Download Licentiate thesis from the Department of Immunology,

Licentiate thesis from the Department of Immunology,
Wenner-Gren Institute, Stockholm University, Sweden
Inflammatory responses due to
Plasmodium falciparum infection during
pregnancy and childhood
Stéphanie Boström
Stockholm 2012
SUMMARY
In areas were malaria is endemic, pregnant women and children are the groups most at risk
of developing severe and life-threatening disease. Malaria during pregnancy may have
detrimental consequences both for the mothers and their offspring as well as affecting infants
during their first years of life.
Cytokines and chemokines are major mediators during an immune response and are crucial
in the defense against pathogens. They regulate immune-cell development, direct cells to sites
of infection and in general modulate the immune system. The balance between pro and antiinflammatory cytokines is crucial in determining the outcome of an infection. Therefore, the
aim of these studies was to evaluate inflammatory responses in pregnant women and children
infected with Plasmodium falciparum. To do this, two separate cohort studies were employed:
1) A longitudinal, prospective study conducted in Tanzania where pregnant women were
enrolled and followed up during pregnancy, 2) A case-control study carried out in two
sympatric ethnic groups, the Dogon and the Fulani, in Mali. The Fulani have been shown to
be less susceptible to malaria as compared to other sympatric ethnic groups.
A panel of selected cytokines, chemokines and antibodies was analyzed in plasma from the
participating pregnant women and children to evaluate the impact of Plasmodium infection.
Our results show that upon infection with P. falciparum, the cytokine and chemokine levels in
the pregnant women are changed. The anti-inflammatory factor IL-10 and the inflammatory
factor IP-10 were found to be increased at all time points when the pregnant women had an
infection and might therefore be important biomarkers for inflammation during pregnancy.
Fulani children exhibited higher inflammatory cytokine levels and humoral immune
responses towards the parasites as compared to Dogon children. When the children were
further subdivided into infected and uninfected individuals, the infected Dogon children
showed increased levels of MCP-1, MIG and IP-10 but decreased levels of RANTES
compared to their uninfected counterparts. In contrast, there were no differences in the levels
between infected and uninfected Fulani. This suggests that the Fulani have a higher
inflammatory response which potentially could protect them against P. falciparum infections
already at an early age. Taken together, our results demonstrate that infections with P.
falciparum induce both inflammatory and anti-inflammatory factors and suggest that the
balance between these factors is important for the outcome with respect to disease severity.
2
LIST OF PAPERS
This thesis is based on the original papers listed below, which will be referred to by their
roman numerals in text:
I.
Stéphanie Boström, Samad Ibitokou, Mayke Oesterholt, Christentze Schmiegelow,
Jan-Olov Persson, Daniel Minja, John Lusingu, Martha Lemnge, Nadine Fievet,
Phillipe Deloron, Adrian JF Luty and Marita Troye-Blomberg. Biomarkers of
Plasmodium falciparum infection during pregnancy in women living in
northeastern Tanzania. Manuscript in preparation.
II.
Stéphanie Boström, Pablo Giusti*, Charles Arama*, Victor Dara, Boubacar Traore,
Amagana Dolo, Ogobara Doumbo, Marita Troye-Blomberg. Changes in the levels
of cytokines, chemokines and malaria-specific antibodies in response to
Plasmodium falciparum infection in sympatric ethnic children living in Mali.
Submitted.
*
These authors contributed equally to this work.
3
TABLE OF CONTENTS
SUMMARY............................................................................................................................................ 2
LIST OF PAPERS ................................................................................................................................. 3
TABLE OF CONTENTS ...................................................................................................................... 4
ABBREVIATIONS ............................................................................................................................... 5
THE IMMUNE SYSTEM..................................................................................................................... 6
INNATE IMMUNITY ........................................................................................................................ 6
ADAPTIVE IMMUNITY ................................................................................................................... 8
THE INFLAMMATORY RESPONSE ............................................................................................. 11
CYTOKINES ....................................................................................................................................... 12
PRO-INFLAMMATORY CYTOKINES .......................................................................................... 13
ANTI-INFLAMMATORY CYTOKINES ........................................................................................ 15
CHEMOKINES ................................................................................................................................. 17
PREGNANCY AND THE IMMUNE SYSTEM .............................................................................. 19
IMMUNE TOLERANCE.................................................................................................................. 19
IMMUNE ACTIVATION DURING PREGNANCY ....................................................................... 20
THE PLACENTA ............................................................................................................................. 21
MALARIA ........................................................................................................................................... 23
MALARIA IS A MAJOR HEALTH PROBLEM............................................................................. 23
THE LIFE CYCLE OF THE MALARIA PARASITE ..................................................................... 23
CLINICAL MANIFESTATIONS OF MALARIA ........................................................................... 24
INNATE AND ADAPTIVE IMMUNE RESPONSES IN MALARIA ............................................ 25
PROJECT BACKGROUND .............................................................................................................. 29
MALARIA DURING PREGNANCY .............................................................................................. 29
MALARIA AND ETHNIC GROUPS IN WEST AFRICA .............................................................. 30
PRESENT STUDY .............................................................................................................................. 32
OBJECTIVES ................................................................................................................................... 32
SUBJECTS AND METHODS .......................................................................................................... 33
RESULTS AND DISCUSSION ....................................................................................................... 34
Paper I ........................................................................................................................................... 34
Paper II.......................................................................................................................................... 37
GENERAL CONCLUSIONS ............................................................................................................. 39
FUTURE PERSPECTIVES ............................................................................................................... 40
ACKNOWLEDGEMENTS ................................................................................................................ 41
REFERENCES .................................................................................................................................... 42
4
ABBREVIATIONS
ADCC
APC
CSA
DAMP
DC
FoxP3
ICAM
IDO
iE
IFN
Ig
IL
IP
IVS
LAP
LBW
LPS
MCP
MHC
MIG
MIP
MS
NF-kβ
NK
NO
PAMP
P. falciparum
PfEMP1
PM
PRR
RA
RANTES
ROS
SOCS
STAT
Tc
TGF
Th
TLR
TNF
Treg
uNK
uPAR
VEGF/Flt1
antibody-dependent cellular cytotoxicity
antigen-presenting cell
chondroitin sulphate A
danger-associated molecular pattern
dendritic cell
transcription factor forkhead box P3
inter-cellular adhesion molecules
indoleamine 2,3-dioxygenase
infected erythrocyte
interferon
immunoglobulin
interleukin
inducible protein
intervillous space
latency-associated protein
low birth weight
lipopolysacharide
monocyte chemotactic protein
major histocompatibility complex
monokine induced by IFN-γ
macrophage inflammatory protein
multiple sclerosis
nuclear factor-kβ
natural killer
nitric oxide
pathogen-associated molecular pattern
Plasmodium falciparum
P. falciparum erythrocyte membrane protein 1
placental malaria
pattern recognition receptor
rheumatoid arthritis
regulated upon activation normal T cell expressed and secreted
reactive oxygen species
suppressor of cytokine signaling
signal transducer and activator of transcription
T cytotoxic cell
transforming growth factor
T helper
toll-like receptor
tumor necrosis factor
regulatory T cell
uterine natural killer
urokinase receptor
vascular endothelial receptor
5
THE IMMUNE SYSTEM
The immune system is crucial for our defense against invading microorganisms and relies
on a complex network of organs, cells and molecules responsible for maintaining the body’s
homeostasis and hindering pathogens from infecting us. It is capable of recognizing self from
non-self and distinguishing what is dangerous and what is not, thus keeping us safe in a
surrounding constantly exposed to environmental microbes. We are equipped with many
effective means for blocking potential pathogens from entering our body. The skin and the
mucus membranes act as physical barriers while lytic enzymes and low pH in the stomach
work as chemical barriers to reduce pathogenic invasion. Mucosal surfaces further extend the
protection against invading pathogens by secretion of antimicrobial peptides. Besides these
physical and chemical barriers, the immune system is equipped with immune cells that
conduct surveillance for pathogens that do manage to break through. These immune cells are
transported in the body, via the blood and lymphatic vessels, into specialized immune
compartments such as the spleen, thymus, and lymph nodes where they expand and multiply
for an efficient immune response. There are two branches of the immune system, the innate
and the adaptive arms, which together act to disarm pathogens but that have major differences
in terms of their specificity, their speed of action and the generation of memory.
INNATE IMMUNITY
The innate immune system ensures rapid limitation of microbial invasion through
phagocytosis, release of inflammatory mediators, activation of the complement system as well
as synthesis of acute phase proteins, cytokines and chemokines. These processes are activated
by specific stimuli, represented by structures that are unique to microorganisms, such as
lipopolysacharides (LPS), mannose and teichoic acids that constitute pathogen-associated
molecular patterns (PAMPs) which are conserved among microorganisms and are commonly
found on the surface of pathogens. The innate immune system recognizes these structures
through pattern recognition receptors (PRRs) among which the toll-like receptor family
(TLRs) are the most studied. Up until today, 10 different TLRs have been identified in
humans.1 Some are located in the cell membrane and others inside the cell, which enables an
effective way of detecting both intra and extra-cellular pathogens. TLR1, TLR2, TLR4, TLR5
and TLR6 are found on the cell surface and recognize structures that are representative for
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both gram positive and gram negative bacteria such as peptidoglycan, LPS and flagellin2.
TLR3, TLR7, TLR8 and TLR9 are located on the endolysosome and recognize viral DNA
and RNA in the cytoplasm.2 Other PRRs are the C-type lectin receptors, nucleotide
oligomerization domain-like receptors and retinoic acid inducible gene-like receptors.
Activation of the PRRs by their ligands initiates signaling cascades in the cell that ultimately
lead to activation of nuclear factor-kβ (NF-kβ) pathways and consequently to the induction of
pro-inflammatory cytokines and increased antimicrobial activities. It has also been proposed
that activation of innate immune system is not only based on the recognition of PAMPs but
also relies on the presence of danger signals or danger-associated molecular patterns
(DAMPs) released by injured cells, for example the release of uric acid.3
The main effector cells in the innate immune system are macrophages, neutrophils,
dendritic cells (DCs) and natural killer (NK) cells. Neutrophils are professional phagocytes
that usually are the first responders to migrate towards the site of inflammation or bacterial
stimuli. Once at the inflammatory site, they start to phagocytose the pathogen and also
enhance their production of reactive oxygen species (ROS) that efficiently kills the pathogen.
NK cells are important in our nonspecific defense and have cytotoxic activity against virusinfected cells or tumor cells. They recruit neutrophils and macrophages and activate DCs, T
and B cells by secreting a variety of potent cytokines. Monocytes circulate in the blood before
they enter tissues where they differentiate into macrophages and DCs. Two major monocyte
subpopulations have been described,4 the classical (CD14++CD16-) and the inflammatory
(CD14+CD16+) subset that differ in terms of effector functions and trafficking potential.5-7
Monocytes and macrophages are efficient phagocytes that engulf microbes and cellular
debris. They are also potent cytokine producers upon bacterial stimuli.
During inflammation, antigen-presenting cells (APCs) that are specialized in the uptake,
processing and presentation of antigens to cells of the adaptive immune system. DCs are the
most specialized APC, capturing and presenting antigens to naïve lymphocytes, and they are
therefore considered to be a bridge between the innate and the adaptive immune system. DCs
are found in peripheral tissue such as the skin, liver and intestine where they capture antigens,
become activated and subsequently migrate to the lymph nodes. Inside the lymph node, they
present antigens to T cells thereby initiating an immune response. The innate immune system
is clearly important in the fight against pathogenic organisms but it has its limitations. It
works rapidly and is immediately initiated whenever sensing a threat, but it is on the expense
of being non-specific and results in no memory. For this reason the innate immune system
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may be sufficient to clear or control an infection, until the adaptive arm of the immune system
takes over to ensure complete elimination of the threat.
