Download Licentiate thesis from the Department of Immunology,

Licentiate thesis from the Department of Immunology,
Wenner-Gren Institute, Stockholm University, Sweden
Innate immune responses in cord blood,
and the influence of pathological pregnancy
Ebba Sohlberg
Stockholm 2011
SUMMARY
Innate immune cells, such as monocytes, are important for early-life immune responses, as
adaptive immunity in the newborn is suboptimal. Immaturity of infection control in newborns
has been related to defective antimicrobial responses and impaired toll-like receptor (TLR)
signaling, findings illustrating the importance of understanding immune maturation in the
neonatal period. Our overall aim was to study innate immunity in the newborn, with focus on
monocytes and monocyte-NK cell collaboration, and how it is affected by exposure to the
pregnancy-related disorder preeclampsia.
In the first study we investigated the two major monocyte subsets, the CD14++CD16- and
CD14+CD16+ cells, in cord blood (CB). Surface expression of activation-related receptors
was assessed. Further, CB and adult peripheral blood (PB) mononuclear cells (MC) were
stimulated in vitro with peptidoglycan (PGN), and phosphorylation of MAPK and production
of intracellular and secreted cytokines was measured. CB monocyte subsets produced high
levels of the early-response cytokines with TNF and IL-12p70 exceeding adult levels, and
also showed high phosphorylation of p38-MAPK. Thus, the monocyte response to a common
bacterial ligand at birth did not appear immature. However, the CD14+CD16+ cells were
present in a higher frequency in PGN-stimulated CBMC cultures and this could reflect a
failure to further differentiate into effector cells following bacterial stimulation. We
concluded that the monocyte subset cytokine responses to PGN in CB are potent, but that the
behavior of CD14+CD16+ cells following PGN stimulation might contribute to a qualitative
difference of the neonatal antimicrobial response as compared to the adult.
In the second study we examined whether the immune-mediated pregnancy disorder
preeclampsia would affect CB monocyte and NK-cell activation. Levels of cytokines were
quantified in maternal and CB serum, CBMC were analyzed for expression of activationrelated surface receptors, and intracellular and secreted cytokines after stimulation with PGN
or PGN+IL-15. Preeclampsia induced an inflammatory serum cytokine profile with
differences for mild and severe preeclamptic subgroups in both mothers and newborns. The
CB monocyte compartment was not affected by preeclampsia, however CB NK cells showed
an altered expression of the activating receptors NKG2D and NKp30. Also, unstimulated CB
NK cells from the preeclamptic group expressed more intracellular TNF and IFN-γ. Together
this suggests that in preeclampsia, an in utero inflammatory priming of neonatal innate
immunity takes place, and possibly indicates an effect on NK-cell function in newborns of
preeclamptic mothers.
2
LIST OF PAPERS
This thesis is based on the original papers listed below, which will be referred to by their
roman numerals in the text:
I.
Sohlberg E, Saghafian-Hedengren S, Bremme K, Sverremark-Ekström E.
Cord blood monocyte subsets are similar to adult and show potent
peptidoglycan-stimulated cytokine responses. Revised and resubmitted.
II.
Sohlberg E, Saghafian-Hedengren S, Bachmayer N, Rafik Hamad R, Bremme
K, Sverremark-Ekström E. Preeclampsia affects cord blood NK-cell activation
patterns. Manuscript.
3
TABLE OF CONTENTS
SUMMARY............................................................................................................................................................. 2
LIST OF PAPERS.................................................................................................................................................. 3
TABLE OF CONTENTS....................................................................................................................................... 4
ABBREVIATIONS ................................................................................................................................................ 5
THE IMMUNE SYSTEM...................................................................................................................................... 6
THE INFLAMMATORY RESPONSE ................................................................................................................ 7
INNATE SENSORS ............................................................................................................................................... 8
INTRACELLULAR SIGNALING FOLLOWING TLR ACTIVATION ......................................................... 10
CYTOKINES ........................................................................................................................................................ 11
EARLY RESPONSE CYTOKINES.................................................................................................................. 12
MONOCYTES / MACROPHAGES................................................................................................................... 15
MONOCYTE HETEROGENEITY................................................................................................................... 15
NK CELLS............................................................................................................................................................ 17
MONOCYTE AND NK-CELL COLLABORATION ...................................................................................... 19
IMMUNE FUNCTION EARLY IN LIFE ......................................................................................................... 19
PREGNANCY AND IMMUNE ACTIVATION ............................................................................................... 21
IN UTERO INFLUENCES ON MATURATION OF NEONATAL IMMUNE RESPONSES ......................... 22
PREECLAMPSIA ................................................................................................................................................ 23
CONTRIBUTING FACTORS TO DEVELOPMENT OF PREECLAMPSIA ................................................. 23
EFFECTS OF PREECLAMPSIA ON OFFSPRING......................................................................................... 25
PRESENT STUDY ............................................................................................................................................... 26
OBJECTIVES.................................................................................................................................................... 26
SUBJECTS AND METHODS .......................................................................................................................... 26
RESULTS AND DISCUSSION ........................................................................................................................ 27
Paper I .......................................................................................................................................................... 27
Paper II ......................................................................................................................................................... 31
GENERAL CONCLUSIONS.............................................................................................................................. 34
FUTURE PERSPECTIVES ................................................................................................................................ 35
PREECLAMPSIA ........................................................................... ERROR! BOOKMARK NOT DEFINED.
NEONATAL IMMUNITY AND VIRAL INFECTIONS ...................................ERROR! BOOKMARK NOT DEFINED.
ACKNOWLEDGEMENTS................................................................................................................................. 37
REFERENCES ..................................................................................................................................................... 38
4
ABBREVIATIONS
APC
antigen-presenting cell
CB
cord blood
CBA
cytometric bead array
CBMC
cord blood mononuclear cells
CLR
C-type lectin receptor
DAMP
danger-associated molecular pattern
DC
dendritic cell
ERK
extracellular signal-regulated protein kinase
FCS
fetal calf serum
IFN
interferon
IL
interleukin
IDO
indoleamine 2,3-dioxygenase
LPS
lipopolysaccharide
MAPK
mitogen-activated protein kinase
MHC
major histocompatibility complex
MICA/B
MHC class I chain-related proteins A/B
NK cell
natural killer cell
NLR
nucleotide oligomerization domain-like receptor
NOD
nucleotide oligomerization domain
PAMP
pathogen-associated molecular pattern
PBMC
peripheral blood mononuclear cells
PGN
peptidoglycan
PRR
pattern recognition receptor
RLR
retinoic-acid inducible gene like receptor
ROS
reactive oxygen species
Tc
T cytotoxic cell
Th1
T helper type 1
Th2
T helper type 2
TLR
toll-like receptor
TNF
tumor necrosis factor
Treg
regulatory T cell
ULBP
UL-16 binding proteins
5
THE IMMUNE SYSTEM
The mammalian immune system is very complex and processes vast amounts of information.
It is capable of distinguishing self from non-self and thus, protects against a multitude of
environmental microbes that we face from the moment of birth. The immune system also has
to be able to tell pathogenic from non-pathogenic organisms, as organs like the gut are
colonized rapidly at birth with a commensal microbial flora. Further, the immune system can
mediate homeostasis and eliminate damaged and/or altered cells.
Physical barriers such as skin and mucosa act in concert with antibacterial molecules and
specialized cells to preserve immunity of the host. Immune cells are present throughout the
body in the circulating blood and lymphatic tissues and in specialized immune compartments
such as the thymus, spleen and lymph nodes. Two intertwined branches of immunity
cooperate to eliminate threats to the host, the innate and adaptive immune system.
Constitutive innate defenses ensure rapid limitation of microbial invasion through preexisting
molecules, such as complement factors and acute phase proteins, and phagocytic cells. Innate
recognition of microbial presence can be mediated through the expression of germ-line
encoded pattern recognition receptors (PRRs) or Natural Killer (NK)-cell associated
receptors. The principal innate effector cells are neutrophils, NK cells, monocytes,
macrophages and dendritic cells (DCs). Neutrophils are professional phagocytes that engulf
and eliminate microbes by production of reactive oxygen species (ROS). NK cells display
cytotoxic activity against virus-infected or tumor cells, and are potent producers of cytokines
that regulate other immune cells. Monocytes are precursors of and replenish macrophages and
DCs in tissues, as well as being potent cytokine producers upon microbial encounter.
Macrophages are specialized in the uptake, digestion and presentation of antigens to adaptive
cells. DCs are the most effective antigen-presenting cells (APCs), they are present in
peripheral tissues where they sample antigen to later migrate to lymph nodes to prime naïve T
cells and activate B cells and NK cells.
