too few subjects for a reliable statistical analysis.
Notably, also non-pathogenic bacteria have been associated with a reduced prevalence in
allergic disorders. A study that compared the gut microflora of two populations with different
incidence of allergic diseases concluded that alteration in the microbial intestinal flora in the
first year of life might affect the development of atopy216. Further, the composition of gut
flora as early as during the first week after birth has been associated with atopic disease up to
the age of 5 years217.
Molecular mechanisms?
When epidemiologic observations were integrated into immunology, the rise in incidence of
allergic disorders was initially explained by a deprived early-life activation of the immune
system that results in a lower boost of Th1 responses and predisposes children to Th2mediated diseases182,194. This immunological framework did not explain the concurrent
increasing incidence in Th1-mediated autoimmune diseases218, which led to a modification of
the hygiene hypothesis to state that interactions with environmental microbes induce the
immunoregulatory mechanisms of Tregs that serve to maintain the balance between T-cell
tolerance and reactivity182,194. Which molecular agents contribute to the protection against
development of allergies? Farmer households contain much more endotoxin than non-farmer
environments, and the highest levels were in seen in farmstables198. In addition, a strong
relationship between endotoxin concentrations, measured in the mattresses of children, and
the protection against development of allergic disorders has been observed197. LPS represents
only a part of the total microbial burden however. Interestingly, it has been shown that
children with higher muramic acid (a major component of PGN) concentrations in their
mattress dust have a lower incidence of asthma and wheezing irrespective of farming status or
LPS exposure219. These findings are in agreement with the previous notion that farmer’s
children have significantly higher amounts of CD14 and TLR2 mRNA in their blood220. This,
along with the capacity of TLRs to influence adaptive responses, has drawn massive attention
to the role TLRs in the field of allergy research over the past years221. Genetic studies have
provided some further clues. For instance, Eder & al found that a TLR2-gene variant among
farmer’s children may revert the protective effect of microbial exposure, as it is associated
with an increased risk of developing asthma and allergic disease222. Furthermore, a variant of
CD14 has shown to modify the allergo-protective effect of farm milk consumption223.
37
Intriguingly, another study found a relation between higher incidence of asthma in Swedish
children that had a specific TLR4 polymorphism and decreased production of IL-12p70 by
their APCs224. There are also studies evaluating associations between additional TLR-gene
polymorphisms including those for TLR3, TLR6, TLR9 and TLR10 and the risk of
allergies225.
Effect of allergic heredity and herpesviruses on early-life innate responses
Maternal allergy
Parental allergy is a strong risk factor for the development of allergic disorders188.
Interestingly, it has been observed that maternal allergy confers a higher risk of allergic
disease in the offspring compared with paternal allergy226 and in general, maternal factors,
placental factors or both are frequently discussed in relation to development of allergies227-229.
Plausible explanations for the increased allergic outcome in relation to maternal
allergy could be the transfer of maternal factors such as antibodies, cytokines or allergens to
the foetus228. There is evidence for a strong relationship between elevated maternal IgE (both
total and allergen-specific) and cord-blood (CB) IgE levels226,230. Actually, IgE has been
detected in the foetal sites of placenta tissue231, and there is evidence demonstrating that
placental IgE is of maternal origin232. Bonnelykke & al suggested that CB IgE could be the
result of maternal transfer to the foetus by demonstrating a strong relation between maternal
specific-IgE and its levels to that of CB’s233. This study further showed a correlation between
CB specific-IgE and IgA, which is a marker for contamination with maternal serum. In
contrast, Pfefferle & al demonstrated that the majority of CB samples with IgE content were
devoid of detectable IgA and that in many IgE-positive CB samples corresponding specificIgE was lacking in the mothers234. Nevertheless, the presence of IgE in CB as a predictor of
later development of allergic disorders is disputable. For example elevated IgE in CB has
been reported to predict allergic disorders235, but it appears however that a combination of
family history of allergy with presence of CB IgE could improve such prediction235-237. Other
studies have not found such associations233,238.
Whether in utero sensitization may occur or not is also a matter of controversy.
The study by Bonnelykke & al suggested that in utero sensitization does not occur233, as
opposed to that of Pfefferle & al234. Allergens have been detected at low levels in CB239 and
at higher levels in placental tissue240. Such findings might explain the observations of
allergen- and antigen-specific T-cell responses in CB cells from newborns241-243, which are in
favour of prenatal priming of the immune system. Kihlström & al showed that children from
38
mothers exposed to high doses of birch pollen during their pregnancy were at a higher risk of
birch-sensitization and development of allergic disease compared with their low-dose exposed
peers244. Nevertheless, maternal exposure during pregnancy, compared with postnatal
exposure, to high levels of birch pollen appeared to be more protective against birchsensitization in the child244. Pregnancy is a complex immunological phenomenon suggested to
tilt newborns towards a Th2 immune profile245 possibly as a result of lower IFN-γ
responses246,247. Indeed, a universal skewing of initial T-cell responses towards Th2 profile
has been found among newborns241. This polarization is suggested to be more pronounced in
neonates with allergic predisposition243, 248-250 and those that subsequently become allergic246,
247
. However, altered CB cytokine levels in general, rather than a pronounced bias, has also
been observed in children with atopic disease251. Whether IL-1, TNF and IL-6, which are
found in the amniotic fluid, are able to travel across the placenta has been investigated. One
report found no transplacental transfer of any of these three cytokines252 whereas another
study demonstrated a bi-directional passage of IL-6253.