ADAPTIVE IMMUNITY
Unlike the innate immune system, the adaptive immune system confers a high degree of
specificity and diversity and is able to remember captured pathogens by the production of
antigen-specific memory cells. The innate arm is said to activate the adaptive immune system
and therefore the initial quality of the innate immune response may direct the proceeding
activation of the adaptive immune responses.8 Traditionally, adaptive immunity is divided
into two different branches; 1) the cell-mediated response, in which the effector cells are
antigen-specific T cells, and 2) the humoral response, which is made up by antibodies
produced by antigen-specific B cells. Antigen recognition occurs through their surface
receptors, which display a high degree of diversity and specificity due to germ line gene
rearrangement. T cells recognize processed antigens when they are presented in association
with the major histocompatibility complex (MHC) I or II on the surface of APCs. When a
specific T cell recognizes the antigen presented by an APC, such as the B cell, the T cell
provides additional help to the B cell, whereafter both the T and the B cells expand and
generate highly specific effector cells and long lasting memory cells.
T cells are essential in the development of the cell-mediated immunity. They originate
from the bone marrow but fully mature in the thymus into two major groups; the T helper
(Th) CD4+ and the T cytotoxic (Tc) CD8+ cells. In general, Tc are important for the clearance
of virus-infected and tumor cells, while the Th cells mainly regulate other cells of the immune
system by secreting cytokines. The Th cells can be further differentiated into Th1 and Th2
cells depending on their cytokine expression profiles. Th1 cells produce pro-inflammatory
cytokines such as tumor necrosis factor (TNF), interferon (IFN)-γ, interleukin (IL)-1 and IL-2.
These cytokines activate Tc cells and macrophages to stimulate cell-mediated immunity and
inflammation and are characterized by the transcription factor T-bet and STAT4. On the other
hand, Th2 cells mainly produce IL-4, IL-5 and IL-13 which are important in regulating B-cell
maturation and subsequent production of antibodies. Th2 cells have a major function in
clearing helminthic infections and they are characterized by transcription factor GATA-3 and
STAT6. A positive feedback loop exists in which the cytokines produced by one subgroup of
the Th cells are inhibiting the differentiation and cytokine secretion of the other subgroup and
8
vice versa. However, recently the Th1/Th2 paradigm had to be revised with the discovery of a
third subset of effector T cells, named Th17 cells which selectively produce IL-17.9, 10 The
Th17 cells are characterized by the transcription factor ROR-α. This population has shown to
play an important role in clearing extracellular pathogens, induce tissue inflammation and has
also been implicated in the pathogenesis of certain diseases, such as rheumatoid arthritis
(RA), psoriasis, multiple sclerosis (MS), Crohn’s disease and allergic asthmas.11, 12 Therefore
the Th1/Th2 concept is a bit simplistic and has nowadays been expanded into a broader
Th1/Th2/Th17 cell paradigm.13
All the functions of the effector T cells are regulated by the CD4+ regulatory T (Treg) cells.
The Treg cells are an important group of T cells, which are involved in the induction and
maintenance of tolerance towards self by suppressing self reactive T cells.
14
They reside in
the thymus as naïve T cells but when they go to the periphery and become activated they
acquire a memory phenotype.15, 16 There are several types of Treg cells distinguished by their
different expression of cell surface molecules and cytokine production. The natural occurring
Treg cells reside in the thymus and are CD4+CD25+ and express high levels of the
transcription forkhead box P3 (FoxP3) and are potent producers of IL-10 and transforming
growth factor (TGF-β). Treg cells can also be induced from peripheral T cells. The induced
Tr1 and Th3 cells are characterized by secretion of IL-10 and TGF-β, respectively.
B cells are key cells in the humoral immune response. They are produced in the bone
marrow as immature B cells that become fully matured in the spleen and one of their
important tasks in the immune system is to generate immunoglobulins (Ig). When a naïve B
cell recognizes a pathogen through the B-cell receptor it engulfs it and internalizes it and
processes it to finally present it in association with the MHC II molecule on its surface. When
this occurs, a T cell can recognize the antigen and bind to the B cell and together with
additional co-stimulatory molecules the B cell becomes activated. Upon activation of a naïve
B cell, clonal expansion occurs which results in the production of antibody-secreting plasma
cells and memory-B cells. The B cell receptors may undergo somatic hypermutations (point
mutations) in the germinal centers, which increases its affinity and specificity for an antigen,
thereby making it more and more prone to capture the antigen it previously has encountered.
Importantly, each B cell can only produce one specific antibody.
The Ig molecules can be membrane bound or secreted and the two forms have different
functions. As a membrane form, the Ig function as a receptor, while the secreted form mainly
9
works as a neutralizing agent that blocks foreign pathogens or activates other mediators. After
activation, the B cells may also undergo class switching in which the Ig production is shifted
from one antibody class to another one that further directs the immune response. There are
five different isotypes: IgM, IgD, IgG, IgA and IgE. The antibodies are effective in clearing
pathogens and their effector functions are mainly to promote opzonisation (by binding to the
surface antigens on the pathogen) to activate the complement system (which is part of the
innate immune system and consists of a complex pathway of proteins and molecules that
interact to mediate direct lysis of cells) or to mediate antibody-dependent cellular cytotoxicity
(ADCC).
IgM is the first antibody that encounters an antigen and is a potent activator of the
complement system. After binding to the pathogen, IgM with or without complement
promotes clearance of the pathogen by activating phagocytic cells, by limiting pathogen
dissemination throughout the host as well as by enhancing the presentation of pathogenderived antigens to the adaptive immune system.17
IgG is one of the key antibody isotypes involved in the humoral immune response. It
represents almost 70% of all isotypes in the blood and is like IgM, a potent activator of the
complement system. IgG is the only antibody that can pass extensively across the placental
barrier to provide passive immunity from a mother to her fetus.18 In humans, IgG can be
divided into 4 different subclasses IgG1-IgG4. IgG1 has the highest prevalence in blood
followed by IgG2, IgG3 and IgG4.19 IgG1 and IgG3 are generally considered to be cytophilic,
while IgG2 and IgG4 are not.20 In terms of activating complement, IgG3 and IgG1 are the
most effective once, while IgG2 is a weak activator whereas IgG4 does not seem to be able to
activate it at all. In humans, a Th1 response induces IgG1 and IgG3 subclasses, whereas a Th2
type of response mediates IgE and IgG4 production.
IgD is co-expressed together with IgM on most B cells and is in general found in very low
levels in blood (less than 1%) and has a very weak capacity to activate complement.21
IgA is a key antibody involved in mucosal immunity.22 This antibody class works together
with nonspecific protective factors, such as the mucus to block microbial adhesion to
epithelial cells without causing a tissue-damaging inflammatory reaction.
IgE is the subtype mostly known for its involvement in allergic reactions and protection
against helmints.23
10
THE INFLAMMATORY RESPONSE
Inflammation is a crucial component of the body’s immune system. It is part of the first
line of defense that takes place when an infectious agent comes into our body or when we
have a tissue injury. It consists of vascular responses, migration and activation of leucocytes,
secretion of soluble mediators and systemic reactions. Inflammation is manifested by redness,
heat, swelling, pain and loss of function. Tissue resident cells such as macrophages, DCs and
mast cells secrete a variety of soluble mediators such as histamine, prostaglandins and
cytokines that enhance cell function at the inflamed site. Local production of the chemokines
IL-8 and monocyte chemotactic protein (MCP)-1 induces chemotaxis and results in
recruitment of neutrophils to the inflamed site and later also monocytes which can
differentiate into macrophages and DCs at the inflamed site. Furthermore, adhesion molecules
of the selectin family, the inter-cellular adhesion molecules (ICAM) and vascular cell
adhesion molecule (VCAM)-1 on endothelial cells, becomes highly expressed and aid in the
recruitment of cells. The whole purpose of the process is to remove the inducing stimulus and
to start local tissue recovery. There is an increased vascular diameter and vascular
permeability of the blood vessels, which enables leukocytes to migrate into the inflamed
tissue supported by the highly expressed adhesion molecules on endothelial cells and guided
by induced chemotaxis. The neutrophils and macrophages are specialized phagocytes, but
they also have an important role in mediating the acute phase response through the production
of IL-1, IL-6 and TNF. Once the pathogen is cleared or when the tissue has been repaired, the
signal that triggered the inflammation needs to be inhibited in order to sustain normal
homeostasis. A failure to regulate this suppression may have detrimental consequences for the
host, leading to chronic inflammation and in the end severe tissue damage. One major
signaling pathway involved in this task is represented by the suppressor of cytokine signaling
(SOCS) proteins that mediate a negative feedback loop to cytokine signaling.24, 25 For the final
resolution of inflammation the immune cells recruited to the site have to be removed, which
may happen by inducing apoptosis followed by clearance by macrophages.
11
CYTOKINES
Cytokines are low-molecular-weight regulatory proteins that are secreted by many cells of
the immune system in response to a number of stimuli. They are involved in virtually all
physiological responses in the body and are key players in coordinating immune responses
between cells, by binding to a variety of receptors and to induce cell-specific immune
responses. A cytokine can work in three different ways; by binding to receptors on the
membrane of the same cell that secretes it (autocrine action), by binding to receptors on a
nearby cell (paracrine action) or by binding to receptors on targets cells in distant parts of the
body (endocrine action) but the last mentioned happens very rarely. There are different
families of cytokines depending on their function. However, any given cytokine can have
different actions depending on which cell it influences or if it works in antagonistic or
synergistic ways with other cytokines. In addition, most cytokines have more than one
biological activity, probably depending on the local context in which the cytokine is
produced. The IFNs are key molecules in the early defense against infections and are in many
cases the first line of resistance to many viral infections. The ILs, which form a large group of
cytokines, are mainly produced by/and deliver signals to leukocytes. They are involved in
regulation of cell division and differentiation. Chemokines are another group of important
chemoattractant cytokines that regulate cell trafficking of various types of leukocyte
populations through seven-transmembrane G-protein-coupled receptors. They are produced in
response to exogenous stimuli, such as viruses and bacterial LPS as well as endogenous
stimuli such as IL-1, TNF and IFNs. Chemokines mainly act on neutrophils, monocytes,
lymphocytes and eosinophils and influence their ability to migrate, especially to inflamed
sites by inducing chemotaxis. Thus, chemokines have an important role in host defense
mechanisms. Chemokines are divided into several families based on the position of their
cysteine residues. The two major families that have been classified are the CXC or α
chemokines (e.g. IL-8, IFN-γ inducible protein (IP)-10, and platelet factor 4), and the CC or
β-chemokines (e.g. MCP-1, macrophage inflammatory protein (MIP)-1α, MIP-1β and
RANTES). Interest in chemokines and their function has grown since it became clear that
besides having chemotactic properties they also play a role in angiogenesis, hematopoeisis
and development.26, 27 A brief description of the cytokines and chemokines of relevance in the
papers will follow.