Innate defenses may be sufficient to clear invading pathogens, but if not they can limit
infection until cells of the adaptive immune branch have expanded enough to ensure
elimination of the threat. The initial quality of the innate response, and which cytokines are
produced therein, will direct subsequent adaptive functions.1 The adaptive effector cells are T
and B lymphocytes. They display a high degree of diversity and specificity to pathogens, due
to somatic rearrangements of genes encoding the B cell- and T cell-receptors. Adaptive
immunity is divided into humoral responses, which lead to the release of antigen-specific
6
antibodies from plasma B cells, or cell-mediated responses where the effectors are antigenspecific T cells. The major T cell populations are the CD4+ T helper (Th) cells and the CD8+
T cytotoxic (Tc) cells. For T and B cells to become activated they need to recognize antigens
presented on major histocompatibility complex (MHC) class I or II, and get additional signals
from co-stimulatory molecules and cytokines produced by innate cells. Upon activation,
clonal proliferation of T- and B cells generates highly specific effector cells but also longlasting memory cells.
THE INFLAMMATORY RESPONSE
Inflammation is critical for both innate and adaptive immunity. It is the process that first takes
place upon infection or tissue injury and is characterized by alterations in vascularity,
secretion of soluble mediators and the migration of leukocytes. Infiltration of leukocytes to an
inflamed site is promoted both by dilation and increased permeability of blood vessels, and
up-regulation of adhesion molecules like selectins and inter-cellular adhesion molecules
(ICAMs) on endothelial cells. Tissue resident cells such as macrophages, DCs and mast cells
secrete inflammatory factors that promote immune cell function in inflamed tissues. Further,
local production of chemoattractants such as the chemokine interleukin (IL)-8 is important in
mediating adherence and leukocyte infiltration. The acute inflammatory response is
characterized by arrival of neutrophils and subsequent recruitment of monocytes that
differentiate into macrophages and DCs on site. Both neutrophils and macrophages
phagocytose microbes, but macrophages have a longer life span. They also have an important
role in orchestrating the immune response through the production of IL-1, IL-6 and tumor
necrosis factor (TNF), cytokines that mediate many of the systemic acute-phase effects. Once
pathogen clearance is achieved, feedback inhibitory loops are initiated to restrain immune
activation and prevent tissue damage associated with excessive inflammation. One major
inhibitory pathway induced by inflammation and microbial recognition, is expression of
suppressor of cytokine signaling (SOCS) proteins that inhibit signal transduction.2 For
resolution of inflammation the immune effector cells should themselves be removed from the
site. This can happen either by recirculation or by uptake by macrophages that in turn die
locally by programmed cell-death. A failure of these or other inhibitory mechanisms to
develop may lead to chronic inflammation and disease.3
7
INNATE SENSORS
Innate immune cells possess a variety of sensors that enable them to distinguish between
different classes of pathogens. They are found both on the cell surface and either free or
membrane-bound in the cytosol, ensuring detection of microbes that employ different modes
of infection. The innate sensors are commonly referred to as pattern-recognition receptors
(PRRs). Manipulation of the PRR signaling pathways seems to be a common theme for
microbial interference with immune responses underlining their importance for host defense.4
PRRs recognize structures that microbes cannot easily change without affecting their fitness
such as components of the bacterial cell wall, or viral nucleic acids. Collectively these
molecules are referred to as pathogen-associated molecular patterns (PAMPs). Also nonimmune cells like epithelial cells express PRRs and can produce factors upon PRR ligation
that stimulate the immune response.5 Therefore it may be possible that an initial sensing of
microbes occurs in tissues that thereafter direct the activation of immune cells.6 Innate sensors
can also respond to endogenous danger-associated molecular patterns (DAMPs) like heat
shock proteins, self nucleic acids or uric acid. The release of DAMPs is a common event
when cells have been exposed to damage, pathogens or toxins or are undergoing necrosis. The
discovery of immune responses initiated by danger signals sparked the debate on whether the
immune system responds to substances that cause damage, rather than to those that are simply
foreign.7
There are several varieties of innate sensors and deficient expression or function of many of
them has been associated with increased susceptibility to infectious and chronic inflammatory
diseases.8-10 The major families of PRRs are C-type lectin receptors (CLRs), nucleotide
oligomerization domain (NOD)-like receptors (NLRs), retinoic acid inducible gene-like
receptors (RLRs) and Toll-like receptors (TLRs). CLRs, like Dectins or DC-SIGN, are
expressed on the cell surface and recognize mannose, fucose and glucan carbohydrate
structures from fungi, but also from mycobacteria and viruses. Their ligation primarily
modulates NF-κB activity and thus production of inflammatory cytokines.11 NLRs are present
in the cytosol and act as sensors of bacterial components. For instance NOD1/NOD2
recognize peptides derived from the degradation of PGN, a bacterial cell wall polymer,12
although it is somewhat unclear how extracellular PGN reaches cytoplasmic NODs.13 Some
of the NLRs are associated with large cytoplasmic complexes called inflammasomes, like the
prototype NALP3, that sense PAMPs and/or DAMPs and metabolic stress. NLR ligation
results in activation of inflammatory caspases, that leads to proteolytic activation of the
8
inflammatory cytokines IL-1β and IL-18.14 RLRs, like RIG-I or MDA5, are present in the
cytosol and recognize viral RNA which leads to production of type I interferons and
inflammatory cytokines.15 TLRs are transmembrane receptors found on the cell surface or on
intracellular vesicles. There are 10 TLRs so far in the mammalian genome that together
display a wide specificity.16 For instance TLR4 recognizes lipopolysaccharide (LPS), TLR5
bacterial flagellin, TLR7 and TLR8 single stranded RNA and TLR9 unmethylated CpG
motifs in DNA. TLR2 recognizes bacterial lipoproteins but also lipoteichoic acid (LTA) and
PGN together with NOD1/NOD2.12
Figure 1. Expression of PRRs at different cellular locations enables detection of pathogens
employing different infection modes. Modified from Werts, Girardin & Philpott.17
9
INTRACELLULAR SIGNALING FOLLOWING TLR ACTIVATION
The importance of the TLR pathway is evident considering the clinical consequences of
defects in TLR-associated signaling molecules, reviewed in Ku et al.18 TLR activation
initiates distinct intracellular signaling cascades, which result in transcription of genes
encoding
inflammatory
cytokines
and
chemokines,
co-stimulatory
molecules
and
antimicrobial compounds. TLRs are composed of various domains including extracellular
ligand-binding leucine-rich-repeats and an intracellular Toll/IL-1 receptor (TIR) signaling
domain. TLRs are continuously recycled from the cell surface in absence of ligand. Upon
ligation the TLRs are stabilized, dimerize and association of adaptor proteins to the TIRdomains is triggered. The adaptors include MyD88, IRAK, TRIF, TRAF and TAK1 and they
serve to transduce and amplify the signal. All TLRs except TLR3 activate the MyD88dependent pathway, making MyD88 one of the key adaptors. Downstream of TAK1,
mitogen-activated protein kinases (MAPKs) are activated through phosphorylation. MAPKs
in turn phosphorylate the transcription factor AP-1 that switches on genes encoding the
inflammatory cytokines.19 There are three major families of MAPKs; the extracellular signalregulated protein kinases (ERK) and the c-Jun NH2-terminal kinases (JNK) and p38-MAPK
that show some overlap in function.20 Stimulation or inhibition of MAPK members can affect
cytokine release and/or expression of co-stimulatory molecules by APCs. For example,
inhibition of p38-MAPK and ERK has been shown to suppress PGN-induced production of
IL-6, IL-10, TNF and IL-12p70 by DCs.21 MAPKs have been under investigation in
connection to inflammatory diseases and implicated as potential targets for therapeutics.22,23
Activation of TLRs is essential for the innate immune response against pathogens, however
signaling needs to be strictly regulated in order to avoid inappropriate inflammatory
responses. Many negative regulators exist including splice variants for adaptor proteins,24
MAPK-phosphatases25 and microRNAs,26 that all target different steps in the signaling
cascade to suppress inflammation.
10
Figure 2. TLR signaling elicits production of inflammatory cytokines and type I interferons.
Adapted from Kumar, Kawai & Akira.19 Reprinted with the permission of Elsevier.
CYTOKINES
In order to coordinate immune responses, cells need to communicate. For this purpose, the
production and secretion of soluble proteins called cytokines is of major importance.