Rather few studies have addressed the functional quality of neonatal innate
responses in relation to maternal factors. Defective TLR responses, for instance TLR2 and
TLR9, in neonates of smoking mothers have been noted254. This study did not find any
influence of maternal allergy on TLR responses except a tendency towards lower IL-10 upon
TLR2 challenge. Others have however demonstrated reduced mRNA expression of CD14,
TLR2 and TLR4 in newborns to allergic mothers255. A study from our group found that
neonates with allergic mother display lower monocyte surface levels of TLR2 and TLR4 in
relation to their mothers256. Although there were no differences in TLR expression between
newborns with allergic and non-allergic mothers, CB mononuclear cells (MCs) from babies
with maternal allergy released markedly lower IL-6 amounts upon PGN challenge256. In
contrast, Prescott & al found no defective IL-6 response in neonates of allergic mothers upon
stimulation with lipoteichoic acid (LTA), an alternative TLR2 ligand257. However, challenge
with this ligand appeared to induce a weak IFN-γ response, which was higher in neonates
with non-allergic mothers. Taken together, these observations indicate that maternal allergy
could potentially influence the allergic disposition of offspring through functional deficiency
of foetal monocytes.
39
Epstein-Barr virus
EBV is a DNA virus that belongs to the Herpesviridae family and is one of the most
successful viruses infecting more than 90% of humans around the world. Although B cells are
the principal targets for EBV, epithelial cells and monocytes can also be productively infected
with this herpesvirus258,259. Upon infection of B cells, EBV establishes latency and persists for
lifetime of the host. EBV-infected individuals chronically or intermittently shed EBV into
their saliva, through which EBV is characteristically transmitted. While primary infection
during early childhood is the rule where hygiene standards are low, many children in affluent
societies appear protected from early-life EBV infection260,261. Delayed primary infection in
adolescence or adults often causes infectious mononucleosis (IM). IM has clinical
manifestations mostly attributed to the forceful proliferation of activated T cells but also
splenomegaly, an absolute increase in peripheral MCs and presence of heterophile
antibodies258. Accordingly, EBV seropositivity appears to relate to mixed T-cell cytokine
responses without a clear Th1 or Th2 dominance, both in asymptomatic 2-year old infants262
and in subjects with IM263. Whereas the antibody profile is polyclonal and heterophile Igs are
readily detected during ongoing symptomatic EBV infection258, these are usually absent in
infants with primary EBV infection264.
Only few studies have attempted to address the relation between EBV infection
and development of allergy. Due to its potent capacity to induce polyclonal antibody
responses, primary infection with EBV has been speculated to result in the manifestation of
atopic phenotypes due to an exaggerated host response265,266. This is based on two studies
where higher EBV antibody titers were found in atopic children, aged between 5 and 18-years
267
and in adults with atopic dermatitis265. Yet, the relationship between EBV and allergy
seems unclear here; is the allergic status itself causing defective control of virus spread and
thus higher EBV antibody titres in a seropositive person? Or does infection with EBV per se
trigger IgE production and the manifestation of allergic phenotypes? Once transformed by
EBV, B cells start producing IL-4 and IL-5268,269, which are central in the regulation of
allergic responses such as the production of IgE and eosinophil activation182,183,185,186. Then
again, EBV reactivation in B cells of allergic individuals could lead to increased antigen loads
and expression that in turn induce higher antibody titres. Sensitization to environmental
allergens requires longer time and develops usually between 1- and 10-years of age270.
Therefore, it is conceivable that the mitogenic effect of EBV would not precipitate an allergy-
40
promoting immune profile at a postnatal age when the immune system is still relatively
antigen-inexperienced.
Is there a role for monocytes in the development of allergies?
Before the introduction of the hygiene hypothesis, the monocyte was a “forgotten” cell in the
field of allergy. As the alter ego of the macrophages and dendritic cells, the monocytes were
also neglected as APCs in allergic responses. Nevertheless, along with the hygiene hypothesis
and the demonstration of TLR2 and TLR4’s involvement in allergies197,198,220, came the
awareness of their importance for proper stimulation and shaping of downstream adaptive
responses. Upham & al found that infants who develop atopy within 2-years of age display
reduced basal levels of monocytic MHC class II at birth271. Alterations by means of a higher
monocyte IL-12-producing ability have also been detected in children with food allergy272.
A few studies have also investigated the role of different monocytic subsets in
relation to allergic disorders. An elevated ratio of CD14+CD16+ during the exacerbation
period in patients who had atopic eczema/dermatitis syndrome (AEDS)171 has been reported.
Others have investigated the functionality of these subsets in subjects with AEDS, and
demonstrated a selective impairment in TLR2 initiated IL-1β and TNF production in
monocytes irrespective in being CD14+CD16+ or CD14+CD16–
273
. In asthmatic patients,
elevated proportion of circulating CD14+CD16+ cells has been reported274, 275 where both of
the monocytic subsets were demonstrated to be responsible for the lower amounts of IL-10
and IL-12 detected after LPS stimulation275. Yet, these results are based on finding from
adults with an ongoing inflammatory condition and the disentangling of the cause-effect
relationship is therefore difficult. For instance, it is plausible that an increased suppressive
action of other immune components could render monocytes less responsive to further
stimulation56,58,59. An understanding of monocyte subset characteristics during early years of
life; for instance before, during or after manifestation of IgE-sensitization, could provide
further insights to whether alterations in monocyte subset ratios and/or changes in their
function could indeed be of importance in the development of allergic diseases.
Currently, there are barely a handful of published papers that have examined the
role of APC (DCs to be more precise) and NK-cell interplay in atopic diseases. Scordamaglia
& al found that allergic patients have a decreased mDC-induced IFN-γ response by their NK
cells276. Buentke & al demonstrated that Malassezia, which is an opportunistic yeast that
contributes to inflammation during AEDS, can modulate NK-cell killing of mDCs277. Further,
41
a study in mice showed that seropositivity with human analogues of EBV and CMV confers
protection against bacterial infection by means of higher macrophage bactericidal activity278.
Here, the general boost of innate immunity was demonstrated to operate via elevated levels of
IFN-γ and TNF. In humans, few studies have addressed the role of herpesviruses in skewing
innate responses during infancy, which could be highly relevant in relation to the
development of allergies.
42
PRESENT STUDY
OBJECTIVES
The overall aim of this thesis was to examine how the developing immune system – with
particular focus on the monocytes – is affected by microbial exposure during early childhood
years.