12
PRO-INFLAMMATORY CYTOKINES
IL-1β and TNF
IL-1β and TNF are two acute phase proteins that are rapidly induced upon tissue injury or
when the immune system senses microbes. They are produced by many cells of the immune
system but mostly by activated monocytes, macrophages and DCs. TNF can also be produced
by activated NK cells, T cells and B cells. Both of these factors are potent pro-inflammatory
cytokines and must therefore be kept tightly regulated. As a consequence, under normal
conditions they are not found in high concentrations in the circulation which otherwise could
be dangerous for the host. They are both involved in systemic and local responses to infection
and inflammation by generating fever, activating lymphocytes and inducing other systemic
acute phase proteins, such as C-reactive proteins and fibrinogen by the liver. IL-1β is
regulated by the NALP3 inflammasome and is kept in an inactive form as a pro-peptide. Upon
recognition of microbes or danger signals by the inflammasome, the caspase-1 enzyme
becomes activated and cleaves the precursor form of IL-1β into its active form, which then
can be secreted.28 IL-1 signals through the IL-1 receptor and ligation to the receptor activates
signal transduction pathways involving, for example, NF-kβ, AP-1 and p38 MAPK.29
Likewise, TNF signals through the TNFR1 and TNFR2 receptors and after binding to the
receptor mediates apoptosis and differentiation or proliferation, by activating signaling
pathways similar to IL-1β.30 As mentioned above, these cytokines are major mediators during
infection and inflammation and elevated levels of these cytokines have been found in
systemic shock, autoimmune diseases and chronic inflammatory diseases. For this reason,
new therapies aiming to block IL-1β and TNF are under investigation. Currently, antibodies
specific for TNF have been approved for the treatment of Crohn´s disease31 and anti-TNF
therapy using soluble TNFR2 have also been approved for treatment of RA.32
IL-6
IL-6 is one of the key cytokines mediating fever and acute phase responses. In many cases
it mediates its effects in combination with IL-1β and TNF. IL-6 plays an important role in the
resolution of inflammation and is believed to be a regulatory switch directing the early innate
defense towards acquired immunity.33 Many cells can produce and secrete IL-6 in response to
IL-1β/TNF stimulation or after triggering by PRRs. Depending on the immune response, IL-6
13
can act both as a pro-inflammatory and as an anti-inflammatory cytokine. During the
initiation of inflammation, IL-6 encourages production of acute phase proteins and activation
of macrophages as well as recruiting immune cells such as lymphocytes to the site of infection
and/or tissue injury. In the late stage of infection, IL-6 takes a protective role and counteracts
the manifestations of certain inflammatory responses by stimulating the development of
adaptive immune responses and inducing apoptosis in neutrophils, thereby down-modulating
production of reactive oxygen species (ROS) and other toxic compounds and aiding in the
resolution of inflammation. IL-6 signaling is mediated by two pathways, either by binding to
the membrane-bound IL-6 receptor (IL-6r) or by binding to its soluble form of the IL-6r. Both
pathways activate the signal transducing glycoprotein gp130 followed by activation of gene
transcription. Because of the many functions of IL-6 it has been implemented in various
inflammatory diseases such as RA, MS, inflammatory bowels disease and Castlemans
disease.34
IL-12p70
The IL-12 family of cytokines has a fundamental role in the induction of Th1-associated
immunity by inducing differentiation of Th1 cells. IL-12 is produced primarily by monocytes,
macrophages, DCs and B cells. The major functions of this cytokine are to induce IFN-γ
production in NK cells and T cells, to enhance NK and Tc-cell cytotoxicity and to induce
differentiation of naïve T cells into Th1-effector cells, thus having a central role in cellmediated immunity. IL-12 is composed of two covalently linked subunits (IL-12p35 and IL12p40) and together they comprise the biologically active form IL-12p70. Two new members
of IL-12 have also been described:35 IL-23 and IL-27, which share the same features as IL-12
but are composed of different subunits. IL-23 contains p40 and p19 subunits, while IL-27
comprises the p28 and EB13 subunits. The main signaling pathway induced by the IL-12
family is the JAK/STAT pathway in which IL-12 mainly activates STAT4, whereas IL-23 and
IL-27 mainly activate STAT3 and STAT1, respectively. The importance of the IL-12 family
is evident from the fact that a deficiency in either IL-12 or its receptors has been shown to
lead to impaired cell-mediated immunity and enhanced susceptibility to diseases.36
14
Interferons
The IFNs are a multigene family of inducible cytokines that exhibit a diversity of
biological functions in the immune system. Their most important functions are their anti-viral
and anti-tumoral activity but they also exert immunomodulatory effects on other immune
cells. They are broadly grouped into type I IFN and type II IFN. Type I IFN include IFN-α
and IFN-β and are induced, for example, by viral infections, while type II IFN include IFN-γ
induced by mitogenic or antigenic stimuli.37 Most virus-infected cells can produce IFN-α/IFNβ when they are grown in cell culture. In contrast, only certain specific cells produce IFN-γ,
including NK cells and activated T cells. The IFNs binds to different receptors, such as the
IFNAR-1/IFNAR-2 that are ubiquitously expressed for type I IFNs and the IFNGR-1/IFNGR2, that are expressed only on activated T cells, macrophages and NK cells, for the type II IFN,
but they all share downstream signaling molecules and regulate the same genes.38 Binding of
the IFNs to their receptors leads to a) increased expression of intrinsic proteins, b) induced
apoptosis by virus-infected cells, c) cellular resistance to virus infection and, d) activation of
NK cells and DCs.
ANTI-INFLAMMATORY CYTOKINES
IL-10
IL-10 is a unique cytokine with regulatory properties and is one of the most important antiinflammatory cytokines known today.39 It works as a negative feedback molecule that
effectively blocks the biological functions of cells such as T cells, monocytes and
macrophages, usually by blocking the NF-kβ transcriptional pathway. The pivotal role of this
cytokine is evident by the fact that the same cells that initiate inflammation also induce the
production of IL-10, thereby limiting the extent of the inflammation to ensure a balanced
immune response. For this reason, many cell types can produce IL-10 including monocytes, B
cells and the majority of all T-cell subpopulations as well as DCs and NK cells. Thus, the
principle function of IL-10 is to limit and ultimately terminate the inflammatory response. In
addition, it also regulates growth and/or differentiation of B cells, NK cells, cytotoxic and Th
cells, DCs, mast cells, granulocytes and endothelial cells. IL-10 signals through the IL-10
receptor, which is composed of at least two subunits of the interferon receptor family which
mostly signal via the JAK/STAT pathway. IL-10 modulates the expression of cytokines,
15
soluble mediators and cell surface molecules with important consequences for the cells’
ability to activate and sustain immune- and inflammatory responses. For example, it potently
suppresses key functions in monocytes/macrophages such as the production of several
cytokines (IL-1β, IL-6, IL-12, IL-18 and TNF), chemokines (MCP-1, RANTES, IL-8 and IP10), the expression of MHC class II molecules and the co-stimulatory molecules CD80 and
CD86.40 Further, IL-10 inhibits the production of IL-12 and the expression of co-stimulatory
molecules on various DC subsets,40 and also exerts its anti-inflammatory properties by
inducing the differentiation of Treg cells.41 The capacity of IL-10 as a suppressive agent is
also evident by the fact that the sequence of IL-10 has close homology to several virus
genomes, including Epstein Barr virus.42
TGF-β
TGF-β belongs to a family of regulatory cytokines that have pleiotropic functions in a
broad range of cell types. It is mostly recognized for its involvement in maintaining tolerance
through the regulation of lymphocyte proliferation, differentiation and survival.43 In addition,
TGF-β plays an essential role in the suppression of inflammation but has lately also shown to
have a positive role in these responses. For example, TGF-β induces Treg cells in the
presence of IL-2, while in the presence of IL-6 it induces pathogenic IL-17 producing Th17
cells.44 TGF-β regulates multiple responses in the immune system, including suppression of
effector Th cell differentiation, inhibition of T and B cell proliferation and effector cytokine
production, such as IL-2, IFN-γ and IL-4 and suppression of NK cells, DCs and macrophages.
In mammals, three isoforms of TGF-β (TGF-β1, TGF-β2, TGF-β3) have been identified,
among which TGF-β1 is the predominant form expressed in the immune system.45 TGF-β is
synthesized in an inactive form composed of the TGF-β dimer in association with the latencyassociated protein (LAP). This latent TGF-β molecule can either be secreted or it can bind to
latent-TGF-β-binding protein that deposits the protein complex in the extracellular matrix. For
TGF-β to become activated it has to bind to the TGF-β activator that triggers LAP
degradation. Binding of the active form of TGF-β to one of its two receptors (TGF-βRI and
TGF-βRII) initiates signaling pathways that primarily activate the Smad transcription factors.
The importance of TGF-β as a master regulator becomes evident by the fact that knock-out
mice lacking TGF-β suffer from extreme inflammatory dysregulation and die prematurely
and.46
16
CHEMOKINES
IL-8
IL-8 (CXCL8) is a chemokine highly involved in inflammation. It is produced by a variety
of cells of the immune system i.e. monocytes, granulocytes, lymphocytes, fibroblasts and
endothelial cells. The primary function of IL-8 is to induce chemotaxis of responsive cells
mostly neutrophils, which is its primary target cell. Neutrophils respond to IL-8 by
chemotaxis to inflammatory sites and subsequent up-regulation of surface adhesion
molecules, increased phagocytosis, production of ROS and exocytosis, which are key
functions during inflammation. While neutrophils are the primary target cell, other cell types
such as endothelial cells, macrophages, mast cells and keratinocytes also respond to IL-8,
which underscores its importance as a chemoattractant factor during inflammation. Like many
other chemokines, IL-8 signals through two G-protein coupled receptors (CXCR1 and
CXCR2) which are expressed mostly on neutrophils. CXCR1 is specific for IL-8 whereas
CXCR2 can bind to other chemokines as well.47
RANTES (CCL5)
RANTES (regulated upon activation normal T cell expressed and secreted) was first
considered a T-cell-specific chemokine but is now known to be produced by a number of
other cell types, such as epithelial cells, fibroblasts, platelets, macrophages as well as
endothelial cells. RANTES induces the migration of leucocytes by binding to specific
receptors in the seven-transmembrane G-protein coupled receptors family, namely CCR1,
CCR3, CCR4 and CCR5. RANTES is a potent chemoattractant for T cells and monocytes, but
also brings other cell types such as DCs, esinophils, NK cells and mast cells to the site of
infection.48 Increased RANTES expression has been found in various inflammatory disorders
and pathologies for example in atherosclerosis, RA and inflammatory airway disorders.49 In
addition, RANTES is important in the defense against viral infections, and has been found to
be degranulated from virus-specific CD8+ T cells.50 Further, the importance of RANTES in
antiviral immunity can be appreciated by the fact that certain viruses have evolved evasion
mechanisms from RANTES. One example is the human cytomegalovirus, which expresses a
chemokine homolog that sequesters RANTES.51 One of the receptors for RANTES (CCR5)
17
has also been discovered to be a co-receptor for HIV. For this reason, therapeutic strategies
targeting RANTES and CCR5 are being tested for treatment against HIV infection.52
MIG (CXCL9) and IP-10 (CXCL10)
MIG (Monokine induced by IFN-γ) and IP-10 (IFN-γ-inducible protein 10) belong to the
CXC or α subfamily of chemokines and are produced by a variety of leucocytes such as
monocytes, endothelial cells and fibroblasts in response to IFN-γ and TNF. They both have
potent chemotactic activities and direct the trafficking to the site of infection of primarily
activated T cells, NK cells and NKT cells. Expression of IP-10 is often seen in many Th1type inflammatory diseases.53 IP-10 and MIG are structurally closely related and share the
same high-affinity receptor; CXCR3.
Monocyte chemotactic protein (MCP)-1 (CCL2)
MCP-1 is produced by a variety of cell types, either constitutively or after induction by
oxidative stress, cytokines or growth factors. Many cells can produce and secrete MCP-1
upon stimulation such as fibroblasts, epithelial, endothelial and smooth muscle cells but the
two most important sources are monocytes and macrophages. MCP-1 signals through the
CCR2 receptor which activates MAPK and ultimately the NF-kβ transcription factor. It is a
potent chemoattractant for monocytes but it is also important for memory T cells and NK
cells. Along with IL-8, MCP-1 is very often found in tissues during inflammation, where it
triggers adhesion of monocytes to vascular endothelium, thus having an important role in
extravasation. MCP-1 expression has been noted in many inflammatory diseases during
progression including athrosclerosis, RA and cancers.54, 55 In these cases, the massive influx of
macrophages to the site of infection is thought to be partly responsible for the tissue damage
and the exacerbation of the disease.