Cytokines bind to a variety of receptors and induce cell-specific responses to regulate the
intensity and duration of an immune response. A given cytokine can have different actions
depending on antagonistic or synergistic effects with other cytokines and whether the target
cell has prior antigen priming. There are different families of cytokines based on function; the
prototype cytokines are called interleukins (ILs), since they are produced by, and deliver
signals to leukocytes. Among other functions, ILs are involved in perpetuation of
inflammation together with the tumor necrosis factor (TNF) family. Interferons (IFNs) are
key molecules in the early defense against viruses and induce an antiviral state in cells.
Chemokines are cytokines that influence the migratory behavior of leukocytes. A brief
description of the cytokines that are produced early on during an immune response, and by
innate cells such as monocytes and NK cells will follow.
11
EARLY RESPONSE CYTOKINES
IL-1β and TNF
Some of the earliest cytokines initiated during inflammation are IL-1β and TNF that are
produced primarily by activated monocytes, macrophages and DCs. In addition NK cells, B
and T cells also produce TNF. Both IL-1β and TNF are very potent pro-inflammatory
cytokines and are therefore not normally found in high levels in circulation in healthy
individuals. Synthesis of IL-1β is tightly regulated by the inflammasome and occurs upon
recognition of PAMPs and DAMPs. This leads to activation of caspase-1 and proteolytic
cleavage of pro-IL-1β into IL-1β.14 TNF is also produced in response to PRR activation by
PAMPs. Further, both IL-1β and TNF induce their own synthesis and that of each other.
The two cytokines synergize to mediate the systemic acute phase response that follows local
inflammation including induction of fever, production of acute phase proteins such as Creactive protein by the liver, and activation of the vascular endothelium. IL-1 signals through
the type 1 IL-1 receptor (IL-1R1) and TNF through TFNR1/TNFR2. Both cytokines act
immunostimulatory and ligation to their cognate receptors launches downstream pathways
that are similar to those elicited by TLR ligands. Overproduction of IL-1β and TNF is
involved in several pathologies such as septic shock, and various autoinflammatory and
autoimmune disorders, reviewed in Kastner et al. and Bradley et al.27,28 Blockade of IL-1 and
TNF signaling through antibodies, soluble receptors or receptor antagonists are currently
being used as therapeutic agents for inflammatory disorders including rheumatoid arthritis
(RA) and Crohns disease.27,29
IL-6
Many of the systemic acute phase effects are mediated by IL-1β and TNF in combination
with IL-6. Further, IL-6 has an important role in the shift from the early innate response to
subsequent adaptive immunity.30 Mainly monocytes, macrophages, DCs, endothelial cells and
B cells produce IL-6 in response to PRR and/or IL-1β/TNF stimulation. IL-6 acts pro- or antiinflammatory depending on the stage of the immune response. In the initial phase of infection
or tissue injury, IL-6 induces acute-phase proteins, activation of macrophages and mediates
lymphocyte arrival to the site of inflammation. In the resolution phase of inflammation, IL-6
favors the onset of acquired responses by e.g. inducing neutrophil apoptosis, whilst acting as a
lymphocyte stimulatory factor. Also, IL-6 can block further pro-inflammatory cytokine and
chemokine production thus affecting leukocyte recruitment.30,31
12
IL-6 signaling is propagated by the glycoprotein (gp) 130 homodimer. It forms a complex
with the membrane-bound IL-6 receptor (IL-6R) or with the soluble (s) IL-6R, when either is
bound to IL-6. The cellular distribution of the IL-6R is rather restricted but the sIL-6R renders
cells that lack expression responsive to IL-6.32 IL-6 driven inflammation is associated with
autoimmune diseases such as RA, multiple sclerosis (MS), and inflammatory bowel disease,
reviewed in Kishimoto et al.33 Recent studies have demonstrated that IL-6 has an important
role in regulating the balance between IL-17-producing Th17 cells and regulatory T cells
(Treg),33,34 thereby possibly providing an important link to the involvement of IL-6 in
pathophysiology.
IL-8
IL-8 (CXCL8) is a chemokine produced during inflammation by a range of cells, e.g.
monocytes, granulocytes, lymphocytes, fibroblasts and endothelial cells. Neutrophils are the
primary targets for IL-8 and respond by chemotaxis and subsequent upregulation of surface
adhesion molecules. IL-8 also enhances generation of ROS and delays neutrophil apoptosis.35
IL-8 signals through two G-protein coupled receptors, CXCR1 and CXCR2, which also
mediate the effect of many other chemokines. CXCR1 and CXCR2 are expressed mostly on
neutrophils and myeloid cells such as monocytes but also on Tc, endothelial and epithelial
cells.36 CXCR1 and CXCR2 have been implicated in various inflammatory disorders such as
RA, MS and asthma.37
IL-12p70
IL-12p70, from here on referred to as IL-12, is a pro-inflammatory cytokine that is closely
involved in both innate and adaptive immune responses. It is composed of two subunits, p40
and p35,38 that together form the biologically active cytokine. Expression of the two subunits
is independently regulated and the rate-limiting step is production of the p35 subunit.39,40 IL12 is predominantly produced by monocytes, macrophages, DCs and neutrophils in response
to activation of PRRs, and/or co-stimulatory signals and cytokines from activated T cells and
NK cells. IL-12 signals through the IL-12 receptor that is expressed mainly by NK cells and T
cells. IL-12 has a crucial role for establishing antigen-specific Th1 responses and enhancing
activation of B cells and the production of Th1-related antibody isotypes, which are essential
to control infections with many microbial pathogens. Although IL-12 might not be required
for Th1 differentiation,41 it induces clonal proliferation of, and maintains already committed
IFN-γ secreting Th1 cells.
13
IFN-γ
IFN-γ belongs to the interferon family whose members are key molecules in the early
antiviral defense. Further, IFN-γ is essential for control of intracellular bacteria and tumor
malignancies. The main IFN-γ producers are NK cells, NKT cells and T cells. IFN-γ directly
promotes antiviral mechanisms by induction of antiviral enzymes but its main function is in
immunomodulation. Over 300 genes are estimated to be upregulated by interferon signaling
including genes coding for molecules involved in adhesion, apoptosis, cell-signaling and
transcription.42 IFN-γ distinguishes Th1 type of immune responses and IFN-γ signaling,
through the interferon gamma receptors IFNGR1/2, is fundamental in the maintenance of
these responses.43 Its production is induced by mitogenic/antigenic stimuli and cytokines that
are secreted by PRR-activated myeloid cells. IFN-γ is also a major activator of APCs and
enhances responses to microbial inducers such as TLR ligands and antigen processing and
presentation capabilities.44
IL-10
IL-10 is a unique cytokine with regulatory properties that can act as a negative feedback
switch on inflammatory responses. This is accomplished by the fact that the same cells that
initiate inflammation also start to produce IL-10. These cells include monocytes and B cells,
but also the majority of T cell subpopulations, DCs and NK cells, which underscores its role
as a critical negative regulator. IL-10 has diverse effects on most hematopoietic cell types for
instance in growth and/or differentiation of T cells, B cells and NK cells. IL-10 dampens the
inflammatory response through down-regulation of genes encoding inflammatory mediators,
and by enhancing expression of molecules that antagonize the inflammatory response.45 IL-10
signals through the IL-10 receptor composed of at least two subunits members of the IFNR
family. IL-10 suppresses a broad spectrum of activated macrophage/monocyte functions,
including cytokine synthesis (for example IL-1β, IL-6, IL-12, TNF) by inhibition of NF-κB,
expression of MHC II molecules and co-stimulatory molecules CD80 and CD86.45 Another
way in which IL-10 puts the brakes on inflammatory responses is by enhancing the
differentiation of IL-10 secreting Tregs, which contribute to control of immune responses and
tolerance in vivo.46 Administration of recombinant IL-10 has only had modest success in
protecting against inflammatory diseases, most probably due to the fine balance between
immuno-suppression and immunopathology. The dose and cellular location is likely to be
important for future attempts to modulate IL-10 activity.47 In the context of vaccination and
14
adjuvancy it is interesting to note that certain TLR stimuli do not elicit IL-10 production
whilst the production of inflammatory cytokines is preserved.48
MONOCYTES / MACROPHAGES
Monocytes are mononuclear phagocytes that belong to innate immunity. They play a crucial
role not only in innate responses but also in priming and maintenance of adaptive responses.