More specifically this thesis:
•
Evaluates how maternal allergy (paper I) and allergic disease (paper II) in children
influence the monocytic response to bacterial components that are commonly found in
the environment.
•
Determines whether the timing of primary EBV infection during childhood could be
an important factor in modifying the risk to develop IgE-sensitization (paper III).
•
Investigates how innate immunity is affected by early-life EBV infection by
examining monocyte-induced NK-cell responses (paper IV).
43
SUBJECTS
Study cohort, evaluation of IgE-sensitization and allergic disease
The objectives of paper I, II and IV were addressed by selecting a total number of 28 (paper
I), 33 (paper II) and 51 (paper IV) subjects from the cohort. Paper III investigated the whole
cohort at 5-years of age (n=281).
Families living in the southern part of Stockholm and expecting a child were asked by
midwifes at the maternity wards if they were interested in participating in the study. The
invitation was addressed to families where only the mother, both or none of the parents were
allergic. Seven hundred and seventeen families showed interest for the study and were further
informed and interviewed by a telephone call. Three hundred and thirty parental couples
fulfilled the settled criteria for participating in the study, and were invited to come to the out
patient ward at Sachs’ Children’s Hospital for further interview and SPT. The same nurse
performed SPT according to the manufacturer’s recommendation (ALK, Copenhagen,
Denmark) against the following inhalant allergens: cat, dog, Dermatophagoides farinae,
birch, timothy, horse, rabbit and mugwort (Soluprick 10 Histamine Equivalent Prick test).
Histamine chloride (10mg/mL) served as the positive control and the allergen diluent as the
negative control. The SPT was considered positive if the weal diameter after 15min was
≥3mm. Only those parents whose SPT results confirmed a positive or a negative history of
respiratory allergy (bronchial asthma and/or allergic rhinoconjunctivitis) to pollen and/or
furred pets were invited to continue (n=281).
All infants were healthy and born full term (>36 weeks of gestation) at hospitals in
Stockholm from September 1997 until August 2000. One hundred and twenty children had
two allergic parents (group dh = double heredity), 84 children had an allergic mother and a
non-allergic father (group mh = maternal heredity) and 77 children had two non-allergic
parents (group nh = no parental heredity). The infants were followed from birth until 5-years
of age and were clinically examined by the same paediatrician (Caroline Nilsson) at 6, 12, 18,
24 and 60 months of age. In accordance with Johansson et al.184 the children were classified
as IgE-sensitized if at least one or more SPT was positive (≥3 mm) and/or if specific IgE
against at least one or more of the selected allergens was ≥0.35kUA/l. Following definitions of
the various allergic diseases were used: AEDS was defined according to Hanifin & Rajka279.
Bronchial asthma was defined as three or more episodes of wheezing, or any episode of
wheezing if combined with a family history of allergic disease or other allergic symptoms in
the child. Allergic rhinoconjunctivitis was diagnosed if rhinitis and/or conjunctivitis appeared
44
at least twice after exposure to a particular allergen and was unrelated to infection. Approval
from the Human Ethics Committee at Huddinge University Hospital, Stockholm, Sweden was
granted. All families provided their informed consent to the study.
Determination of EBV and CMV serostatus at 2- and 5-years of age
The presence of plasma IgG against EBV capsid antigen (EBV VCA) was determined
according to previously published immunofluorescence assays280 where a specific
fluorescence in dilution 1/20 was regarded as a sign of seropositivity. Plasma samples from 5year olds were also tested for EBV nuclear antigen 1281. IgG seropositivity to CMV was
determined by ELISA using purified nuclear antigens from CMV cultivated in human fetal
lung fibroblasts282, where samples with an absorbance above 0.2 at a dilution of 1/100 were
considered as positive. Serostatus could not be determined in all 5-year olds since
measurement of circulating IgE was given higher priority and the volume of plasma samples
was sometimes too small (described in more detail in paper III’s methods). A few of the EBV
analyses were indeterminate despite repeated examination or due to insufficiency of plasma
for performance of additional analyses.
Earlier cohort findings
Gabrielsson & al have previously evaluated the influence of atopic heredity on the newborn
T-cell cytokine responses in this particular cohort248. Markedly higher ratios of IL-4/IFN-γproducing CBMCs were found in neonates with two allergic parents after stimulation with
phytohaemagglutinin (PHA). Further, IL-12+ cells were more frequent in CBMCs from
children with non-allergic parents after allergen stimulation, which collectively suggests that
children with allergic heredity might have a more pronounced Th2 profile248. Nilsson & al
further assessed whether cytokine responses to allergens at birth could be related to
manifestation of allergic disease at the age of 2283. Here there were no differences in the ratios
of IL-4/IFN-γ-producing CBMCs between IgE-sensitized and non-sensitized 2-year old
infants or between those with or without allergic disease. However, infants who had AEDS
within their 2-years of life had less IFN-γ+ cells upon allergen challenge (ovalbumin, cat and
birch) at birth. Subjects with AEDS did not differ in their IL-12 response to allergens whereas
IgE-sensitization was related with fewer IL-12+ CBMCs after stimulation with cat allergen283.
Of note, the allergens were not tested for endotoxin contaminations, which may rather reflect
that IgE-sensitization as well as atopic heredity may associate with a defective innate
45
response. Larsson & al investigated the relation between cytokine responses of mothers and
their children 2-years after birth284. They found more IL-4+ cells after PHA challenge and
more IL-10+ as well as IL-12+-cells after OVA challenge in IgE-sensitized 2-year olds.
Further, a strong correlation between maternal and infant cytokine profiles was demonstrated,
which suggested that increased cytokine response to allergens might be a better predictor of
childhood IgE-sensitization284.
The cohort has been examined for possible relations between viral infections
(respiratory tract and herpesviruses) and IgE-sensitization at 2-years of age213. There was a
strong association between EBV seropositivity and a lower prevalence of IgE-sensitization,
which appeared to be further enhanced by CMV co-infection. The underlying mechanism
behind this protection could not be explained by a Th1-skewed profile, as EBV+ infants
tended to have an overall boost in both Th1 and Th2 responses to PHA262. Seropositivity with
CMV on the other hand was related with a Th1-biased profile, as shown by higher numbers of
IFN-γ producing cells262.