18
PREGNANCY AND THE IMMUNE SYSTEM
IMMUNE TOLERANCE
A fundamental role of the immune system is to protect the host from pathogens. Maternal
immunity during pregnancy is therefore somewhat complex since it both needs to
accommodate and accept fetal antigens and at the same time maintain protection against
microbes and other pathogens. In other words, the maternal immune system requires
substantial regulation for fetal survival but not to such an extent that it compromises
protection of the mother. The mechanisms involved in tolerance of the fetus by the maternal
immune system remain somewhat elusive. Studies by Owen,56 who showed that dizygotic
cattle twins retain cells from the other twin post-natally led Billingham, Brent and Medawar
to formulate the idea of immunological tolerance.57 However, Sir P. Medawar was the first to
recognize the idea of fetal-maternal tolerance and proposed the concept of the fetal allograft
to explain the special immune relationship between the mother and the fetus.58 The idea he
proposed was that the semi-allogeneic fetus was able to survive because the immunological
interaction between the mother and the fetus was suppressed, possibly due to lack of
presentation of fetal antigens to maternal cells due to a physical separation of maternal and
fetal tissue by the placenta. Although his explanations were not fully correct, their hypothesis
had a profound influence on this research area during the following years. These original
ideas were later extended and revised by Wegmann et al,59 who postulated that pregnancy is
purely a Th2 phenomenon meaning that the Th1 cells that mediate cytotoxic responses are
inhibited, while the number of Th2 cells are increased to support the production of antibodies
and sustained pregnancy. This Th2-biased hypothesis has been the belief for many years and
received support from clinical evidence of women with RA who experience a temporary
remission of their symptoms during pregnancy,60 while systemic lupus erythematosus, in
which the principal pathology is mediated by excessive autoantibody production (humoralmediated), tends to flare up during pregnancy.61,
debate.63,
64
62
The Th2 hypothesis is currently under
However, it has been clearly shown that the Th1/Th2 ratio is changed in the
decidua during pregnancy, which is probably supported by increases in local progesterone
levels (a potent Th2 inducer) and secretion of Th2 cytokines, such as IL-4, IL-5 and IL-10.65
However, a strict Th2 dominance throughout pregnancy does not seem to be essential and
recent data indicate that a successful early pregnancy requires help of cytokines which
traditionally are associated with Th1 responses. For example, IFN-γ has previously been
19
considered as a potentially harmful cytokine during pregnancy, since it could lead to
pregnancy complications through embryotoxic activity or damage of the placental tissue, but
in murine models it has shown to be needed for proper vascularisation during placental
development.66 Th1 cytokines are thought to be detrimental for a sustained pregnancy since
administration of IL-2, IFN-γ and TNF-α to pregnant mice has been shown to lead to fetal
resorption,67 and high levels of Th1 cytokines have been found in women who develop
spontaneous abortions.68-70 For this reason, anti-inflammatory factors such as IL-10 and TGFβ, are needed to sustain pregnancy. In addition, others have suggested that the Th1 cytokines
might be irrelevant to pregnancy and that the appearance of Th1 cytokines may be a reflection
of cellular immunity that becomes activated as a response to pregnancy complications, such
as pre-eclampsia and spontaneous fetal loss.71 Therefore the original prediction that pregnancy
is a Th2 phenomenon might have been too simplified. A recent review by Mor et al,72
suggests that pregnancy is both a pro- and anti-inflammatory condition, depending upon the
stage of gestation. Furthermore, one important immune evasion strategy by the fetus is the
absence of classical MHC class I (HLA-A and HLA-B) and class II molecules on the
trophoblast. The expression of non-classical HLA-G does not appear to stimulate Tc activity73
and actively inhibit NK cells74 and is therefore believed to be involved in implantation
through modulation of maternal decidual leucocytes.75 Other factors suggested to help in
sustaining maternal tolerance of the fetus include expression of Fas ligand on fetal cells to
eliminate activated T cells76 and local production of the T-cell suppressive substance
indoleamine 2,3-dioxygenase (IDO) expressed by fetal syncytiothrophoblasts and decidual
macrophages.77 Also, placental exosomes, which display proteins that down regulate the
cytotoxicity of T and NK cells, have been found in maternal circulation.78, 79
IMMUNE ACTIVATION DURING PREGNANCY
Increasing knowledge has confirmed that both the innate and the adaptive immune system
are essential for a successful pregnancy. The maternal immune system changes both locally in
the placental environment and in the peripheral circulation. Maternal adaptive immune
responses are generally suppressed, while the innate components of the immune system are
activated systemically.80 During normal pregnancy, the human decidua, or implantation site, is
populated by large numbers of immune-competent cells such as macrophages, NK cells and
Tregs,81-83
84
but there are no B cells present. These cells (macrophages, NK and Tregs) have
20
shown to be essential during the first trimester since depletion of them results in termination
of the pregnancy,85 which suggests that they have effects on placental development,
implantation or decidual formation. A special NK-cell subset exists, named the uterine NK
(uNK) cell, which is the major immune cell type in the decidua representing almost 70 % of
all decidual leucocytes. uNK cells are phenotypically and functionally different from
peripheral NK cells86 and have been shown to be important for the vascularisation early
during pregnancy87, and decreased numbers of uNK cells have been associated with
intrauterine growth restriction.88 Macrophages represent about 20-25 % of the decidual
leucocytes but their numbers may vary depending on hormones affecting the influx of
leucocytes and monocytes into the decidua. Also, cytotrophoblasts may stimulate migration of
macrophages by local production of MIP-1.89 Since there are very few immune defense
systems within the placental tissue it is thought that the macrophages residing in the decidua
have an important role in non-specific host defense within the placenta. They could facilitate
clearance of apoptotic cells and debris as well as secreting cytokines and immunosuppressive
prostaglandins, which could hamper the function of Tc cells and uNK cells.
THE PLACENTA
The placenta is a highly specialized organ, primarily of fetal origin, that develops after the
successful implantation of the blastocyte into the endometrium (Fig 1). The outer layer of the
blastocyst becomes the trophoblasts that form the outer layer of the placenta. This can be
further divided into two layers the underlying cytotrophoblasts and the overlaying
syncytiotrophoblasts. Soon after the implantation, fetal blood vessels are established called
villi, which are surrounded by the syncytiotrophoblasts. Maternal blood will perfuse the
decidua, via the spiral arteries, and into the intervillous space (IVS) at around 10 weeks of
gestation.90 Failure in the remodeling process of the vessels might lead to pregnancy
complications such as early pregnancy loss, intrauterine growth restriction and preeclampsia.91, 92 As a result of this remodeling, fetal villi are bathed in maternal blood which
creates the platform for maternal-fetal exchange of nutrients, respiratory gases and waste
products. The placenta further stimulates production of hormones, growth factors and
cytokines that aid in this uptake and delivery system. Thus, the primary function of the
placenta is to supply the fetus with oxygen and nutrients throughout delivery, while
discarding waste products. The placenta also serves as an interface between the mother and
21
the fetus and separates the fetus blood supply from the mothers. However, this barrier is not
complete and small molecules can passively diffuse through the placenta. One exception is
antibodies that are actively transported across the placental barrier to give passive protection
to the fetus from the mother that usually persists for up to 6 months after birth. The main
antibody class involved is IgG, but small amounts of IgA and IgM antibodies are also thought
to be able to cross the placenta.93 The placenta also produces high levels of several mediators
that are thought to drive Th2 immunity, while dampening Th1 responses, such as IL-4, IL-10,
prostaglandins and progesterone94-96 to support fetal tolerance.
Figure 1. The structure of the human placenta. Adapted from:
http://php.med.unsw.edu.au/embryology/index.php?title=Placenta_Development
22
MALARIA
MALARIA IS A MAJOR HEALTH PROBLEM
Malaria remains one of the most common infectious diseases in the world. In 2009, an
estimation of 225 million cases of malaria were reported by the World’s Health Organization
and despite endless efforts to prevent its spread almost one million deaths still occurs
annually.97 Malaria is caused by a protozoan parasite of the genus Plasmodium and currently
five species (P. falciparum, P. malariae, P. vivax, P. ovale and P. knowlesi) are known to
cause human malaria. P. falciparum is the deadliest of the five known species, causing the
most severe form of the disease and is responsible for most of the deaths. The parasites are
widely distributed in tropical and subtropical regions making African countries especially
affected. In 2009, malaria was reported to be endemic in 35 African countries.97 In malariaendemic areas, it is mostly children under the age of five and pregnant women who
experience the most severe complications associated with the disease. Complications such as
severe anemia and cerebral malaria are thought to be the major cause of morbidity and
mortality in children, but the host’s immunological response could also be contributing to the
pathology. Following repeated infections, immunity increases with age, however, sterile
immunity is almost never achieved and parasites can still be found in the blood circulation
without the infected person displaying any clinical symptoms.
THE LIFE CYCLE OF THE MALARIA PARASITE
The life cycle of P. falciparum is complex and involves both human and mosquitoes as
hosts to be able to replicate and survive. The lifecycle starts when an infected Anopheles
mosquito takes a blood meal and injects sporozoites, via the saliva, into the human host (Fig.
2). Within 2-30 minutes the sporozoites migrate to the liver, where they infect hepatocytes
and the asexual stage of the life cycle is started. After approximately one week, the infected
hepatocytes burst and fully developed merozoites are released which starts to invade
erythrocytes in the blood circulation, thereby initiating the intra-erythrocytic stage of the life
cycle. This stage is responsible for all the clinical manifestations often seen during a malaria
episode. Inside the erythrocytes, young parasites undergo a series of maturation steps
resulting in the formation of trophozoites and ultimately into schizonts. When the schizonts
rupture, newly made merozoites are released ready to infect new erythrocytes. This cycle of
23
rupture and reinfection of erythrocytes has a periodicity of 48-78 hours, depending on the
Plasmodium species, and gives rise to the typical fever episodes characteristic for malaria.
Some parasites differentiate into female and male gametocytes that can be picked up by a new
feeding mosquito. These gametocytes fuse inside the mosquito’s midgut and after a series of
developmental steps new sporozoites are formed and migrate to the salivary glands, thereby
completing the parasite’s life cycle.
Figure 2. The life cycle of P. falciparum. Adapted from Jones MK et al.98 Reprinted with
permission from NPG.
CLINICAL MANIFESTATIONS OF MALARIA
The majority of P. falciparum infections cause flu-like symptoms such as fever, headache,
malaise and nausea that may resolve by itself without the patient taking any drug-treatment. In
some cases however, the infection can lead to severe and life threatening disease such as
cerebral malaria, severe malarial anemia and acute respiratory distress, which mostly occur in
young children and in the pregnant women. The outcome of an infection is never the same for
24
two individuals and disease severity may vary depending on the host genetics, age, previously
acquired immunity as well as transmission rate in the area and parasite strain. For that reason,
the disease can vary from being mild asymptomatic, to symptomatic and to severe disease. In
high malaria-endemic areas, infants and young children are more susceptible to develop the
life threatening diseases, while in low-malaria transmission areas, even adults may develop
severe symptoms.
INNATE AND ADAPTIVE IMMUNE RESPONSES IN MALARIA
Innate and adaptive immune responses can together limit the peak of parasitemia and
reduce the load of circulating parasites. How this may be achieved is presented in Figure 3.
An immune response to the malaria parasite begins when the mosquito injects sporozoites into
the skin and/or blood stream. Antibodies against antigens on the sporozoites surface, such as
the circumsporozoite protein, are able to limit infection by inhibiting invasion of hepatocytes.
In addition, early interactions between circulating parasites and cells of the innate immune
system including DCs, monocytes, macrophages, NK cells, NKT cells and γδ T cells are
important in the initial limitation and control of parasite loads. These cells can directly destroy
infected hepatocytes through perforins and granzymes or by cross linking death receptors,
thereby inducing apoptosis.99 The major role of the innate immune system during infection
with P. falciparum appears to be the production of cytokines such as IL-12 and IFN-γ, which
are critical for the development and regulation of type 1 immune responses involving CD4+ T
cells, B cells and other effector cells. The APC, especially the DCs, become activated by
parasite ligands that are recognized by TLR leading to subsequent up-regulation of their costimulatory molecules and adhesion molecules and their capacity to secrete cytokines, in
particular IL-12. CD4+ T cells are of major importance for the induction of blood-stage
immunity, while the CD8+ subset has been shown to be cytolytic against liver stages of the
parasite. CD4+ T cells produce cytokines which are involved in further activation of the innate
immune system but also in the support of anti-malaria-specific antibody production. They also
produce IL-2 that further activates NK cells to produce IFN-γ, which activates macrophages
to efficiently kill parasites by secretion of NO and ROS. In addition, IFN-γ-producing CD4+ T
cells also assist in the induction and regulation of CD8+ T cells and of NK and NKT cells.