Besides acting as an important source of early cytokines following infection, circulating
monocytes are precursors of macrophages and DCs that act as highly efficient APCs.49
Monocytes originate from hematopoietic progenitor cells in the bone marrow. Their growth
and differentiation depend on lineage determining cytokines such as M-CSF and GM-CSF,
and upon interactions with cells in the local niche. After differentiation, monocytes enter the
blood stream where they circulate for a few days and constitute about 5-10% of the leukocyte
pool. Under steady-state conditions, monocytes can replenish tissue-resident macrophages by
homing to different tissues, although to what extent this occurs has been debated.49,50
Differentiation of tissue-specific macrophages or DCs is to a large extent dictated by the local
microenvironment, signals from neighboring cells and the extracellular matrix. Stromal cells
within tissues can exert immune regulatory functions by directing development towards
regulatory phenotypes.51 When pathogen invasion occurs, there is a shift in the homeostatic
pathway and monocytes act as efficient effector cells with antimicrobial activities. They are
drafted from the blood into inflamed tissues promoted by signals such as monocyte
chemotactic protein-1 (MCP-1/CCL2) and adhesion molecules, such as integrins and selectins
that control migration. At the inflamed site monocytes will differentiate into recruited
macrophages and/or DCs.
MONOCYTE HETEROGENEITY
The blood monocyte population is heterogeneous and the subpopulations most likely play
different roles in antimicrobial responses. The two major subsets are distinguished by the
presence or absence of CD16 (FCγRIII) and the degree of expression of CD14 (LPS coreceptor). The CD14++CD16– subpopulation constitutes 80-90% of total monocytes in
comparison to the minor subset of CD14+CD16+ monocytes.52
15
CD14++CD16– monocytes and CD14+CD16+ monocytes have distinct trafficking potential
due to differential expression of chemokine receptors, and are recruited into tissues via
distinct molecular mechanisms. CD14+CD16+ cells express high levels of CX3CR1 and
migrate towards the chemokine CX3CL1. CD14++CD16− cells express CCR2 and CD62L and
can migrate in response to CCL2.49,53-55
With regard to PRRs, monocytes in general express high levels of NOD1/NOD2 and TLR2
and TLR4.56,57 CD14+CD16+ cells in turn display the highest levels of TLR2 and TLR4.58,59
In response to the TLR4 ligand LPS, CD14++CD16– cells produce high levels of the cytokines
IL-6, IL-8, CCL2 and CCL3, whereas CD14+CD16+ cells produce IL-1β, TNF, IL-6 and
CCL3. Both CD16+ and CD16- cells produce IL-10.58,60,61 Further, the CD14+CD16+ cells
have increased capacity for antigen presentation and phagocytosis.62,63
The two subsets may also differentiate into macrophage and DC subpopulations with distinct
phenotypes. One study found that TLR2/1-induced differentiation of monocytes into CD1b+
DCs was restricted to CD14+CD16+ cells, while differentiation into DC-SIGN+ cells was
enhanced in the CD14++CD16– subset.64 The frequency of CD14+CD16+ cells is increased in
adult and neonatal sepsis65 and other inflammatory and infectious conditions.66-68
Table 1. Some receptors differentially expressed by the two major monocyte subsets.
The developmental relationship between CD14++CD16− and CD14+CD16+ cells is currently
unknown but the two subsets have been suggested to represent sequential stages of monocyte
differentiation.69,70 Support for this theory has come from transcriptome analysis69 and studies
where CD16−CX3CR1− monocytes became CD16+CX3CR1+ under the influence of TGF-β,
IL-10, M-CSF and CCL2.71,72 Thus the CD14++CD16− cells could give rise to the more mature
macrophage and DC-like CD14+CD16+ cells,72,73 although this remains to be proven.
16
To make matters more complicated, CD14+CD16+ cells can be further subdivided into
CD14highCD16+ and CD14dimCD16+ cells.60 A recent proposal suggests a common
nomenclature for monocytes naming these 3 types classical (CD14++CD16-), intermediate
(CD14hiCD16+), and nonclassical monocytes (CD14dimCD16++).74 In contrast to classical and
intermediate monocytes, CD14dimCD16++ cells produce low amounts of inflammatory
cytokines in response to bacterial ligands, but high amounts to measles virus and TLR7/8
ligands and could therefore be involved in the inflammatory response to viruses.60
NK CELLS
NK cells are innate cells that are widely distributed in lymphoid and non-lymphoid tissues
and constitute approximately 5-20% of the blood lymphocyte population.75 NK cells are CD3CD56+ cells and express a range of other receptors, of which NKp46 has also been suggested
to define NK cells in order to facilitate cross-species comparisons.76 The NK-cell
developmental pathway is not fully clear but commitment to the NK cell lineage, education of
NK cells towards self-markers and establishment of functional competence occurs in the bone
marrow.77 NK cells may also develop in peripheral sites such as secondary lymphoid
organs.78 NK cells contribute to host defense through cytolysis-mediated killing of virusinfected cells and tumor cells. This is accomplished by release of pre-formed cytoplasmic
granules that contain lytic proteins like granzyme and perforin, and/or by FAS ligand-FAS
interactions. NK cells are also an important source of cytokines and chemokines, like IFN-γ,
GM-CSF, IL-10, IL-13 and TNF, which contribute to inflammation and polarization of T-cell
responses.
The NK cell population is heterogeneous and there are two major NK-cell subsets with
functional differences and distribution between anatomical sites. In blood, the majority of NK
cells belong to the CD56dimCD16+ cytolytic NK subset and express homing receptors for
inflamed sites. The minor circulating CD56brightCD16– NK cell subset displays low cytolytic
activity but high production of IFN-γ and express homing receptors for secondary lymphoid
tissues.79
NK cells are not simply ‘on’ or ‘off’; rather they vary in their responsiveness. Activation
status is regulated in a rather complex fashion, and is the net result of simultaneous signals
from a set of inhibitory and activating receptors. Central to NK-cell recognition of aberrant
cells is the presence or absence of MHC class I molecules on the cell surface (“missing-self”
17
recognition).80 The two major families of MHC-specific inhibitory receptors are the Ig
superfamily (KIR and LIR) and the C-type lectin (CD94/NKG2A) receptor superfamily.
These prevent NK-cell activation upon encounter of cells displaying normal MHC class I
expression. Stressed, transformed or virally infected cells can lack normal MHC expression
and thus fail to provide the inhibitory signal. These cells will be lysed provided that they also
express activating ligands. NK-cell activation receptors include CD16, NKG2D, 2B4, and
NKp80 and the natural cytotoxic receptors (NCRs) NKp46, NKp44, and NKp30.
The main activating receptors constitutively found on all NK cells in peripheral blood are
NKG2D and the natural cytotoxicity receptors (NCRs) NKp30 and NKp46.81 NKG2D and
NKp30 are highly expressed on cord blood NK cells,82 suggesting that they play important
roles for NK-cell activation very early in life. NKG2D recognizes molecules that are
upregulated on stressed and/or transformed cells such as MHC class I chain-related proteins A
and B (MICA/MICB) and the UL-16 binding proteins (ULBP) that are induced by DNA
damage, oxidative stress and inflammation.83 NKG2D-ligand interaction triggers cytotoxicity,
cytokine production and cell proliferation.84,85 The ligand specificity of the activating receptor
NKp30 is currently unknown, but has been speculated to include viral and apoptosis-related
proteins.86,87 Ligation of NKp30 is involved in NK-cell induced DC maturation through NKcell killing of immature (i)DCs, and induction of DC maturity by IFN-γ and TNF secretion,
suggesting an immunoregulatory role.88,89
18
MONOCYTE AND NK-CELL COLLABORATION
Accessory cells are required to provide both soluble and contact-dependent activation signals
for proper pathogen mediated NK-cell activation. NK cells become fully active only when
additional stimuli such as monocyte secreted IL-12, IL-15 and IL-18 are present. IL-12
induces proliferation in pre-activated NK cells and enhances NK-cell cytotoxicity and IFN-γ
secretion. Further, myeloid cell activation by IFN-γ amplifies production of IL-12/IL-18,
which in turn potentiates IFN-γ secretion thus creating a feed forward loop.43 IL-15 is
important to maintain NK cell development and proliferation.90,91 IL-18 seems to direct
differentiation into a distinct NK-cell “helper” pathway characterized by expression of several
mature DC-associated surface markers.92 Moreover, it seems there is close relation between
the three cytokines in mediating activation of NK-cell effector functions. For example, in
combination with IL-12, both IL-18 and IL-15 induce potent IFN-γ production.93-95
Interestingly, the monocyte subsets seem to have the capacity to differentially affect NK-cell
activation. Gene set enrichment analysis has shown that CD14+CD16+ cells are enriched in
genes related to NK-cell mediated cytotoxicity,69 indicating a close relationship between these
cells in battling infection. Further, our group have found that PGN-stimulated CD14+CD16+
cells induce higher NK-cell IFN-γ production than CD14++CD16- cells,96 most probably
connected to a higher capacity for IL-12 production (Paper I).