46
METHODS
All methods that were applied in each study are found in the corresponding papers. Here,
there will be a brief description of how p38-MAPK phosphorylation was assessed in
monocytes, as this method was quite new when study I was initiated.
Phospho-Specific Flow Cytometry
Based on results from our previous study256 we wanted to address whether the altered activity
of a particular TLR-mediated intracellular target could be the underlying factor behind the
defective IL-6 response in children with allergic heredity. Owing to the very limited amounts
of PBMCs from the subjects, we chose to apply a phospho-specific flow cytometry based
method to analyze p38-MAPK phosphorylation in monocytes. This technique has several
advantages. For example, phosphorylation events are displayed in single-cells within the same
or heterogeneous populations since this technique simultaneously allows surface marker
analysis. It allows minimal manipulation of samples before analysis, and in contrast to
Western-blot which requires relatively large number of cells to detect phospho-epitopes, the
flow cytometry-based method does not require as many cells285.
Detection
of
phospho-p38-MAPK
TM
manufacturer’s instructions (BD
was
performed
according
to
the
Phosflow Phosphorylation State Analysis, BD Biosciences
Pharmingen). Briefly, CBMCs or PBMC were stimulated with LPS or PGN during 30
minutes followed by immediate fixation for 10min at 37°C with 5% CO2. Cells were washed
three times with permeabilization buffer and thereafter labelled with titrated amounts of
monoclonal mouse anti-human CD14 and monoclonal mouse anti-phospho-p38-MAPK. In
our hands, phospho-p38-MAPK tagged, non-stimulated cells served as an appropriate control
since the isotype control for phospho-p38-MAPK did not display any unspecific binding in
non-stimulated, LPS or PGN stimulated cells. However, low levels of phospho-p38-MAPK
were detected within non-stimulated CD14+ cells, which suggested that monocytes had a low
degree of p38-MAPK activity under basal conditions. Therefore, all values were presented as
stimulation index of GeoMFI of phosphorylated p38-MAPK in stimulated CD14+ monocytes
divided by GeoMFI of phosphorylated p38-MAPK in non-stimulated CD14+ monocytes.
47
RESULTS AND DISCUSSION
Paper I & II
Maternal allergy and allergic disease in the child differentially alter the monocytic responses
to microbes, and these alterations appear during defined time-periods after birth
A lower IL-6 response after PGN challenge in CBMCs from children with allergic mothers256
most likely reflects defective monocyte activity and may therefore have implications for the
development of allergies197,220,222. Paper I examined whether this attenuated IL-6 response
persisted through infancy by investigating the same subjects, with or without allergic mothers,
at birth and at 2-years of age. Since the impaired IL-6 response was found despite similar
levels of surface TLR2 between the neonates of allergic and non-allergic mothers
256
, we
hypothesized that monocytes from neonates of allergic mothers may have a defective
signalling in their TLR-pathway that leads to a lower IL-6 release. p38-MAPK was
considered to be a suitable target for several reasons; it is activated by microbial compounds
such as LPS93 and PGN286, it has a crucial role in the maturation and function of
APCs102,103and is implicated as a potential target for therapeutics100, 287.
The major finding from this study was the observation of decreased p38-MAPK
phosphorylation and IL-6 response in 2-year old infants with allergic mothers (Figure 2b and
3b, paper I). The degree of phospho-p38-MAPK was highly correlated with IL-6 levels
(rs=0.72, p<0.001 for LPS and rs=0.81, p<0.001 for PGN). To evaluate which of the factors,
maternal allergy or IgE-sensitization, that was related to a lower monocytic response, we
further divided the 2-year olds of each heredity group into IgE-sensitized and non-sensitized
subgroups. Interestingly, at 2-years of age maternal allergy appears to still have a significant
influence on the child’s monocyte functionality; comparisons between allergic status of the
mother, as opposed to IgE-sensitization of the child, revealed significant differences in p38MAPK phosphorylation capacity (Table 2, paper I).
Evaluation of allergic disease at the age of 2 is difficult and therefore the
findings in paper I were related to IgE-sensitization. This is because many children with
wheezing before the age of 2 are not allergic and in general, atopic eczema is only associated
with the occurrence of specific IgE antibodies in 1/3 of children (personal communication,
Caroline Nilsson). Manifestation of allergic symptoms is usually established at pre-school
ages270,288, which probably results from a more antigen-experienced immune system with
48
increasing age. Therefore, in paper II we wanted to investigate how allergic disease could
influence monocyte TLR2 and TLR4 regulation and microbial response in 5-year old
subjects. As perturbations in monocyte-subset balance have been found in inflammatory
conditions169-171, we were also interested in determining whether such imbalance could be
noted during early manifestation of allergic disorders and if the subsets were differentially
affected by the allergic condition.
There was neither any difference in the proportion of monocyte subsets nor in
their basal expression of surface TLR2 and TLR4, when comparing allergic with non-allergic
children. Notably, allergic 5-year olds showed a markedly lower up-regulation of surface
TLR2 on their monocytes in response to PGN, which was apparent in both monocyte subsets
(Figure 2, paper II). The altered regulation of TLR2 after PGN challenge was, however, not
accompanied by changes in p38-MAPK activity or pro-inflammatory cytokine production
whereas spontaneous IL-6 release was found to be significantly higher in allergic subjects
(Figure 3, paper II). We also assessed the role of allergic heredity in the above-investigated
parameters but found no clear associations (personal communication, Petra Amoudruz).