IFN-γ is a key cytokine during P. falciparum infection and is associated with reduced
susceptibility to malaria.100-102
25
Figure 3. Linking innate and adaptive immunity to P. falciparum infection. Adapted from
Stevenson M.M. & Riley M.E.103 Reprinted with permission from NPG.
Role of cytokines
Cytokines are major mediators during P. falciparum infection but many contradictory
results have been obtained regarding their involvement in protection and/or pathology as a
result of using different animal models and different Plasmodium strains. However, available
data are consistent with a requirement for an early production of pro-inflammatory Th1
cytokines, including TNF, IL-12 and IFN-γ that may limit the progression from
uncomplicated malaria to severe and life-threatening complications. High levels of IFN-γ as
part of a Th1-driven immune response have been associated with a more favorable outcome in
most animal models of malaria,104,
105
an effect that has been attributed to the monocyte-
macrophage activating capacity of IFN-γ, that rapidly kills malaria blood stage parasites by
NO and ROS production. Likewise, in humans, high IFN-γ levels have been associated with
resistance to reinfection in children102 and to be increased in uncomplicated compared to
severe malaria cases,106 further supporting a critical role for IFN-γ in limiting progression of
the disease. Furthermore, an important role for IL-12 in early responses against Plasmodium
26
has been proposed. IL-12 appears to be critically linked to IFN-γ production, thereby allowing
an early and sustained Th1 response.107 Injection of IL-12 into mice before infection with
Plasmodium provides full protection through an IFN-γ dependent mechanism.108 In young
African children who presented with mild or severe P. falciparum infection, the plasma levels
of IFN-α and IL-12 were found to be lower in children with severe rather than mild malaria,
and IL-12 correlated inversely with parasitaemia.109 A similar picture as for IFN-γ emerges for
TNF-α in early responses against Plasmodium. Treatment with anti-TNF-α antibodies results
in a tendency towards longer times for parasite clearance. Furthermore, increased plasma
levels of TNF-α together with IFN-γ, IL-1β and IL-6 and reduced production of IL-4 and
TGF-β have been reported in patients with cerebral malaria.110 Elevated levels of IL-1 have
also been associated with increased severity of the disease.111,
112
Paradoxically, the same
cytokines that are implicated in protection against malaria have also been found to be
involved in the pathogenesis of the disease. For example, TNF seems to be important in the
early responses to malaria and in late pathological manifestations. Excess production of TNF
is likely to be involved in the appearance of symptoms such as fever and headache associated
with severe disease.113 In addition, low levels of IL-10 or a low IL-10/TNF ratio are associated
with malarial anaemia in African children,114-116 which suggests that the clinical course of
severe malaria is influenced by a relative imbalance between TNF and IL-10 levels. It is
widely accepted that Th2 cytokines down-regulate Th1-derived cytokines. Elevated levels of
IL-10 have been reported in severe malaria.117 In vitro studies and studies in mice have shown
that IL-10 inhibits TNF-α production and also down regulates the MHC class II expression on
monocytes/macrophages.118 Thus, IL-10 appears to have important roles in counteracting the
potentially harmful host pro-inflammatory response to malarial antigens. Overall, the outcome
of P. falciparum infection may depend on the fine balance between pro and anti-inflammatory
cytokines.
The role for chemokines during infection is to direct the innate and adaptive cells to sites
were they are needed.119 A range of chemokines are elevated during malaria infection,
especially some pro-inflammatory chemokines such as MIP-1α, MIP-1β and IL-8.120-123 MIP1α and MIP-1β are critical chemoattractants for monocytes and after recruitment they activate
monocytes/macrophages to produce TNF-α, IL-1 and IL-6.124
27
Role of antibodies
The most direct evidence that antibodies are important mediators in protective immunity to
malaria comes from old classical passive transfer experiments, in which antibodies from
malaria-immune adults were given to children with severe malaria.125 The treatment was
found to successfully reduce the parasite loads to nearly undetectable levels in these children.
Additional studies in mice deficient in their Fcγ receptors further supported a role for
antibodies in protection.126 Several mechanisms may explain how these antibodies are thought
to neutralize the parasite.127 Antibodies reactive to malaria antigens may inhibit hepatocyte
and erythrocyte invasion by binding to the surface of free sporozoites and merozoites. They
can also bind to surface antigens on already infected erythrocytes, thereby inhibiting the
intraerythrocytic development of the parasite. They may also facilitate phagocytosis of whole
infected erythrocytes or merozoites by promoting opzonisation and increase the phagocytic
capacity of monocytes and macrophages.128 In addition, antibodies may, beside their direct
role in inhibiting parasite invasion and/or growth, also induce ADCC via the FcR on the
effector cell and the induction of toxic mediators such as TNF or NO.127 Further, antibodies
may also block parasites from binding to scavenger receptors, such as CD36 or ICAM-1 on
endothelial cells, which the parasites commonly use as binding sites to avoid clearance by the
spleen and establish an infection.
Several epidemiological studies have underlined the importance of having high circulating
levels of malaria-specific antibodies, especially levels of IgG, in relation to protection against
severe malaria. This protection seems to be largely dependent on the balance between the
cytophilic i.e IgG1 and IgG3 and the non-cytophilic i.e IgG2 and IgG4 antibodies.129 Of note,
the cytophilic IgG1 and IgG3 subclasses have been associated with protection against P.
falciparum malaria in various endemic areas130-134 and some have even suggested that the noncytophilic antibodies could possibly compete with the cytophilic types, thereby blocking their
protective effects. Interestingly, in some areas IgG2 antibodies in combination with certain
genetic mutations in the FcγRIIa have found to be protective133 and especially in combination
with low IgG4 levels.135 In contrast, IgG4 does not seem to have any protective role.
28
PROJECT BACKGROUND
MALARIA DURING PREGNANCY
25 million pregnant women are currently at risk to develop malaria and malaria during
pregnancy accounts for almost 10.000 maternal and 200.000 neonatal deaths every year.136
However, these figures might be underestimated when considering the significant impact of
malaria on perinatal morbidity and mortality as well as exaggeration by co-infections, such as
HIV.
The malaria parasites seem to have evolved ways to evade the immune system and
especially the complex placental environment. That the pregnant women down-regulates her
cell-mediated immunity is known, but the only evidence for increased susceptibility to
infections during pregnancy is for intracellular parasites, such as Plasmodium. This
suppressive state of Th1 immunity and up-regulation of Th2 responses has been used as an
explanation for the women’s increased susceptibility. However, this explanation can not by
itself explain some of the features associated with malaria during pregnancy. For example,
why the prevalence is higher in primigravidae compared to secundigravidae or multigravidae
or why the severity of the disease including symptoms and other complications tends to
decrease over successful pregnancies is not known. The understanding of why pregnant
women become more susceptible to develop malaria is still incomplete, but one possible
reason is that P. falciparum infected parasites are able to bind to special proteins in the
intervillous spaces of the placenta, creating the condition known as placental malaria (PM).137
This condition is probably one of the most complex interactions between a host and a
pathogen, taking place in the placenta known today.
One reason for P. falciparum´s ability to invade and infect the host so efficiently is the
remarkable ability of these parasites to adhere and sequester. During the erythrocytic stage,
young ring-stage parasites circulate in the blood and mature into trophozoites, which start to
express adhesion molecules that are displayed on the erythrocyte membrane. These adhesive
ligands make it possible for the parasites to adhere to endothelial tissue. One of the major
family of proteins that have been identified as one of these adhesive ligands on the
erythrocyte is the P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the
var multigene family.138 Adults residing in endemic areas acquire a wide range of antibodies
towards these proteins after multiple exposures and are therefore said to be semi-immune.
29
However, in the pregnant women, the development of a new organ, the placenta, represents a
new niche to which infected erythrocytes (iE) can and will adhere.139 These parasites express a
unique repertoire of PfEMP1 proteins that the primigravidae women have not encountered
before. A particular variant of PfEMP1 has been named var2CSA which has the capacity to
bind to chondriotin sulphate A (CSA) expressed on the surface of trophoblasts in the IVS.140
Binding of iE in the placental tissue leads to physiological changes, such as thickening of the
trophoblast membrane, intervillous infiltrates of immune cells,
141-143
and deposition of
malarial pigment, called hemozoin, in phagocytic cells.144 As a consequence of placental
malaria, babies are usually born with a low birth weight (LBW)141 and the babies have been
shown to have a reduced chance of surviving their first 2 years of life.145, 146 LBW can be
caused by intrauterine growth retardation or pre-term delivery as a result of placental
insufficiency and impaired blood flow,147 but the exact mechanism is still not known. What
really occurs inside the IVS once the parasites adhere is not completely clear. The binding of
iE to CSA in the placental is thought to stimulate syncytiotrophoblasts and cells in the
placenta to release various chemokines. Elevated levels of chemokines have been described,
especially levels of MCP-1, MIP-α and β as well as IP-10 in placentas from infected
women.121,
148, 149
MIP-1α and β recruits additional macrophages and MCP-1 plays an
important role in recruiting monocytes to the infected placenta.121, 148 Overall these factors are
responsible for the massive infiltration of inflammatory cells into the placenta. The
inflammatory milieu with increased plasma cytokine levels causes a shift of the placenta
cytokine balance which is necessary for elimination of parasites.150 On the other hand,
excessive levels may also contribute to pathology, especially high TNF levels have been
shown to be a risk factor for LBW.151 Thus, the balance between pro and anti-inflammatory
factors seems to be important.
MALARIA AND ETHNIC GROUPS IN WEST AFRICA
The genetic and cultural background of the two sympatric ethnic groups Fulani and Dogon
are by now very well established.152, 153 In brief, the Fulani are nomadic pastoralists that have
been settled in the study area in Mali (where samples included in paper II were taken from)
for the last 200 years, but they have also settled in other countries of West Africa. The Dogon
are on the other hand, traditional farmers who migrated to this particular study area more than
50 years ago. There is no intermarriage between the two ethnic groups. The Fulani of West
30
Africa is a particular interesting ethnic group because of their lower incidence of malaria
compared to other sympatric ethnic groups living in Mali152 and in Burkina Faso.153 These
tribes live under similar social, cultural and geographical conditions but the Fulani have been
shown to be less susceptible to developing malaria. They suffer less clinical symptoms, have
fewer parasites in their blood152, 153 and have been found to have a stronger humoral immune
response as compared to other ethnic groups132-134, 154. Furthermore, they also have increased
spleen rates155 and increased numbers of malaria-specific IL-4 and IFN-γ producing cells154
but lower frequencies of Tregs156 compared to their neighboring ethnic groups. In addition,
APC subsets from Fulani and Dogon children has also been shown to respond differently to
different TLR stimulations,157 suggesting that there is a difference in the immune regulation
between these two groups. However, the fundamental reason for the Fulani’s relative
resistance to P. falciparum is as yet unknown.
31
PRESENT STUDY
OBJECTIVES
The overall aim of this work is to examine the impact of P. falciparum infection on
inflammatory responses in pregnant women and children residing in African countries.
More specifically we aimed to:

Carry out comprehensive analysis of inflammatory markers in pregnant women
throughout their pregnancy and at delivery to find possible biomarkers for pregnancyassociated malaria (Paper I).

Examine cytokine, chemokine and antibody levels in children from two African ethnic
groups, which differ in their susceptibility to P. falciparum infection to investigate
their involvement in protection and/or pathology of malaria (Paper II).
32
SUBJECTS AND METHODS
Subject demographics, information of the study areas and methods used are found in detail
in the corresponding papers. In this section, there will only be a brief description of the study
subjects and experimental procedures.
The first study was a longitudinal cohort study of pregnant women and their babies,
conducted in Tanzania and Benin from 2008 until 2010. At both study sites, 1000 pregnant
women were enrolled and followed up during pregnancy with a series of scheduled antenatal
visits up until delivery. Here we present data from the cohort in Tanzania on a subgroup of
121 pregnant women (42 infected and 79 uninfected). The infected women were selected
based on having a positive RDT and came to the hospital for all three antenatal visits and at
delivery. Infected women were individually matched to 2 separate uninfected controls of
similar age (±4), gestational age (±2 weeks) and the same gravidity.