IMMUNE FUNCTION EARLY IN LIFE
The development of the immune system starts in utero in an environment normally free from
microbes. The immune system of the newborn is therefore largely antigen inexperienced and
lacks memory cells, something that may contribute to the high frequency and severity of
microbial infections that afflict both newborns and infants. Although it is becoming
increasingly clear that neonatal cells are capable of mounting fully mature immune responses
to certain antigens, the neonatal immune system does exhibit a number of functional
immaturities as compared to the adult.
Naïve B cells express lower levels of activation-related markers such as CD40 and
CD80/CD86, and there is limited production of IgG to a range of T-cell dependent and –
independent antigens. Further, there seems to be a bias for formation of memory instead of
plasma B cells all together hampering efforts for early-life immunization, reviewed in
19
Siegrist.97 With regard to the neonatal T cell compartment, Tc cells can mount mature
cytotoxic responses under certain circumstances,98,99 but both Th1 and Th2 cell responses are
often reduced in magnitude. The most pronounced and recognized defect is in T-cell
production of IFN-γ.100,101 Nonetheless, the tuberculosis vaccine BCG can be administered at
birth and induces potent Th1 responses.102,103 This seems to depend on BCGs’ capacity to
engage multiple TLRs on DCs.104
Immaturities in DCs and their ability to further support e.g. Th1 differentiation may be an
important factor underlying neonatal susceptibility.105 This is probably related to a reduced
DC production of IL-12 in response to LPS and a number of other microbial stimuli.106-108
Neonatal monocytes have been extensively studied, as they are an integral part of the early
innate defense, and have been described to give both immature and mature responses. Still,
very little is known regarding the developmental expression of RLRs, NLRs and CLRs or
their signaling intermediates, however TLR2 and TLR4 are expressed at an adult level on
neonatal monocytes.109,110 Regarding various downstream TLR adaptors, findings have
included lower111 or adult-level112 basal MyD88 expression and adult-level110-112 or
deficient110,112 ERK1/2 and p38-MAPK activation in response to LPS, LTA and/or R-848 (a
TLR7/8 ligand).
At birth there are adult-level NK-cell percentages, including the CD56dim and CD56bright
subpopulations.113 Cytotoxicity and capacity for production of IFN-γ after IL-12+IL-18
stimulation are at adult levels.114 However, neonatal NK cells have high expression of
inhibitory NKG2A/CD94 receptors, and low expression of granzyme B that might contribute
to low cytolytic activity of cord blood NK cells without prior cytokine stimulation.115
Overall the neonatal immune response exhibits a qualitatively different cytokine production
profile than that of adults, which in turn could affect infection susceptibility of the newborn.
The dogma has long been that newborns have a Th2-biased immunity and this has recently
developed to incorporate a Th2/Th17-type of immunity over Th1-type of immunity.116-118
Although Th1 type of immunity appears to be down-regulated it is also clear that the neonatal
response strongly depends on the stimulus used and the setting in which the cells are
cultured,116,118-121 and a global Th2 bias at birth is not evident.122 During ontogeny maturation
of various immune response takes place and a recent longitudinal study showed slow agerelated development, from cord blood to the age of 5, in cytokine production capacity to
multiple TLR stimuli.123 Further, upregulation of CD80 and HLA-DR on monocytes and DCs
following TLR stimulation is also increased during the first year of life.119
20
PREGNANCY AND IMMUNE ACTIVATION
Much has happened since Medawar, Billingham and Brent suggested mechanisms behind the
tolerance to the semi-allogeneic fetus.124 The thought of the fetus as a graft probably
hampered progress in the field of reproductive immunology as from an evolutionary
perspective, the co-existence of mother and fetus should in fact be desirable. Immunological
detection of pregnancy is now recognized to be important for maintaining gestation
successfully. The maternal immune system changes both locally in the intrauterine
environment and in the peripheral circulation. The implantation site is filled with immune
cells such as NK cells, macrophages and T cells. A phenotypical and functionally unique NKcell subset named uterine (u)NKs are the pre-dominant cell population in the uterus and make
up some 70% of all decidual leukocytes. The uNKs contribute to vascularisation early in
pregnancy by production of angiogenic cytokines.125
Some factors that are proposed contribute to tolerance to the allogeneic fetus include
trophoblast expression of Fas ligand and subsequent clonal deletion of activated T cells, and
suppression of T-cell activation in placenta by local production of indoleamine 2,3dioxygenase (IDO) by APCs.126 In the presence of IDO, Treg are promoted and in its absence
there is a re-programming towards more inflammatory Th17 cells.127,128 Interestingly recent
findings reveal that CD14+ myelomonocytic cells communicate with NK cells in the decidua
and that the crosstalk results in the induction of IDO and subsequent induction of Tregs.129
Moreover, macrophages in decidua secrete high spontaneous levels of IL-10 and thus
contribute to regulatory environment.130 In addition, placental exosomes found in maternal
circulation can act immunosuppressive by inhibition of T- and NK cell signaling.131,132
In circulation the maternal adaptive immune system is suppressed with a down-regulation of
T-cell functions whereas components of innate immunity are systemically activated, and this
dysregulation is thought to be crucial for gestation.133 What type of immune response is
beneficial overall seems to depend on the location, e.g. blood or fetal-maternal interface and
on the stage of pregnancy. As the T cell repertoire is expanding, so is the debate surrounding
the role of different T cells for reproductive success, discussed in Saito et al.134 In contrast to
prior belief, a strict Th2 dominance throughout gestation does not appear to be essential. IFNγ was traditionally considered harmful to pregnancy, but has been shown in murine models to
be needed for proper vascularization in the early stages of placental development.135 In
addition, there are increased numbers of Tregs in circulation in pregnancy.136 With regard to
the innate system, monocytes are activated and display increased surface expression of
21
molecules mediating adhesion (CD11a and CD11b, CD54) and phagocytosis (CD64), and
have an increased ability to produce pro-inflammatory cytokines (IL-1β, IL-6, IL-12) and also
IL-10.137,138 NK cells numbers are reduced, and so is cytotoxicity and IFN-γ production.133
IN UTERO INFLUENCES ON MATURATION OF NEONATAL IMMUNE
RESPONSES
That the conditions we face in the womb may influence future health and disease is at the
heart of an emerging scientific frontier focused on the intra-uterine environment. In
pregnancy there is a continuous exchange of maternal and fetal factors. Maternal antibodies,
mainly IgG, readily cross the placenta to enhance neonatal immunity.139,140 Whether there is
trans-placental cytokine transfer is less clear but at least IL-6 has been reported to cross the
placenta.141,142 It was shown early on that fetal cells like trophoblasts and leukocytes can be
found in the maternal circulation.143,144 Also maternal cells can be found in the fetal
circulation, including most subsets of immune cells.145
Tregs, that can influence other effector cell responses, may play an important role in
maintaining the balance of maternal and fetal immune responses. One study found that
maternal cells that had crossed the placenta induced generation of Tregs that suppressed fetal
antimaternal responses. This finding reveals a form of antigen-specific tolerance induced in
utero.145 In addition, it appears that in utero exposure to allergens or infectious pathogens can
lead to priming of antigen-specific fetal T lymphocytes.146,147
Moreover, maternal immune activation and development of neonatal immunity could be
intertwined. Maternal allergy affects functional responses of cord blood mononuclear cells
(CBMC) in terms of a lower monocyte derived IL-6 production,109 and this defective response
persists into childhood.148 This connection was not seen for paternal allergy indicative of a
pre-natal influence, thereby raising the question if other maternal immunological conditions
may also affect neonatal immunity.