The above findings suggest that monocyte function during infancy, as opposed
to later in childhood, may be under a strong influence of maternal allergy. This could have
consequences for a proper initiation and shaping of adaptive responses, and thus, the
development of allergies. Indeed, maternal allergy has been suggested to confer a higher risk
for offspring to develop atopic disease226,289. Allergic mothers have been found to have
substantially higher levels of systemic IL-10 at the time of delivery290, which could potentially
be a dampening factor on the child’s monocyte responses to microbial components. Up to
date however, transplacental transfer of IL-10 has not been examined. In the previous study
by Amoudruz & al, allergic and non-allergic mothers did not differ in their IL-10 production
after microbial challenge in vitro256. Actually, it appears that the allergic status of the mother
is not related with changes in innate cell cytokine production, including IL-10, during
pregnancy291.
Since neonates with high exposure to muramic acid have increased IL-6producing capacity in comparison to their peers with low muramic acid exposure292, a
differential microbial exposure may have influenced our findings on IL-6. However, this is
rather unlikely since exposure factors such as number of siblings, day-care attendance or
having pets at home were similar between the heredity groups (Table 1, paper I). We chose to
further quantify sIL-6R and sgp130 to be able to evaluate IL-6-mediated effects in more
49
detail. There were no differences in sIL-6R or sgp130 in CBMC cultures, before or after
microbial challenge. Studies addressing relations between sIL6-R or sgp130 levels and
disease severity have measured these factors in body fluids at the local site of inflammation or
in the serum of patients125,141,293,294, which may explain why no differences were seen. An
alternative explanation could be that the 2-year old sensitized subjects do not have an
established inflammation due to undetectable allergic symptoms at the time of blood
sampling.
So what could be the consequence of a lower monocyte activity and IL-6
response during the very earliest years of life? IL-6 released by APCs after microbial
stimulation has been shown to inhibit the suppressive action of Tregs139. Similarly, both the
numbers and the function of Tregs are partially increased by blockage of IL-6140. Furthermore,
p38-MAPK activation is critical in driving Th1 responses67,101,102, and highly related with
monocyte activity (paper I). A maternal allergy-related defective IL-6 response, possibly
accompanied by additional APC-derived cytokines such as IL-12248,295 in postnatal years,
might lead to an insufficient Th1 differentiation. Indeed, the newborns with allergic heredity
in the cohort were shown to have higher IL-4/IFN-γ ratios along with fewer IL-12+ in their
CBMCs, which suggests that their immune system is more Th2-skewed at birth248. Since the
innate IL-12 producing capacity upon bacterial challenge is not fully matured during early
childhood296,297, a proper IL-6 response following microbial challenge might be necessary to
inhibit Treg suppression and thereby support adequate differentiation of Th1-responses. Of
note, higher levels of IL-6 have also been detected in CBMCs from farmers’ newborns298,
who are protected from developing allergic disorders later in childhood197,200,201. Nevertheless,
the same babies with increased IL-6 response at birth display rising ratios of IFN-γ/IL-6
production capacity with increasing age298, suggesting that perhaps it is a matter of balance
between individual cytokines after microbial exposures in infancy that sets the stage for
protection against development of allergies.
In contrast to the 2-year olds, 5-year old allergic children had markedly elevated
levels of spontaneous IL-6 (Figure 3, paper II) and slightly higher basal IL-8 and IL-1β. This
increase might well reflect the initiation point where the pathophysiological effects of IL-6
and possibly IL-1β and IL-8 begin to emerge in allergic individuals. It should be noted that
none of the allergic children included in paper II had acute symptoms at the time of blood
sampling, which could explain the missing differences in monocyte subset composition
between the groups. The expansion of CD14+CD16+ cells is associated with ongoing
inflammation and their proportion decreases along with recovery from symptoms171. In
50
agreement with Tacke & al, this suggests that elevated CD14+CD16+ numbers may appear
secondary to disease development and are not necessarily the initial cause of inflammation299.
The were too few number of allergic subjects in this study (n=16) to allow the division into
subgroups on the basis of multiple symptoms, which could have provided further information
on whether imbalanced subset ratios or changes in inflammatory cytokine levels could
thereby be observed.
The higher spontaneous levels of IL-6 in the allergic 5-year olds along with
dysregulated TLR2 expression after microbial challenge may suggest an improper monocyte
activity in allergic individuals. The altered TLR2 expression was, however, not paralleled by
a defective cytokine response, which points towards compensatory mechanisms beyond
surface PRRs for PGN recognition. An example of such mechanism is the activation of
NOD1 and NOD2, which recognize different structural core motifs from PGN and trigger
signalling and gene expression profiles similar to those mediated by TLR2300,301,302. Yet, in
allergic adults, monocytes release less TNF when stimulated with Pam3Cys, a synthetic
agonist of TLR2, which indicates that a selective impairment of TLR2 signalling could be
present in atopic individuals273.
It is necessary to acknowledge some limitations. In contrast to the previous
study by Amoudruz & al256, a recent study performed on a larger population of newborns
could not confirm a defective IL-6 response after TLR2 challenge in those born to allergic
mothers257. The reason for this discrepancy could well be due to our examination of a smaller
number of subjects. However, the smaller group size does not explain why similar trends on
lower PGN induced IL-6 response were found again in paper I, which investigated another
cohort than Amoudruz & al. Furthermore, the use of different TLR2 ligands might have
contributed to the study discrepancies, as the ligand specificity for LTA and PGN appears to
differ300,301,303. Whether a lower PGN induced IL-6 response could be due to defects in the
NOD-triggered pathway rather than in that of TLR2 remains to be elucidated.
TLRs may potentially become targets of allergy therapeutics304 and TLR4 as
well as TLR2 stimulation (with both PGN and Pam3Cys) have been shown to ameliorate
allergic responses in mice305. If similar trials would be initiated for humans, it should be
considered that the outcome of such interventions may become affected by 1) maternal
allergy rather that the infant’s own IgE-sensitization within the first 2 years of life and by 2) a
defective monocyte TLR2 regulation in pre-school aged children with allergic disease.
51
Paper III & IV
Postnatal EBV infection protects against the development of chronic IgE-sensitization during
childhood and associates with a suppressed IFN-γ response by NK cells
Viruses and man have co-evolved for millions of years, during which the human immune
system has developed several strategies to keep the persistent invaders at bay306.