The second study was conducted from October to November 2008 in a rural area of the
Dogon valley in Mali where people from the Dogon and Fulani ethnic groups live together in
sympatry. In total, 77 children (age 2-10) were randomly included in the study and were
divided according to their ethnicity and slide reading result. Out of these children, 40 children
belonged to the Dogon ethnicity of whom 20 were slide positive for P. falciparum and 20
were negative. Consequently, 37 children belonged to the Fulani ethnic group of whom 14
were slide positive and 23 were negative.
From both study sites, venous blood from the participating pregnant women and children
presented with or without P. falciparum infection was obtained. For the pregnant women,
venous blood was obtained from each time point the women came to the hospital. Plasma was
collected and the samples were shipped back to Sweden and analyzed for inflammatory
markers (IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF, IFN-α, IFN-γ, RANTES, MIG, MCP-1,
IP-10, vascular endothelial growth factor (VEGF/Flt1), urokinase receptor (uPAR),
angiopoietin (Ang)-1 and Ang-2) and antibody titers (malaria-specific total IgG, IgM and IgG
subclasses (IgG1-IgG4)) using ELISAs or CBA.
33
RESULTS AND DISCUSSION
Paper I
Pregnancy is a unique state in life in which the women carries, nourishes and supports the
growing fetus until it is time for delivery. The mother’s immune system is strongly modulated
to perform this task considering that it has to be able to accept fetal antigens in her womb
without attacking it. The placenta has a key function during this process as a source for
immunoregulatory molecules. It sustains fetal tolerance and at the same time supports the
fetus with nutrients, while working as a barrier for invading pathogens. The P. falciparum
parasite has evolved ways of evading the immune system during pregnancy and uses the
placenta as a hiding place to avoid clearance by the spleen by binding to proteins in the
placental tissue.139, 140 For this reason, and due to the many difficulties in developing a vaccine
in combination with the wide spread drug resistance, it leaves the pregnant women and her
fetus vulnerable. Therefore there is a major need for malaria prevention and rapid diagnosis
followed by accurate treatment of pregnant women. Over the past century important studies
have accumulated and have laid the groundwork for the knowledge we possess regarding
placental malaria. Many have characterized the effect of infection with P. falciparum and/or
placental malaria on the mother and her fetus at enrollment or specifically at delivery using
cross-sectional designs.158 In this study we aimed to assess aspects of the timing of infection
during pregnancy by using a longitudinal design.
We started out to investigate selected markers in peripherl blood plasma from pregnant
women without malaria since such data are currently lacking in an African population. The
factors we analyzed were IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF, IFN-α, IFN-γ, RANTES,
MIG, MCP-1, IP-10, VEGF/Flt1, uPAR, Ang-1 and Ang-2. We found that most of the factors
were stable during the course of pregnancy, from inclusion all the way up until delivery,
suggesting that many of the factors are tightly regulated. Especially factors previously
associated with poor pregnancy outcomes such as IFN-γ and IFN-α were very stable.
Interestingly, some factors that we measured, especially levels of IL-6, IL-8, IP-10, uPAR and
VEGF/Flt1 increased at delivery compared to the other time points. These findings are in
agreement with those of others159 who have reported increased levels of some of these factors
at the end of a normal pregnancy, probably as a result of initiating labor.
34
To be able to investigate the influence of an infection on the selected markers we analyzed
the factors in pregnant women with or without a P. falciparum infection. It is widely accepted
that infection with P. falciparum during pregnancy can alter the cytokine and chemokine
balance both in the placenta as well as in the periphery121, 148, 150, 151, 160, 161 and that this might
lead to poor pregnancy outcomes.141,
143, 151, 160, 161
As a result of infection, we observed
increased levels of MIG, MCP-1 and IP-10 in infected women compared to uninfected
controls. One fundamental feature often seen during placental malaria is the massive
inflammatory infiltrates of monocytes and macrophages into the placenta, probably as a cause
of parasite adherence.141-143 The increased concentration of the measured chemokines might
aid the recruitment of these cells into the placenta as a means for the phagocytic cells to move
to the infected site and efficiently kill the parasites. Increased levels of several chemokines
have previously been reported during placental malaria121,
122, 148
and especially IP-10 have
been shown to be produced by intervillous blood mononuclear cells isolated from infected
placentas149 further supporting this. Of all the molecules we quantified, the level of RANTES
was the only one that decreased in association with infection. RANTES appears to be
important in various inflammatory diseases, however the role for RANTES during malaria is
not completely known. Low levels of RANTES have previously been reported in severe
malaria cases123,
162
and of note in association with cerebral malaria.163 Studies in mice
deficient in RANTES production have shown a dysfunction of virus-specific CD8+ T cells
with imbalanced cytokine production and viral control.164 RANTES is considered to be a
chemoattractant for T cells but also act on monocytes and DCs. These studies suggest that
RANTES has an important role in regulating and/or sustaining immune responses during viral
infections.
We also confirmed increased levels of IL-10 in the infected women compared to controls,
which has been reported earlier.165 However, in this study by using a longitudinal design, we
also showed that IL-10 together with IP-10 levels increases in the infected women irrespective
of their gestational age. IL-10 is a cytokine that has been involved in both protection and in
the pathogenesis of malaria. High levels might be beneficial for the host by reducing
exaggerated harmful inflammation, but may also be detrimental by suppressing the antiparasitic specific responses that are needed for parasite clearance. On the other hand, IP-10 is
pro-inflammatory and might be induced when schizont ruptures and malaria antigens are
released. Thus, this factor has previously been associated with mortality in P. falciparum
mediated cerebral malaria.166, 167 In addition, IP-10 has also been shown to be produced in high
35
amounts of uNK cells.168 Both IP-10 and IL-10 might be important markers for inflammation
during placental malaria.
From a clinical point of view, it is known that primigravidae women are more susceptible
to malaria infection with more severe symptoms and clinical complications when infected as
compared to multigravidae women.169,
170
In line with this, we show that babies born from
infected primigravidae women have a lower birth weight compared to secundigravidae and
multigravidae women. Likewise, we could also show that primigravidae women have lower
heamoglobulin levels and white blood cell count when infected as compared to
secundigravidae and multigravidae women. This may suggest that the infection is more severe
during the first pregnancy and that this could affect the health of both the mother and of her
fetus.
Overall, to the best of our knowledge there are no comprehensive prospective, longitudinal
studies that describe cytokine and chemokine profiles during pregnancy and at delivery. In
this study, we evaluated a panel of selected markers in plasma from infected and uninfected
pregnant women and found that levels of IL-10 and IP-10 might be valuable for diagnostic
purposes when investigating inflammation during pregnancy malaria.
36
Paper II
Studies in African countries comparing different aspects of the immune responses elicited
by the malaria parasite have been conducted in the Fulani and their sympatric ethnic
groups.152, 153 In these studies it has been shown that the Fulani are less susceptible to develop
malaria compared to their neighboring groups, although they live under the same parasite
pressure.153 However, most of the studies performed on these ethnic groups have been done on
asymptomatic adults and very little is known about these responses in children from these
ethnic groups. The focus of our study has therefore been to investigate the inflammatory and
antibody responses in infected and uninfected children from the Fulani and Dogon ethnic
groups living in Mali. More specifically our study aimed to investigate whether these
differences that have already been established in adults could also be evident during
childhood.
Cytokines and chemokines are important mediators during P. falciparum infection and the
balance between pro and anti-inflammatory cytokines may be important in terms of the
clinical outcome of the disease. In this study the inflammatory cytokines; IL-1beta, IL-6, IL-8,
IL-10, IL-12p70, TNF-α, IFN-α and IFN-γ and the chemokines; RANTES, MIG, MCP-1 and
IP-10 were analysed in plasma samples from children belonging to the two ethnic groups. For
IL-1β, IL-10 and TNF no conclusive results could be found due to the fact that many samples
had levels of these cytokines below the detection limit. When comparing the Fulani and the
Dogon children, regardless of infectious status, we found higher levels of all tested cytokines
in the Fulani children compared to Dogon. The same pattern of increased cytokine levels were
also found in infected Fulani children when compared to infected Dogon children. This
indicates that Fulani children have higher baseline levels of these tested cytokines compared
to Dogon children.
Interestingly, when comparing infected children between the ethnic groups, only IFN-γ was
significantly higher in infected Fulani. This is in agreement with what has been reported by
McCall and colleagues171 who reported that mononuclear cells from Fulani are able to produce
much more IFN-γ after stimulation with parasite antigens compared to cells from the Dogon.
High levels of IFN-γ are crucial early during an infection to limit the first wave of
parasitemia. The increased levels seen in infected Fulani compared to infected Dogon might
be one reason for Fulani’s relative better protection against malaria.
37
Chemokines are essential mediators in directing cells in the immune system. However,
chemokines and their distribution in plasma have never been investigated in Dogon and
Fulani ethnic groups. Our results showed that the Fulani children have higher basal
chemokine levels compared to the Dogon. Further, upon infection the levels increased in
infected Dogon compared to uninfected counterparts, while this marked up-regulation was not
evident in infected Fulani compared to their uninfected peers. The stable levels of MIG,
MCP-1 and IP-10, all of which are potent chemoattractants for immune cells, such as
monocytes, macrophages and T cells further suggests that Fulani children already are more
prepared to combat infections compared to their neighboring ethnic group.
We also measured the anti-malaria specific antibodies; IgG, IgM and IgG subclasses
(IgG1-IgG4) in plasma from these children. We confirmed the higher titers of anti-malariaspecific antibodies in Fulani compared to Dogon children, as previously reported by others in
adults.132-134 In fact, our study showed significantly higher levels of anti-malaria specific total
IgG, IgM as well as higher levels of the IgG1, IgG2 and IgG3 but not IgG4 subclasses in
Fulani compared to Dogon children. Interestingly, when children were divided into infected
and uninfected indviduals, we observed decreased levels of IgG3 antibodies in infected Fulani
as compared to their uninfected counterparts. This result may suggest that the IgG3 antibodies
have bound parasite-derived antigens and formed immune complexes and hence are therefore
found in lower levels in circulation.
Taken together, our results clearly show that the Fulani children have a stronger
inflammatory response compared to the Dogon. This was evident from the fact that Fulani had
higher baseline levels of several cytokine and chemokine levels in their circulation. We also
confirmed the higher titers of malaria-specific antibodies in the Fulani children compared to
Dogon children. Furthermore, upon infection with P. falciparum the infected Dogon children
had increased levels of several chemokines tested compared to their uninfected counterpart,
and these differences were not evident between infected and uninfected Fulani children. These
findings suggest that Fulani are more prone to combat pathogens than Dogon children and
that these differences that we observed already are established at an early age.
38
GENERAL CONCLUSIONS
In this work, we investigated the inflammatory response due to infection with P.
falciparum in pregnant women and children in two African cohorts. We show that an
infection during pregnancy causes a change in the cytokine profile of several of the factors we
investigated. When the women’s cytokine profile was assessed longitudinally we found
transient increases in the levels of IL-10 and IP-10, regardless of the gestational age of the
women. These results suggest that IL-10 and IP-10 might be important biomarkers for
inflammation during placental malaria. We also confirmed that primigravid women are more
affected by the disease when infected, as reflected by lower haemoglobulin levels and white
blood cell count as well as more often giving birth to low birth weight babies, compared to
infected secundigravidae and multigravidae.
We also show that the inflammatory response differs in children from two ethnic groups
with known different susceptibility to contract malaria. We show that Fulani children, like
their adult population, have increased cytokine and malaria-specific antibody titers as
compared to their sympatric ethnic group Dogon. We also show, for the first time, that Fulani
children have stable chemokine levels in their circulation upon an infection, while this was
not the case in Dogon children. These findings suggests that there are marked differences in
the cytokine and chemokine levels as well as antibody titers between Fulani and Dogon that
are established already at an early age.