22
PREECLAMPSIA
Preeclampsia is the most common complication associated with pregnancy. This multisystem
disorder is potentially life threatening and occurs in around 3-5% of all pregnancies. The
pathophysiology is not completely understood, but is characterized by insufficient placental
development during early pregnancy, endothelial-cell dysfunction and an excessive maternal
systemic inflammatory response. There are many risk factors for developing preeclampsia;
first and foremost having had preeclampsia in a previous pregnancy but also pre-existing
hypertension, diabetes, autoimmune disease, family history of preeclampsia, obesity and
multiple gestations, reviewed in Redman et al.149 Preeclampsia is a heterogeneous disorder
and the varied manifestations may arise from maternal, placental, or shared etiologies.150,151
The clinical stage of preeclampsia manifests after 20 weeks of gestation with hypertension
(above 140/90 mmHg) and proteinuria (above 0.3 g/l).152,153 Based on these two clinical
parameters preeclampsia can be divided into mild and severe subgroups. Severe preeclampsia
is often, but not always, related to an early onset of the disorder (delivery necessary before 34
weeks). Severe and early onset preeclampsia is associated with more adverse maternal and
perinatal outcomes.154-156
CONTRIBUTING FACTORS TO DEVELOPMENT OF PREECLAMPSIA
Preeclampsia is believed to progress in three stages with the last stage representing the fullblown clinical disorder. The first stage involves abnormal placentation due to maternal
immune maladaptation at the implantation site.157 Normally fetal trophoblasts invade deep
into the maternal spiral arteries to create a high flow, low resistance vascular bed that can
supply the fetus with nutrients, oxygen and carry out waste exchange. In preeclampsia this
process is impaired leading to narrow spiral arteries of the placenta and ensuing oxidative
stress.151 The trophoblast expresses non-classical HLA-E, -G and –C. HLA-C is the dominant
ligand for inhibitory KIR receptors expressed by uNK cells, and some KIR/HLA-C
combinations have been found to be unfavorable to trophoblast invasion due to the overall
signals that the NK cell receives.150
The second stage of preeclampsia may result from the endoplasmic reticulum and oxidative
stress of the preeclamptic placenta occurring in addition to the already inflammatory
environment of pregnancy.157 Placental immune cells are activated; for instance there is
increased IL-6 production by decidual cells and increased number and altered receptor
23
expression of placental NK cells.158-160 The preeclamptic placenta further releases proinflammatory debris such as microparticles and cell-free fetal DNA into the maternal
circulation, which provokes elevation of the systemic inflammatory response.161,162
In general, a Th1 type of immune profile is predominant in preeclampsia and is believed to
be part of the immunopathology.151,163,164 In circulation there is a higher percentage of IFN-γ+
NK cells161 and monocytes spontaneously produce IL-1β, IL-6 and IL-8.165 Further, secreted
IL-18 and IL-12 that drive IFN-γ production and Th1 responses are also enhanced.161,166
Enhanced Th1 type of immunity may affect the tolerance system. Circulating Treg cells are
reported to be significantly lower than those in normal pregnancy, however their suppressive
function is unaltered.136
Recently, molecules related to maternal endothelial cell dysfunction have been suggested as
biomarkers for predicting and diagnosing preeclampsia. The best studied is FMS-related
tyrosine kinase 1 (sFlt-1), a soluble form of the vascular endothelial growth factor (VEGF)
receptor. Increased levels of sFlt-1 appear in circulation before clinical manifestation of
preeclampsia.167
Figure 3. The different stages and contributing factors for the development of preeclampsia.
Adapted from Redman & Sargent.151 Reprinted with the permission of AAAS.
24
EFFECTS OF PREECLAMPSIA ON OFFSPRING
Preeclampsia is the most common reason for pre-mature births as the only treatment is
delivery and removal of the placenta. Apart from being born too early, children of
preeclamptic mothers run an increased risk of intra-uterine growth restriction due the underdeveloped placenta. Placental morphology and growth has been identified as a predictor of
cardio-vascular health in the offspring,168 and later in life the children of preeclamptic
mothers more often suffer from childhood hypertension.169 They also have a higher incidence
of conditions such as arthritis, asthma and diabetes.170-172 From previous studies it has been
hard to separate the effect of being born too early from that of having been exposed to the
inflammatory environment in a preeclamptic pregnancy. However, a recent study by Wu et al.
showed that these adverse effects were indeed related to preeclampsia itself and not prematurity by using a cohort of children born at term to preeclamptic mothers.170 Still very little
is so far known regarding the possible underlying mechanisms and the state of the immune
system in these children.
25
PRESENT STUDY
OBJECTIVES
The overall aim of this work was to examine the response capacity of the neonatal innate
immune system, with particular focus on monocytes and monocyte-NK cell collaboration. We
also wanted to investigate how the neonatal monocyte and NK-cell compartments were
influenced by a maternal immune-mediated disorder like preeclampsia.
More specifically we aimed to:
•
Carry out a comprehensive analysis of monocyte subsets in cord blood and investigate
cytokine responses to a common bacterial ligand at birth (Paper I).
•
Examine production of inflammatory mediators by, and activation-related receptors
on, monocytes and NK cells in cord blood obtained after healthy or preeclamptic
pregnancies (Paper II).
SUBJECTS AND METHODS
Subject demographics and methods are found in detail in the corresponding papers. In this
section there will however be a brief description of the experimental procedure. Peripheral
venous blood was collected from healthy adult volunteers, mothers or from the umbilical cord
right after delivery. Serum was separated and mononuclear cells were isolated from blood by
Ficoll-Paque gradient centrifugation. Cells and serum were frozen until simultaneous analysis
of adult/neonatal or healthy/preeclamptic groups. Upon analysis, cytokine levels in serum
were assessed by cytometric bead array (CBA). Cells were cultured in medium or were
stimulated with the bacterial ligand PGN or PGN+IL-15. Expression of surface markers,
MAPK-phosphorylation, intracellular and secreted cytokines were assessed by flow
cytometry, ELISA and/or CBA. Also, experiments were carried out substituting fetal calf
serum (FSC) in culture medium for neonatal/adult or healthy/preeclamptic serum prior to
phenotypic or functional assessment.
26
RESULTS AND DISCUSSION
Paper I
Immunological responses at birth are under intense investigation to shed light on neonatal
infection susceptibility. Besides suboptimal adaptive immunity,173 the reduced ability to cope
with infectious challenge appears to be associated with qualitative differences in neonatal
innate immune responses that seem to preferentially favor Th2- and Th17- over Th1-type of
immunity.116-118 Monocytes can be divided into two major subpopulations, CD14++CD16- and
CD14+CD16+ cells, which are suggested to play different roles in antimicrobial responses.
Among other characteristics, CD14+CD16+ cells have a higher expression of TLR2 and TLR4
and secrete more inflammatory cytokines upon stimulation with certain TLR ligands,58,59 than
CD14++CD16- cells. The monocyte subsets have not previously been investigated in
newborns, and we hypothesized that the characteristics of the neonatal monocyte
compartment might differ from the adult. Altered ratios of the monocyte subsets, and/or
behavioral diversity of these cells, could contribute to the qualitative difference between the
neonatal and adult cytokine profile.
Firstly, we found that the monocyte subsets in CB and adults were present in similar
frequencies, with CD14++CD16- representing approximately 93% and CD14+CD16+ cells 7%
of the total monocyte population. Secondly, in both groups high expression of CD11c,
CD80/CD86 and CD163 characterized the CD14+CD16+ cells, and in adults also high
expression of HLA-DR. There were no differences in the levels of expression of any of the
markers between CB and adult monocytes indicating an overall similar basal phenotype.
Innate immune responses in the newborn are not generally impaired, as CBMC are capable of
producing sizeable amounts of selected cytokines when stimulated with a variety of TLR
agonists.116,118-120 In this study a limited amount of CBMC necessitated the choice of one
stimulus for functional studies. We chose the bacterial ligand PGN that is a common target for
immune recognition, since it is an essential part of the cell wall of both Gram positive and
Gram negative bacteria.12 PGN is recognized by the PRRs TLR2 and NOD1/NOD2 that are
highly expressed by monocytes.56,57 When CBMC were stimulated with PGN we noted
secretion of equal (IL-1β, IL-6, IL-8, IL-10) or even greater (TNF) cytokine amounts
compared with adult levels. Also intracellular CB monocyte TNF levels exceeded adult levels
along with higher intracellular IL-12p70. With regard to the monocyte subsets, the
CD14+CD16+ cells expressed more IL-12p70, but not more TNF, than CD14++CD16- cells. In
a previous study we provided indirect evidence for increased IL-12 from CD14+CD16+ cells
27
as they induced higher NK-cell IFN-γ production than CD14++CD16- cells,96 and here we
show it directly in both CB and adults. The CD14+CD16+ have previously been described as
the major TNF producers, however this was not the case in our study. The TNF response may
be stimulus-specific or data could be confounded by the fact that the CD14+CD16+ cells can
be further subdivided into CD14highCD16+ and CD14dimCD16+ cells, where the latter produce
lower amounts of TLR2-induced TNF than CD14highCD16+ cells.60
After initial recognition of PGN, a cascade of intracellular events culminate in the production
of cytokines and differences in regulation and/or activation level of mediators in this pathway
could have an impact on the level of cytokine production. PGN-induced signal transduction is
well known to activate the three major families of MAPKs.174 As TLR expression on CB
monocytes is at an adult level,109,110 we searched for an intracellular explanation for elevated
TNF and IL-12p70 production from CB monocytes by investigating activation of two MAPK,
ERK1/2 and p38-MAPK. ERK1/2 was activated by PGN but we saw no difference in
phosphorylation between CB and adults. However, CB monocytes had a higher
phosphorylation of p38-MAPK that correlated with levels of intracellular IL-12p70
production. Even though both MAPK are activated following PGN recognition, they appear to
differentially regulate APC maturation pathways. Inhibition of p38-MAPK seems to confer
the most pronounced effects on cytokine release by APCs and thus in development of Th1
responses,21,175 whereas inhibition of ERK1/2 seems to have only partial or negative effects
on DC maturation.176,177
Cytokine responses of neonatal cells to different innate and adaptive ligands have been
extensively studied. Although some common patterns have emerged, results are often
inconsistent between studies using whole blood or MCs as the source of cells. A contributing
factor could be that levels of adenosine are reported to be high in neonatal serum. Adenosine
is believed to preferentially down-regulate certain cytokines and neonatal cells are described
as more sensitive to adenosine-mediated cytokine suppression than adult cells.121 Therefore
one explanation for the divergent findings on neonatal cytokine production could lie in the
distinction between the inherent capacities of neonatal cells as opposed to the influence of
external factors. In our study we tried to separate the two and firstly showed that the inherent
capacity of CBMC for production of many inflammatory cytokines was not impaired.