Notwithstanding the fact that herpesvirus latency can be parasitic, as it leaves
immunocompromised individuals at risk for viral reactivation and serious disease258,307,308, the
tantalizing question is: what happens if our immune system stops encountering viruses? A
higher incidence of allergic disorders might be one consequence, as delayed primary infection
with herpesviruses in affluent societies260,261,307 coincides with increased prevalence of
allergies309. Furthermore, EBV seropositivity in early life has been shown to relate with a
lower occurrence of allergic sensitization212,213. However, these findings are contradicted by
studies that have emphasized a pathological role for EBV in older children or adults with
atopy212,265,267. Therefore paper III re-examined the study cohort at 5-years of age to
determine whether the timing of primary EBV infection could alter the risk of developing
IgE-sensitization.
The results of paper III were quite interesting; 5-year old children who were EBV
seropositive already at the age of 2 were at a significantly lower risk of being chronically IgEsensitized, i.e. IgE-sensitized at both 2- and 5-years of age. In contrast, acquisition of EBV
after 2-years of age was associated with a higher risk of being IgE-sensitized at the age of 5.
Further, infants who were EBV+ at 2-years of age but not IgE-sensitized were not protected
against later IgE-sensitization, which suggests that the allergy-protective effect of early EBV
infection does not protect against later IgE-sensitization (all in table 2, paper III). Moreover,
chronically IgE-sensitized children had significantly elevated allergen-specific IgE levels
compared to those who developed later IgE-sensitization (Figure 1, paper III) indicating that
the immune capacity to mount allergic responses is different between these groups. That is;
even if early EBV infection cannot protect against later IgE-sensitization, it might still be
beneficial against a pronounced Th2-skewed immune system. The findings from paper III
support the indication that a delayed primary EBV infection may contribute to a higher
prevalence of allergies212,213.
Mice latently infected with human homologues of EBV and CMV have boosted innate
effector functions by means of higher systemic IFN-γ and macrophage bactericidal
52
capacity278. Paper IV investigated whether early-life EBV infection in infants was also related
with higher innate characteristics, which could explain the lower likelihood of IgEsensitization213 (paper III). The focus here was on monocyte and NK-cell interactions, which
have a great impact on the regulation of T-cell differentiation310,311.
Surprisingly, EBV seropositive 2-year olds had a significantly lower proportion of
+
IFN-γ NK cells that also contained decreased levels of IFN-γ, when compared to their
seronegative counterparts (Figure 1c-d, paper IV). Quantified IFN-γ in culture supernatants
paralleled the intracellular findings (Figure 2, paper IV) and in vivo IFN-γ levels were also
markedly lower in EBV seropositive infants (Figure 5, paper IV). Given that the in vitro
findings of attenuated NK-cell IFN-γ responses were monocyte dependent (Figure 4a, paper
IV), we examined whether altered monocyte characteristics could have a role in these
observations. EBV infected subjects tended to have lower proportions of CD14+CD16+ cells
in a non-stimulated condition. Since, these monocytes are potent producers of inflammatory
cytokines159, including IL-12p70 (Sohlberg & al, unpublished data), their capacity to induce
IFN-γ production by NK cells was assessed. Notably, CD14+CD16+ cells were found to
induce NK-cell IFN-γ release more potently than CD14++CD16- monocytes (Figure 4b, paper
IV), which might provide an explanation for the lower innate IFN-γ levels seen in EBV
seropositive infants.
In addition, co-infection with CMV seemed to enhance the EBV-related effects; the
lowest levels of IFN-γ, in vitro and in vivo, were detected in infants who were seropositive for
both EBV and CMV. The explanation for this is unknown. However, there are indications that
primary CMV infection might induce the reactivation of latent EBV, as demonstrated by
serological profiles of EBV reactivation312. Myeloid cells express the NK-cell inhibitory
receptor LIR-1313, which is targeted by UL18, a CMV-derived protein that has been found to
induce a higher production of cytokines including IL-6 and IL-10 by mDCs314. This contrasts
the results of paper IV, as IL-6 levels were strongly correlated with the lower amounts of
IFN-γ (rs=0.66, p<0.001, paper IV). It should, however, be considered that paper III focused
only on a plausible effect modification by CMV in EBV seropositive infants213 since no clear
associations have previously been found for CMV alone213,315.
Whether dampened innate responses in infants are a consequence of herpesvirus
infection, or may itself cause higher susceptibility to infection, remains an open question. A
differential predisposition for acquiring infections may exist316,317, and a very potent innate
reaction towards viruses could potentially block active infections and preclude adaptive
53
responses. Indeed, NK-cell IFN-γ release induced by DCs can effectively restrict primary
EBV infection in human tonsils318. However, the fact that the frequency of EBV
seropositivity increased in our cohort between age 2 and 5 (from 20% to 38%) argues against
a differential disposition to contract EBV. Data from paper IV rather provides support for the
idea that EBV might suppress the innate immune system of infants, and in the co-infected
children; CMV may intensify EBV-induced immune scarring.
What could then be the plausible mechanisms behind the protective effect of
postnatal EBV infection? The polyclonal effects of EBV on the immune system may provide
a likely explanation. While primary, symptomatic, EBV-infection is accompanied by a
vigorous T-cell response and production of a large amount of antibodies258,307, the strength of
such infection in asymptomatic children is unknown. However, increased levels of Th1 and
Th2 cytokines has been demonstrated in 2-year old EBV+ infants262, which could be a marker
of EBV’s polyclonal effect. This mitogenic effect of EBV appears to have time-dependent
dual roles in the modification of the risk to develop IgE-sensitization. During ongoing,
symptomatic EBV-infection, heterophilic antibodies are readily detected while these are
usually absent in young children with primary EBV-infection264. The reason for this might be
the absence of primed-B cells due to an antigen-inexperienced immune system at an early
age. Similarly, sensitization to environmental allergens requires longer time and develops
usually between 1- and 10-years of age270. In the absence of an allergen-specific memory pool
during postnatal years, as opposed to later in childhood, the mitogenic effect of EBV would
not precipitate an allergy-promoting immune profile.