39
FUTURE PERSPECTIVES
Our results from both paper I and II showed that the cytokine and chemokine levels
changed upon P. falciparum infection. However, the malaria transmission in Tanzania (where
the first study was conducted) is very low. Therefore, we will continue to investigate these
responses in pregnant women living in areas where they are more frequently exposed to
malaria parasites (such as the site in Benin) to see if we can repeat the findings we observed
in Tanzania in women with multiple infections during pregnancy. To further evaluate this,
cells from infected and uninfected pregnant women will be phenotypically analyzed for cell
surface markers and co-stimulatory molecules and their association with maternal plasma
levels and occurrence of P. falciparum infection will be evaluated. These analyses will be
performed on inclusion and delivery samples and prospective analyses will be conducted.
Furthermore, from the study site in Tanzania, we have biochemical and haematological
data from each participating women from all time points when they came to the hospital. A
follow up analysis with the same women as in paper I will be performed to investigate the
pathophysiology of P. falciparum infection during pregnancy. Different parameters such as
heamoglobulin, white blood cell counts, platelets, alanine transaminase and creatinine etc.
will be analyzed in these women. We will use internal control values from the women´s own
antenatal visit at which they were uninfected to create a second control group and determine
which factors that might be associated with P. falciparum infection. We will also evaluate if
primigravidae differ compared to secundigravidae or multigravidae of these parameters.
In addition, the results from paper I showed an increase in the levels of IL-10 and IP-10
when the pregnant women had an infection. It would be interesting to set up in vitro assays in
which cell populations know to be important in placental malaria (for example monocytes) are
stimulated with these two factors. The aim would be to investigate how the inflammatory
milieu (caused by the parasites) influences these cells migration, co-stimulatory capacity and
secretion of cytokines to broaden the picture on how the parasites would influence these cells.
Buffy coats will be used to purify monocytes (and their subsets) that will be put in co-cultures
together with IP-10 or IL-10 (to mimic the inflammatory milieu in the pregnant women) and
later challenge these cells with parasite antigens to see how this would affect these cells
ability to migrate but also to investigate their cell surface receptors, co-receptors and what
type of cytokines they produce at protein (from supernatants) or mRNA level.
40
ACKNOWLEDGEMENTS
My warmest thanks to my supervisor Marita Troye-Blomberg, for accepting me in her
group and by doing so, giving me a chance to grow in my work as well as in life. The
challenges have been many and even though I spent my first PhD months away in Tanzania,
you have always supported me and helped me solve my troubles.
I would also like to give my profound thanks to my co-supervisor Adrian JF Luty, for great
support during my writing process, giving me constructive feedback and always encouraging
me to do better.
Thanks to all the senior scientists: Carmen Fernandez, Klavs Berzins, Eva SverremarkEkström and Eva Severinson for your great knowledge and expertise.
To Maggan, Gelana and Anna-Lena for your unbearable help at the department, making
sure everything works smoothly.
To all my co-authors and collaborators in my project, especially to Christentze
Schmiegelow, Mayke Oesterholt, Daniel Minja and Samad Ibitokou for great support and
willingness to always help me. Thanks for all the wonderful time together, especially while
living in Tanzania, both at work and off work.
A special thanks to Pablo Giusti, for endless help during my studies, beginning from my
internship to degree work and all the way up to my current PhD studies. Thank you for your
friendship and your big heart.
To Charles Arama, for your kindness and willingness to always help me whenever I have
questions. You truly are a wonderful person.
To my fantastic roommate Ioana Bujila, for never-ending support and smile through times
when everything is up-side-down. I am really happy to have crossed your path in life and are
looking forward to many more smiles and happy moments together with you.
To all the past and present students at the Immunology department.
Lastly I would like to thank my dearest family, my mother, brothers and especially to my
sister. Thank you for believing at me, I would never have come this far without your help.
Massor med kramar till er!
41
REFERENCES
1. Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immunity: update
on Toll-like receptors. Nat. Immunol. 11, 373-384 (2010).
2. Kumar, H., Kawai, T. & Akira, S. Pathogen recognition by the innate immune system. Int.
Rev. Immunol. 30, 16-34 (2011).
3. Matzinger, P. The danger model: a renewed sense of self. Science 296, 301-305 (2002).
4. Passlick, B., Flieger, D. & Ziegler-Heitbrock, H. W. Identification and characterization of a
novel monocyte subpopulation in human peripheral blood. Blood 74, 2527-2534 (1989).
5. Geissmann, F., Jung, S. & Littman, D. R. Blood monocytes consist of two principal subsets
with distinct migratory properties. Immunity 19, 71-82 (2003).
6. Weber, C. et al. Differential chemokine receptor expression and function in human
monocyte subpopulations. J. Leukoc. Biol. 67, 699-704 (2000).
7. Ziegler-Heitbrock, L. The CD14+ CD16+ blood monocytes: their role in infection and
inflammation. J. Leukoc. Biol. 81, 584-592 (2007).
8. Iwasaki, A. & Medzhitov, R. Regulation of adaptive immunity by the innate immune
system. Science 327, 291-295 (2010).
9. Harrington, L. E. et al. Interleukin 17-producing CD4+ effector T cells develop via a
lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 6, 1123-1132 (2005).
10. Park, H. et al. A distinct lineage of CD4 T cells regulates tissue inflammation by
producing interleukin 17. Nat. Immunol. 6, 1133-1141 (2005).
11. Kimura, A. & Kishimoto, T. Th17 cells in inflammation. Int. Immunopharmacol. 11, 319322 (2011).
12. Crome, S. Q., Wang, A. Y. & Levings, M. K. Translational mini-review series on Th17
cells: function and regulation of human T helper 17 cells in health and disease. Clin. Exp.
Immunol. 159, 109-119 (2010).
42
13. Peck, A. & Mellins, E. D. Precarious balance: Th17 cells in host defense. Infect. Immun.
78, 32-38 (2010).
14. Sakaguchi, S. Naturally arising CD4+ regulatory t cells for immunologic self-tolerance
and negative control of immune responses. Annu. Rev. Immunol. 22, 531-562 (2004).
15. Baecher-Allan, C., Brown, J. A., Freeman, G. J. & Hafler, D. A. CD4+CD25high
regulatory cells in human peripheral blood. J. Immunol. 167, 1245-1253 (2001).
16. Stephens, L. A., Mottet, C., Mason, D. & Powrie, F. Human CD4(+)CD25(+) thymocytes
and peripheral T cells have immune suppressive activity in vitro. Eur. J. Immunol. 31, 12471254 (2001).
17. Ehrenstein, M. R. & Notley, C. A. The importance of natural IgM: scavenger, protector
and regulator. Nat. Rev. Immunol. 10, 778-786 (2010).
18. Kristoffersen, E. K. Placental Fc receptors and the transfer of maternal IgG. Transfus.
Med. Rev. 14, 234-243 (2000).
19. Papadea, C. & Check, I. J. Human immunoglobulin G and immunoglobulin G subclasses:
biochemical, genetic, and clinical aspects. Crit. Rev. Clin. Lab. Sci. 27, 27-58 (1989).
20. Heiner, D. C. Significance of immunoglobulin G subclasses. Am. J. Med. 76, 1-6 (1984).
21. Chen, K. & Cerutti, A. The function and regulation of immunoglobulin D. Curr. Opin.
Immunol. 23, 345-352 (2011).
22. Cerutti, A., Chen, K. & Chorny, A. Immunoglobulin responses at the mucosal interface.
Annu. Rev. Immunol. 29, 273-293 (2011).
23. Johansson, S. G. The History of IgE: From discovery to 2010. Curr. Allergy Asthma Rep.
11, 173-177 (2011).
24. Dimitriou, I. D. et al. Putting out the fire: coordinated suppression of the innate and
adaptive immune systems by SOCS1 and SOCS3 proteins. Immunol. Rev. 224, 265-283
(2008).
43
25. Dalpke, A., Heeg, K., Bartz, H. & Baetz, A. Regulation of innate immunity by suppressor
of cytokine signaling (SOCS) proteins. Immunobiology 213, 225-235 (2008).
26. Baggiolini, M., Dewald, B. & Moser, B. Human chemokines: an update. Annu. Rev.
Immunol. 15, 675-705 (1997).
27. Rollins, B. J. Chemokines. Blood 90, 909-928 (1997).
28. Martinon, F., Mayor, A. & Tschopp, J. The inflammasomes: guardians of the body. Annu.
Rev. Immunol. 27, 229-265 (2009).
29. Arend, W. P., Palmer, G. & Gabay, C. IL-1, IL-18, and IL-33 families of cytokines.
Immunol. Rev. 223, 20-38 (2008).
30. Aggarwal, B. B. Signalling pathways of the TNF superfamily: a double-edged sword. Nat.
Rev. Immunol. 3, 745-756 (2003).
31. Suenaert, P. et al. Anti-tumor necrosis factor treatment restores the gut barrier in Crohn's
disease. Am. J. Gastroenterol. 97, 2000-2004 (2002).
32. Feldmann, M. & Maini, R. N. Anti-TNF alpha therapy of rheumatoid arthritis: what have
we learned? Annu. Rev. Immunol. 19, 163-196 (2001).
33. Jones, S. A. Directing transition from innate to acquired immunity: defining a role for IL6. J. Immunol. 175, 3463-3468 (2005).
34. Kishimoto, T. IL-6: from its discovery to clinical applications. Int. Immunol. 22, 347-352
(2010).
35. Hunter, C. A. New IL-12-family members: IL-23 and IL-27, cytokines with divergent
functions. Nat. Rev. Immunol. 5, 521-531 (2005).
36. Gee, K., Guzzo, C., Che Mat, N. F., Ma, W. & Kumar, A. The IL-12 family of cytokines
in infection, inflammation and autoimmune disorders. Inflamm. Allergy Drug Targets 8, 4052 (2009).
37. Koyama, S., Ishii, K. J., Coban, C. & Akira, S. Innate immune response to viral infection.
Cytokine 43, 336-341 (2008).
44
38. Takaoka, A. & Yanai, H. Interferon signalling network in innate defence. Cell. Microbiol.
8, 907-922 (2006).
39. Ouyang, W., Rutz, S., Crellin, N. K., Valdez, P. A. & Hymowitz, S. G. Regulation and
functions of the IL-10 family of cytokines in inflammation and disease. Annu. Rev. Immunol.
29, 71-109 (2011).
40. Moore, K. W., de Waal Malefyt, R., Coffman, R. L. & O'Garra, A. Interleukin-10 and the
interleukin-10 receptor. Annu. Rev. Immunol. 19, 683-765 (2001).
41. Barrat, F. J. et al. In vitro generation of interleukin 10-producing regulatory CD4(+) T
cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and
Th2-inducing cytokines. J. Exp. Med. 195, 603-616 (2002).
42. Moore, K. W., O'Garra, A., de Waal Malefyt, R., Vieira, P. & Mosmann, T. R.
Interleukin-10. Annu. Rev. Immunol. 11, 165-190 (1993).
43. Li, M. O., Wan, Y. Y., Sanjabi, S., Robertson, A. K. & Flavell, R. A. Transforming
growth factor-beta regulation of immune responses. Annu. Rev. Immunol. 24, 99-146 (2006).
44. Yoshimura, A., Wakabayashi, Y. & Mori, T. Cellular and molecular basis for the
regulation of inflammation by TGF-beta. J. Biochem. 147, 781-792 (2010).
45. Li, M. O. & Flavell, R. A. TGF-beta: a master of all T cell trades. Cell 134, 392-404
(2008).
46. Kulkarni, A. B. et al. Transforming growth factor beta 1 null mutation in mice causes
excessive inflammatory response and early death. Proc. Natl. Acad. Sci. U. S. A. 90, 770-774
(1993).
47. Nasser, M. W. et al. CXCR1 and CXCR2 activation and regulation. Role of aspartate 199
of the second extracellular loop of CXCR2 in CXCL8-mediated rapid receptor internalization.