Secondly, we added CB and adult sera to both CBMC and PBMC to investigate whether this
would influence cytokine production. In comparison to FCS, both types of human sera
conferred changes in cytokine production, with an increase in production of IL-6 and IL-8 but
a decrease in TNF and IL-10. Overall it did not matter if the cells came from CB or adults for
28
the observed effects of serum substitution. Only TNF production from PBMC was
differentially regulated by factors in CB and adult serum, as CB serum was far more
suppressive. This was not observed with CBMC indicating that adult cells were more
sensitive to these effects in contrast to previous observations.121 That TNF production in
particular is down-regulated in monocytes, might be due to adenosine-induced accumulation
of cAMP that preferentially targets TNF through posttranscriptional mechanisms.178 The
benefit of in utero suppression of neonatal and/or maternal TNF responses is perhaps easy to
conceive given the connection of TNF to spontaneous abortion and miscarriage.179 In
connection to this, TNF blockade has been suggested as a promising treatment for patients
with immune-mediated fertility problems.180
As newborns have a heightened susceptibility to infections and a poor ability to mount
protective vaccine responses, immune maturation is of great importance to study. Newborn
immunity seems unable to support Th1-type of responses which has been related to impaired
synthesis of e.g. IL-12.106-108 However results regarding IL-12, and other pro-inflammatory
cytokines, from our study and others suggests no impairment in neonatal production.116,118-120
If CB monocyte activation of p38-MAPK and production of IL-12p70 is not deficient but in
fact higher than in adult cells, then why should neonatal innate immunity be unable to
generate successful Th1 responses? The answer might be found within the neonatal DC
compartment, as DCs are the primary cells that activate T lymphocytes. Early experiments
showed that CB DCs were poor stimulators of the mixed lymphocyte reaction whereas CB T
cells responded normally to adult allogeneic DCs.181 It was subsequently shown that CB DCs
have lower levels of co-stimulatory CD80/CD86 and MHC II181 and incomplete maturation in
response to stimulation with single TLR ligands as compared to adult cells.182 Although DC
capacity for IL-12 synthesis is reduced,106-108 it was shown that this impairment was not an
inherent property of CB monocyte-derived DCs, as they synthesized adult-level IL-12 when
matured with GM-CSF and IL-4.106 This might implicate that precursors of neonatal DCs may
lack the appropriate maturation signals to allow for proper differentiation. Collectively, one
might speculate that the neonatal deficiency in infection control stems mainly from
suboptimal interactions between DCs and (also immature) T cells, which leaves the newborn
vulnerable to sustained infections. The monocyte response per se could on the other hand be
effective to certain strong immune stimuli such as PGN, and contribute to initial protection.
Also, PGN-stimulated CB monocytes are able to induce CB NK-cell IFN-γ (Paper II),
indicating that this important monocyte-NK cell collaboration is also functional at birth.
29
Some limitations in the study design should also be discussed here. Although we conclude
that inherent CB monocyte subset responses to the bacterial ligand PGN are potent and not
suppressed, other innate ligands such as viruses, may elicit a different response from neonatal
monocyte subsets, which remains to be defined. Further, we assessed cytokine production
after 24 hrs to ensure maximal production, particularly with IL-12p70 in mind. However,
TNF is one of the first cytokines to appear, peaking around 4-6 hrs, and its production then
declines.110 Any differences in kinetics of CB and adult TNF production are not visible when
only assessing one time point, and thus adult levels could be higher earlier on as suggested by
previous studies.110 Nonetheless, the intracellular cytokine assay shows accumulated levels
over 24 hrs and therefore we clearly show that when stimulated with PGN, the CB monocyte
TNF response is not deficient.
In this study we provide a comprehensive analysis of monocyte subsets in the human
newborn – a field where very little is known. These findings suggest that at birth, monocytes
have a high inherent cytokine production capacity when challenged with a common bacterial
ligand. However their TNF and IL-10 responses appear to be suppressed by factors in serum.
Interestingly, we found that the percentage of the CD14+CD16+ monocyte subset after PGN
stimulation was higher in CB than in adult cultures, suggesting that they may fail to undergo
further differentiation into macrophage or DC effector cells. Previous studies of monocyte
subpopulations are mainly from mice, where the subsets do not directly correspond to human,
or from adults, and therefore these data may give insights for future understanding of neonatal
monocyte responses at birth.
30
Paper II
During pregnancy the maternal immune system goes through complex adaptations with the
establishment of the hemochorial placenta and tolerance generation to a semi-allogeneic fetus.
In preeclampsia, a major pregnancy-related disorder, immune maladaptation at the
implantation site leads to a poorly developed placenta unable to sustain the needs of the
growing fetus.157 Pregnancy in itself is characterized by a mild inflammatory state with a
systemic activation of innate immune components, and in preeclampsia this response is
excessive.137,183 A relation between maternal immune status and neonatal immunity has been
suggested, but the impact of a preeclamptic pregnancy on the developing neonatal immune
system has not been well studied. Our hypothesis was that the increased inflammation and
exaggerated activation of immune cells at the fetal-maternal interfaces in preeclampsia might
affect activation status of neonatal innate cells.
We started by confirming that preeclampsia induces a maternal inflammatory serum cytokine
profile, by finding elevated systemic IL-1β, IL-6, IL-8 and IFN-γ levels. Depending on the
two major features used for clinical diagnosis, blood pressure and the amount of protein in the
urine,152,153 preeclampsia can further be divided into mild and severe subgroups. The two
subgroups of mothers showed different cytokine profiles, where IL-8 was higher in the severe
group and IL-1β in the mild. For CB there were higher soluble (s)CD163 levels in the group
with mild preeclamptic mothers whereas IL-8 and IL-10 levels were elevated in the severe
group. Inflammation and hypoxia both drive IL-8 production, and the increased levels in CB
serum could originate from epithelial and/or endothelial (placental) cells or CB leukocytes.
Increased IL-10 levels might indicate a regulatory response to inflammation in these neonates.
This theory was supported by the fact that the highest individual CB serum levels of IL-8 and
IL-10 coincided. Furthermore, there was a correlation between levels of IL-6 and IL-10
between healthy and mild preeclamptic mothers and respective CB group, something that
could suggest similar responses to an unknown pregnancy-related factor or possibly selective
transfer over placental surfaces akin to transport of IgG antibodies.139,140 If so, either of these
mechanisms could be disrupted by the exaggerated abnormalities in placental morphology
observed in severe preeclamptic pregnancies, however this remains a speculation.
As monocytes and NK cells are activated in the preeclamptic mother,161,165,166 we more
closely examined these cells in the newborn. The CB monocyte compartment seemed
generally unaffected by preeclampsia, and there were similar expression levels of CD11c,
CD80/CD86, HLA-DR and CD163 on monocytes in the two CB groups. The expansion of
31
CD14+CD16+ cells is associated with ongoing inflammation65 and in the preeclamptic CB
group CD14+CD16+ cells were slightly increased although the difference was not significant.
We also assessed production of IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF, but spontaneous
and PGN-stimulated secretion from MCs (all cytokines) and intracellular monocyte
production (IL-12p70 and TNF) was similar for the two CB groups.
The major finding of this study was the observed alterations in the CB NK-cell compartment.