An alternative explanation, that does not mutually exclude the previous one, is
that a suppressed innate immunity following EBV infection may pave the way for encounter
of additional microbial agents by adaptive immunity, possibly initiating a further boost of the
immune system. Little is known about the direct effect of EBV infection on phagocytes319.
However, EBV has been shown capable of infecting, and replicating in, monocytes259, which
can affect their functionality320,321. EBV has also been demonstrated to interfere with the
generation of DCs by inducing apoptosis in their monocyte precursors322. As CD14+CD16+
cells have a higher potential to differentiate into DCs165, they might be the primary targets of
EBV induced cell death. This could transiently delay the initiation of specific immune
response and enable efficient EBV spread into target cells319.
Furthermore, a study by Silins & al revealed that acute-EBV infection in
asymptomatic adults occurs in the absence of excessive T-cell expansion in spite of high
blood viral-loads323 This could point towards yet unidentified regulatory mechanisms that
54
bring the T-cell response under control during silent EBV infection. EBV can induce elevated
levels of TGF-β324, IL-10325 or IL-10’s viral homologue326. The IL-10 homologue can inhibit
or modulate DC maturation, which results in suppression of their T-cell allostimulatory
capacity327,328. There were no differences between seropositive and seronegative infants in
plasma IL-10, or culture supernatant IL-10 and TGF-β, levels possibly indicating that
differential regulation by cytokines are not likely to account for the lower innate activity in
EBV+ infants. However, this cannot be ruled out until proper monitoring of Treg-cytokine
production has been performed. Furthermore, the 2-year old seropositive infants have higher
polyclonal cytokine production262, which is typical for a productive EBV infection, thus
arguing against an inhibited T-cell response327,328. Tregs can also exert their suppressive
function through cell-to-cell contact57,58 and whether they preferentially target CD14+CD16+
cells remains merely a speculation. Similarly, NK cells can act regulatory by killing immature
DCs329 or over-activated macrophages33, but whether preferential elimination of the
CD14+CD16+ cells could be an explanation behind decreased proportions in EBV+ children
remains also elusive.
Taken together, data from paper III & IV demonstrate that 1) postnatal EBVinfection protects against the development of persistent IgE-sensitization during childhood,
which seems to have implications in altered allergen-specific immune reactivity and that 2)
the mechanism behind the EBV allergo-protective effect appears to involve a suppressed
innate IFN-γ response.
55
GENERAL CONCLUSIONS
How come that maternal allergy is a risk factor for development of allergy, whereas early
EBV infection seems protective against development of allergy, when both associate with a
lower monocyte microbial response? The lower monocyte activity from infants with EBV
infection, as opposed to those with maternal allergy, may reflect an active state of innate
suppression by the virus and should not be confused with a lower monocyte activity seen in
infants with allergic heredity. Indeed, EBV infection promotes a polyclonal adaptive
response, and at the end of the day, the outcome of adaptive immunity is fundamental for the
regulation of allergic responses. The property of adaptive T-cell responses may differ between
these children and assessment of T-cell cytokine profiles in relation to maternal allergy could
provide insight to whether this holds true.
An immature immune system during postnatal years in children with
predisposition to allergy has lead to the suggestion that they need a strong and adequate earlylife immune stimulation. Data from this thesis is in agreement with this proposal and 1)
highlights EBV as a potential immunostimulatory candidate 2) stresses that maternal allergy
should be considered as a factor influencing microbial responses during the earliest years of
life and 3) underscores the importance of early-life microbial exposure, as opposed to later on
in childhood, for the most effective period for the shaping of proper allergy-protective
immune profile. In particular, findings from this thesis suggest that monocytes could have a
role in allergic sensitization as their functionality appears to be influenced by all the
aforementioned aspects.
56
FUTURE PERSPECTIVES AND PRELIMINARY RESULTS
Study I & II
A lower PGN-induced p38-MAPK activation and IL-6 response, seen in monocytes from
infants with allergic mothers, generated a number of new questions. Could the altered
response be due to a defective NOD-signalling pathway? And at which level of the p38MAPK pathway is this defect? Deactivation of MAPK-pathway intermediates for instance by
deposphorylation of kinases by MAPK phosphatases (MKPs), have been suggested to play an
important role in the regulation of immune responses96. For instance, an increased amount of
MKP1 accelerates p38-MAPK inactivation and decreases the production of TNF and IL-6
after LPS stimulation330. In contrast to NOD1 that is ubiquitously expressed, NOD2
expression is restricted to immune cells such as monocytes and macrophages302. Interestingly,
a recent study in mice demonstrated that CARD2 is an essential intermediate of NOD2induced p38-MAPK activation by intracellular pathogens331. Currently, these aspects are
being considered in the follow-up studies of paper I and II.
Study III & IV
Based on the findings of a dual, and age-dependent, role of EBV infection in modulating the
risk of allergic sensitization, and that we observed a decreased innate IFN-γ in EBV
seropositive 2-year olds, we further investigated the innate-cytokine profile of EBV
seropositive and seronegative 5-year olds. Plasma was tested for IFN-γ and a panel of APC
derived cytokines including TNF, IL-1β, IL-6, IL-8, IL-10, IL-15, and IFN-α. We found that
EBV seropositive 5-year olds have markedly higher levels of systemic IFN-α (Figure 4,
p=0.02). The elevated IFN-α levels appear not to be related with a defective control of EBV
infection in terms of EBNA-1/EBNA-2 ratios (data not shown).
57
Figure 4. Systemic IFN-α levels in EBV seropositive (EBV+) or seronegative (EBV-) 5-year olds.
P=0.02. Saghafian-Hedengren & al.