Firstly we examined surface levels of the constitutively expressed NK-cell activation
receptors NKG2D and NKp30. Their levels are elevated on CB NK cells,82 indicating that
they could be important for NK-cell activation early in life. In the mild preeclamptic group we
observed lower NKG2D and higher NKp30 expression. In terms of factors that could affect
these receptors, TGF-β and IFN-γ can down-regulate NKG2D,184,185 and NKp30 can be upregulated by IL-15.186 Levels of IL-15 and TGF-β were not measured directly in serum, but
there were no differences in IFN-γ between the mild preeclamptic CB group and the severe or
healthy group. Interestingly, both NKG2D ligands (MICA/MICB and ULBP) are expressed in
placenta and released on placental exosomes.131 These exosomes are mainly secreted towards
the maternal side of the placenta but are also observed on the fetal side (Lucia MinchevaNilsson, personal communication). In vitro experiments showed that they exerted
immunosuppressive functions by down-regulating NK-cell NKG2D expression and
subsequent NK-cell cytotoxicity.131 Furthermore, an increase in shedding of microparticles
from the preeclamptic placenta has been reported,161,187 together suggesting that an eventual
increase in microparticle burden in CB serum (and/or alterations in cytokine levels), may
provide a link to altered preeclamptic CB NK-cell receptor expression. To test this hypothesis
we incubated healthy CBMC with medium containing CB sera from the healthy, mild or
severe preeclamptic group, and subsequently measured expression of NKG2D and NKp30.
However, levels of the two receptors were similar in all three settings suggesting that there
were no differences in serum factors that could influence NK-cell receptors between the three
CB groups. One might therefore speculate that the observed changes in NK-cell receptor
expression may persist after birth and the immediate effect of the in utero environment, thus
potentially affecting the delicate regulation of NK-cell activation in response to NKG2D and
NKp30 ligands in these neonates.
Secondly, we examined cytokine production and found that unstimulated CB NK cells from
the mild preeclamptic group expressed more intracellular TNF and IFN-γ than CB NK cells
from the healthy group. That both receptor expression and pre-activation of CB NK cells was
32
seen primarily in the mild preeclamptic group might be a result from common factors acting
on these cells. Monocyte-induced NK-cell production of TNF and IFN-γ was not influenced
by preeclampsia, however this does not rule out that cytokine production and/or cytotoxicity
induced via NKG2D or NKp30 by their cognate ligands might be altered. This and other
aspects of the functional relevance of altered CB NK-cell characteristics remain to be
investigated.
In preeclamptic mothers both the monocyte and the NK-cell compartment are activated,
however in CB only NK-cell activation was observed. The factors that are proposed to drive
inflammatory changes in the mother, like systemic oxidative stress, growth factors, leptin and
syncytiotrophoblast debris,188 are not likely to be as prominently present in the circulation of
her developing fetus. Since monocytes and NK cells are activated in very different ways, one
could envision that their triggering factors might not be uniformly present in preeclamptic
mothers and CB alike. One could also speculate that NK cells might be more sensitive to
inflammation-induced changes or reacting to an up-regulation of stress ligands e.g.
MICA/MICB and ULBP in the preeclamptic placenta, although this has not been
substantiated.
One interesting finding from this study was the pronounced difference between the mild and
severe subgroups of preeclampsia, both for mothers and newborns, something that could
relate to potential differences in management of these patients. To our knowledge however
there were no major differences in administration of medications for mild or severe
preeclamptic patients. Cortisol is given to women who develop early onset preeclampsia in
order to promote fetal lung development. As cortisol is an immunosuppressive drug it could
have effects on the immune activation status of mothers and possibly also of their neonates. In
our study population only 3 women were characterized as early preeclampsia, and the mothers
did not significantly differ from the other preeclamptic cases in our data set. CB was obtained
from only one of these deliveries but showed no difference to other preeclamptic CB in our
assays. The pronounced difference between the subgroups that we observed here should
warrant future studies to observe this sub-grouping when attempting to elucidate how
preeclampsia affects immune parameters both in infants and mothers.
In summary the findings from this study show that an in utero inflammatory priming of
neonatal innate immunity takes place in preeclamptic pregnancies, as shown by altered
systemic and NK-cell specific cytokine levels and altered NK-cell activation receptor
expression. The fetal origin of adult diseases hypothesis states that the health and disease of
an individual is influenced by intrauterine life, and indeed children born after preeclamptic
33
pregnancies have an increased incidence of various diseases in childhood. To elucidate
potential functional consequences of changes in the NK-cell compartment, or whether
preeclampsia-associated inflammatory activation is observed into childhood, would require
more studies and a follow-up of the same children.
GENERAL CONCLUSIONS
Although the newborn immune response is deficient in many aspects and shows an altered
cytokine profile compared to adults after stimulation with various TLR ligands, the CB
monocyte response to PGN, a cell-wall component present in virtually all bacteria, is potent,
in particular with regards to TNF and IL-12p70 production. Moreover, cytokine production in
CB and adult cells is under the regulation of serum factors that favor certain cytokine
responses over others. Monocyte capacity for production of early-response cytokines does not
seem to be altered in newborns of preeclamptic mothers despite the increased inflammatory
propensity of their mothers. However, CB NK cells show altered phenotype and increased
spontaneous cytokine production after preeclamptic pregnancies. Further, preeclampsia was
associated with an inflammatory serum cytokine profile in both mothers and newborns.
Therefore a pathological pregnancy may skew maturation of neonatal immunity and should be
considered as a factor influencing innate responses and/or immune cell activation during the
earliest years of life.
34
FUTURE PERSPECTIVES
PREECLAMPSIA
With respect to the findings from Paper II, we will continue to investigate CB NK-cell
functionality in terms of cytotoxicity and cytokine production induced through NKG2D
and/or NKp30 ligation in vitro.
Investigations of the same parameters in children later in the neonatal/infant period should
also be carried out to further clarify the relevance of the alterations in NK-cell characteristics
at birth. A follow-up of a cohort of children born to preeclamptic mothers would also enable
examination of whether the inflammatory serum milieu induced by preeclampsia persists.
However, at present we are unable to collect more samples or enroll patients for long-time
studies. Therefore future work will focus on monocytes and monocyte-NK cell functions and
collaboration, particularly in relation to viral exposures.
NEONATAL IMMUNITY AND VIRAL INFECTIONS
In paper I and II we investigated CB antibacterial responses with focus on monocytes and NK
cells. However newborns are also subjected to viral infections early in life, towards which NK
cells are important effector cells. Members of the herpesvirus family are widely spread
viruses in human populations. Primary infection with Epstein-Barr virus (EBV) and
cytomegalovirus (CMV) often occur during early childhood and the viruses then persist in a
latent form. In a previous study of a cohort of 2-year old children we examined the relation
between herpesvirus seropositivity and the quality of innate responses.96 At this age only half
of the children had acquired EBV and/or CMV infection although all should most likely have
been exposed. Further, the innate response was markedly different, with seropositive children
showing lower NK-cell IFN-γ production as well as lower serum IFN-γ levels. Also adaptive
responses were modulated by herpesvirus infection, as PBMC from 2-year-old EBV
seropositive children tended to have an overall increased cytokine production after PHA
stimulation.189 In connection to this, we have found that early acquisition of EBV infection
protects against persistent IgE sensitization, suggesting that early-life microbial exposure
could contribute to an allergy protective immune profile.190
Possibly an initial difference exists between individuals in immune response factors which
could contribute to viral susceptibility, thus explaining why not all exposed children were
35
seropositive. Also, herpesvirus infection might modulate the immune system during early life,
thus explaining the differences in immune responses we observed in 2-year old children. To
elucidate this we plan to infect CBMC, and possibly cells from children later in childhood,
with EBV and determine differences in antiviral responses, both innate and adaptive. Further,
we will relate serostatus to immune function at different ages in children.
36
ACKNOWLEDGEMENTS
To Eva Sverremark-Ekström for excellent supervision and for allowing me to work freely yet
never stray.
To my co-authors and collaborators Shanie Saghafian-Hedengren, Nora Bachmayer, Rangeen
Rafik Hamad and Katarina Bremme.
To all midwifes and nurses, especially to Mari Jonsson, at the delivery ward and Agneta
Nordwall at the Department of Cardiology, both at the Karolinska University Hospital, Solna
for their kind assistance with sample collection.
To the seniors scientists, my co-supervisor Marita Troye-Blomberg, Carmen Fernandez, Eva
Severinson and Klavs Berzins. Maggan, Gelana and Students at the department for being
friendly and helpful.
To my roommates and friends, Maria Johansson and Shanie Saghafian-Hedengren, for neverending support.
Lastly to my late father who inspired me to go into the field of research.
37
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