TLR9 contributes to immunity against EBV infection332 where pDCs have a prominent role in
the early viral recognition. In addition to its effects on NK-cell cytotoxicity36, IFN-α has also
been shown to induce monocyte differentiation into DCs that have a potent capacity to induce
Th1 cytokine and chemokine responses333,334, as well as activating EBV-specific CTLs335.
A way forward is the examination of pDCs from the cohort’s PBMCs in order to
determine if pDCs have a role in the observed plasma levels of IFN- α and to assess
monocyte characteristics in the 5-year old children with and without previous EBV infection.
Also, the subdivision of 5-year old subjects according to early and late EBV contraction could
provide further insights to if modified innate cell activity could have a role in the
development of allergic sensitization.
58
ACKNOWLEDGEMENTS
Roughly four years have passed since I started my PhD studies at the department of
Immunology at Stockholm University. During this period I have had the opportunity, and the
pleasure, to work with students, scientists and others who have in one way or another
contributed in making this thesis possible. Especially, I would like to express my sincere
gratitude to:
My supervisor Eva Sverremark-Ekström for giving me the opportunity to work in your
group. I truly treasure you competence and when looking back, I realize how patient you must
have been with me! I will never forget the progress from knocking on your door every 5
minutes (study I), to once every day (study II), once a week (study III) and once a month
(study IV), which certainly means that you have helped me to become independent as a
scientist. I wish you all the best with your research.
My co-supervisor Marita Troye-Blomberg, for all your help and advice during these years.
My student co-authors Petra Amourdruz, Ulrika Holmlund, Ebba Sohlberg and Yvonne
Sundström for all the moments we shared in the lab, our teamwork and discussions.
My senior co-authors; Louise Berg at MTC, although many of my thoughts and questions
have been far-fetched, you have been incredibly pedagogic and never made me feel ignorant.
Annika Linde, at SMI for your endless enthusiasm and dedication, and for being so generous
in sharing your knowledge. Although I have only had the opportunity to work with you during
a short time, it has been very educational. You truly inspire me! Gunnar Lilja and Caroline
Nilsson at Sachs´ Children´s Hospital. If it wasn’t for you, none of this would have been
possible. Thank you for letting me use the precious cohort samples and for all your inputs
regarding clinical aspects.
All the children and their families participating in the study cohort.
Jan-Olov Persson at the Division of Mathematical Statistics for providing excellent
statistical support.
59
Monica Nordlund at Sachs´ Children´s Hospital for assistance with plasma sample handling
and Mona Hedenskog at SMI for performance of anti-viral serology.
The seniors Klavs Berzins, Carmen Fernández, Eva Severinson, and to the former senior
Manuchehr Abedi-Valugerdi for your encouragement.
The technicians Maggan and Ann and secretary Gelana at the department, for all practical
help you have offered me during these years and our chats during the coffee breaks.
All former and present students at the department: Alice, Amre, Anna, Ariane, Camilla,
Halima, Jacob, Jacqueline, John, Jubayer, Judith, Karin, Khosro, Lisa, Manijeh, Maria,
Masashi, Mukti, Nancy, Nina-Maria, Nnaemeka, Nora, Olivia, Pablo, Salah, Stefania,
Valentina, Ulrika, Ylva and Yvonne.
The Persian students Samaneh, Khosro, Manijeh and Shiva for our chats and laughs.
Ebba “Sparre” Sohlberg, you can’t imagine how happy I am that you started as a PhDstudent here. By being a curious and dedicated student, you have taught me so much about
myself as an educator, and my potential as an author. It has been a true pleasure working with
you. P.S. Remember to apply “good enough” every now and then ;)
Anna, Nora, Yvonne and Ylva. How I have enjoyed our conversations over dinners, and the
endless wine drinking.
Petra, all the best with your carreer whether it is with science, horses or other beloveds.
My friends Stella & Johan (& Alma), Linda & Micke & the Ceraolos for your precious
friendship and for showing true interest in my PhD-studies. Stella, I wish you all the success
with your own thesis.
Mina snälla bonusbarn (:-D) Maria, David & Adam.
Charles Buster….
60
My mother for having been and being such a great support. Whether it was in comforting me,
drying my tears and boosting my self-esteem when I experienced hard times during my PhDstudies or in cooking the dinner for my dissertation party. I love you so much.
Father, for you sharing your true love of nature with me, and the early introduction to
medicine and life science and how fascinating it can be. I really wish that you could be here
and share this moment with me…
My brother Babak “Bob” and his wife Toktam as well as little Arasto, for all the support,
although far away, you are all kept close to my heart.
My brother Arash for being such a kind brother as well as a dear friend of mine. You can’t
imagine how grateful I am for all the help, both in big and small, you have offered me during
this time.
And most important of all, my husband Uriel “Butti”. No words in the world could express
my happiness for being with you. Thank you for all the encouragement during my educational
period; 8 years of ups-and -downs, and you stood by my side. I look forward, and am proud,
to be the mother of your child.
Lastly, my baby boy. My closest companion during the preparation of this thesis. How you
have been patient with me. I can almost feel your body growing all bigger beneath my palms.
It is amazing how I can love someone I haven’t even met, but I do and can’t wait to hold you
in my arms….
Supported by grants from the Swedish Asthma and Allergy Association’s Research foundation; the Swedish
Research Council grants 74X-15160, 57X-15160-05-2, 74X-15160-03-2 and K2006-74X-15139-03-02;
European Union Grants LSHP-CT 2004 and 503578; the Swedish Medical Society; the Golden Jubilee
Memorial Foundation; Apotekare Hedberg-, the Konsul Th C Berg-, the Samariten-, the Mjölkdroppen-, the
Heart and Lung-, the Crown-princess Lovisa & Axel Tielman-, Golje-, Hesselman-, Vardal-, Swärd/Eklund- and
Åhlén foundations; Glaxo Smith Kline, Brio AB and the Karolinska Institute. Phadia AB supplied with reagents
for plasma IgE-analyses. Shanie Saghafian Hedengren was partly supported by The BioMalPar European
Network of Excellence (LSMP_CT-2004-503578).
61
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