J. Biol. Chem. 282, 6906-6915 (2007).
48. Levy, J. A. The unexpected pleiotropic activities of RANTES. J. Immunol. 182, 39453946 (2009).
45
49. Appay, V. & Rowland-Jones, S. L. RANTES: a versatile and controversial chemokine.
Trends Immunol. 22, 83-87 (2001).
50. Wagner, L. et al. Beta-chemokines are released from HIV-1-specific cytolytic T-cell
granules complexed to proteoglycans. Nature 391, 908-911 (1998).
51. Bodaghi, B. et al. Chemokine sequestration by viral chemoreceptors as a novel viral
escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected
cells. J. Exp. Med. 188, 855-866 (1998).
52. Fatkenheuer, G. et al. Efficacy of short-term monotherapy with maraviroc, a new CCR5
antagonist, in patients infected with HIV-1. Nat. Med. 11, 1170-1172 (2005).
53. Dufour, J. H. et al. IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice
reveal a role for IP-10 in effector T cell generation and trafficking. J. Immunol. 168, 31953204 (2002).
54. Melgarejo, E., Medina, M. A., Sanchez-Jimenez, F. & Urdiales, J. L. Monocyte
chemoattractant protein-1: a key mediator in inflammatory processes. Int. J. Biochem. Cell
Biol. 41, 998-1001 (2009).
55. Yadav, A., Saini, V. & Arora, S. MCP-1: chemoattractant with a role beyond immunity: a
review. Clin. Chim. Acta 411, 1570-1579 (2010).
56. Owen, R. D. Immunogenetic Consequences of Vascular Anastomoses between Bovine
Twins. Science 102, 400-401 (1945).
57. BILLINGHAM, R. E., BRENT, L. & MEDAWAR, P. B. Actively acquired tolerance of
foreign cells. Nature 172, 603-606 (1953).
58. MEDAWAR, P. B. Some immunological and endocrinological problems raised by
evolution of viviparity in vertebrates. Symp. Soc. Exp. Biol. 7, 320-328 (1953).
59. Wegmann, T. G., Lin, H., Guilbert, L. & Mosmann, T. R. Bidirectional cytokine
interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon?
Immunol. Today 14, 353-356 (1993).
46
60. Da Silva, J. A. & Spector, T. D. The role of pregnancy in the course and aetiology of
rheumatoid arthritis. Clin. Rheumatol. 11, 189-194 (1992).
61. Khamashta, M. A., Ruiz-Irastorza, G. & Hughes, G. R. Systemic lupus erythematosus
flares during pregnancy. Rheum. Dis. Clin. North Am. 23, 15-30 (1997).
62. Wong, K. L., Chan, F. Y. & Lee, C. P. Outcome of pregnancy in patients with systemic
lupus erythematosus. A prospective study. Arch. Intern. Med. 151, 269-273 (1991).
63. Marzi, M. et al. Characterization of type 1 and type 2 cytokine production profile in
physiologic and pathologic human pregnancy. Clin. Exp. Immunol. 106, 127-133 (1996).
64. Matthiesen, L., Ekerfelt, C., Berg, G. & Ernerudh, J. Increased numbers of circulating
interferon-gamma- and interleukin-4-secreting cells during normal pregnancy. Am. J. Reprod.
Immunol. 39, 362-367 (1998).
65. Lin, H., Mosmann, T. R., Guilbert, L., Tuntipopipat, S. & Wegmann, T. G. Synthesis of T
helper 2-type cytokines at the maternal-fetal interface. J. Immunol. 151, 4562-4573 (1993).
66. Ashkar, A. A., Di Santo, J. P. & Croy, B. A. Interferon gamma contributes to initiation of
uterine vascular modification, decidual integrity, and uterine natural killer cell maturation
during normal murine pregnancy. J. Exp. Med. 192, 259-270 (2000).
67. Chaouat, G. et al. Control of fetal survival in CBA x DBA/2 mice by lymphokine therapy.
J. Reprod. Fertil. 89, 447-458 (1990).
68. Hill, J. A., Polgar, K. & Anderson, D. J. T-helper 1-type immunity to trophoblast in
women with recurrent spontaneous abortion. JAMA 273, 1933-1936 (1995).
69. Rezaei, A. & Dabbagh, A. T-helper (1) cytokines increase during early pregnancy in
women with a history of recurrent spontaneous abortion. Med. Sci. Monit. 8, CR607-10
(2002).
70. Raghupathy, R. et al. Cytokine production by maternal lymphocytes during normal human
pregnancy and in unexplained recurrent spontaneous abortion. Hum. Reprod. 15, 713-718
(2000).
47
71. Trowsdale, J. & Betz, A. G. Mother's little helpers: mechanisms of maternal-fetal
tolerance. Nat. Immunol. 7, 241-246 (2006).
72. Mor, G., Cardenas, I., Abrahams, V. & Guller, S. Inflammation and pregnancy: the role of
the immune system at the implantation site. Ann. N. Y. Acad. Sci. 1221, 80-87 (2011).
73. Sargent, I. L. Maternal and fetal immune responses during pregnancy. Exp. Clin.
Immunogenet. 10, 85-102 (1993).
74. Munz, C. et al. Human histocompatibility leukocyte antigen (HLA)-G molecules inhibit
NKAT3 expressing natural killer cells. J. Exp. Med. 185, 385-391 (1997).
75. Kovats, S. et al. A class I antigen, HLA-G, expressed in human trophoblasts. Science 248,
220-223 (1990).
76. Guller, S. & LaChapelle, L. The role of placental Fas ligand in maintaining immune
privilege at maternal-fetal interfaces. Semin. Reprod. Endocrinol. 17, 39-44 (1999).
77. Munn, D. H. et al. Prevention of allogeneic fetal rejection by tryptophan catabolism.
Science 281, 1191-1193 (1998).
78. Hedlund, M. et al. Human placenta expresses and secretes NKG2D ligands via exosomes
that down-modulate the cognate receptor expression: evidence for immunosuppressive
function. J. Immunol. 183, 340-351 (2009).
79. Mincheva-Nilsson, L. & Baranov, V. The role of placental exosomes in reproduction. Am.
J. Reprod. Immunol. 63, 520-533 (2010).
80. Sacks, G., Sargent, I. & Redman, C. An innate view of human pregnancy. Immunol.
Today 20, 114-118 (1999).
81. Aluvihare, V. R., Kallikourdis, M. & Betz, A. G. Regulatory T cells mediate maternal
tolerance to the fetus. Nat. Immunol. 5, 266-271 (2004).
82. Bulmer, J. N., Pace, D. & Ritson, A. Immunoregulatory cells in human decidua:
morphology, immunohistochemistry and function. Reprod. Nutr. Dev. 28, 1599-1613 (1988).
48
83. King, A., Loke, Y. W. & Chaouat, G. NK cells and reproduction. Immunol. Today 18, 6466 (1997).
84. Heikkinen, J., Mottonen, M., Alanen, A. & Lassila, O. Phenotypic characterization of
regulatory T cells in the human decidua. Clin. Exp. Immunol. 136, 373-378 (2004).
85. Sasaki, Y. et al. Decidual and peripheral blood CD4+CD25+ regulatory T cells in early
pregnancy subjects and spontaneous abortion cases. Mol. Hum. Reprod. 10, 347-353 (2004).
86. Le Bouteiller, P. & Piccinni, M. P. Human NK cells in pregnant uterus: why there? Am. J.
Reprod. Immunol. 59, 401-406 (2008).
87. Hanna, J. et al. Decidual NK cells regulate key developmental processes at the human
fetal-maternal interface. Nat. Med. 12, 1065-1074 (2006).
88. Williams, P. J., Bulmer, J. N., Searle, R. F., Innes, B. A. & Robson, S. C. Altered decidual
leucocyte populations in the placental bed in pre-eclampsia and foetal growth restriction: a
comparison with late normal pregnancy. Reproduction 138, 177-184 (2009).
89. Drake, P. M. et al. Human placental cytotrophoblasts attract monocytes and CD56(bright)
natural killer cells via the actions of monocyte inflammatory protein 1alpha. J. Exp. Med. 193,
1199-1212 (2001).
90. Jaffe, R. First trimester utero-placental circulation: maternal-fetal interaction. J. Perinat.
Med. 26, 168-174 (1998).
91. Whitley, G. S. & Cartwright, J. E. Trophoblast-mediated spiral artery remodelling: a role
for apoptosis. J. Anat. 215, 21-26 (2009).
92. Lyall, F. Priming and remodelling of human placental bed spiral arteries during
pregnancy--a review. Placenta 26 Suppl A, S31-6 (2005).
93. Ben-Hur, H. et al. Transport of maternal immunoglobulins through the human placental
barrier in normal pregnancy and during inflammation. Int. J. Mol. Med. 16, 401-407 (2005).
94. Roth, I. et al. Human placental cytotrophoblasts produce the immunosuppressive cytokine
interleukin 10. J. Exp. Med. 184, 539-548 (1996).
49
95. Hilkens, C. M. et al. Differential modulation of T helper type 1 (Th1) and T helper type 2
(Th2) cytokine secretion by prostaglandin E2 critically depends on interleukin-2. Eur. J.
Immunol. 25, 59-63 (1995).
96. Szekeres-Bartho, J. & Wegmann, T. G. A progesterone-dependent immunomodulatory
protein alters the Th1/Th2 balance. J. Reprod. Immunol. 31, 81-95 (1996).
97. World health organization. World malaria report 2010. (2010).
98. Jones, M. K. & Good, M. F. Malaria parasites up close. Nat. Med. 12, 170-171 (2006).
99. Hermsen, C. C. et al. Circulating concentrations of soluble granzyme A and B increase
during natural and experimental Plasmodium falciparum infections. Clin. Exp. Immunol. 132,
467-472 (2003).
100. Artavanis-Tsakonas, K. & Riley, E. M. Innate immune response to malaria: rapid
induction of IFN-gamma from human NK cells by live Plasmodium falciparum-infected
erythrocytes. J. Immunol. 169, 2956-2963 (2002).
101. Dodoo, D. et al. Absolute levels and ratios of proinflammatory and anti-inflammatory
cytokine production in vitro predict clinical immunity to Plasmodium falciparum malaria. J.
Infect. Dis. 185, 971-979 (2002).
102. Luty, A. J. et al. Interferon-gamma responses are associated with resistance to
reinfection with Plasmodium falciparum in young African children. J. Infect. Dis. 179, 980988 (1999).
103. Stevenson, M. M. & Riley, E. M. Innate immunity to malaria. Nat. Rev. Immunol. 4,
169-180 (2004).
104. Favre, N., Ryffel, B., Bordmann, G. & Rudin, W. The course of Plasmodium chabaudi
chabaudi infections in interferon-gamma receptor deficient mice. Parasite Immunol. 19, 375383 (1997).
105. Clark, I. A., Hunt, N. H., Butcher, G. A. & Cowden, W. B. Inhibition of murine malaria
(Plasmodium chabaudi) in vivo by recombinant interferon-gamma or tumor necrosis factor,
and its enhancement by butylated hydroxyanisole. J. Immunol. 139, 3493-3496 (1987).
50
106. Torre, D. et al. Role of Th1 and Th2 cytokines in immune response to uncomplicated
Plasmodium falciparum malaria. Clin. Diagn. Lab. Immunol. 9, 348-351 (2002).
107. De Souza, J. B., Williamson, K. H., Otani, T. & Playfair, J. H. Early gamma interferon
responses in lethal and nonlethal murine blood-stage malaria. Infect. Immun. 65, 1593-1598
(1997).
108. Sedegah, M., Finkelman, F. & Hoffman, S. L. Interleukin 12 induction of interferon
gamma-dependent protection against malaria. Proc. Natl. Acad. Sci. U. S. A. 91, 10700-10702
(1994).
109. Luty, A. J. et al. Low interleukin-12 activity in severe Plasmodium falciparum malaria.
Infect. Immun. 68, 3909-3915 (2000).
110. de Kossodo, S. & Grau, G. E. Role of cytokines and adhesion molecules in malaria
immunopathology. Stem Cells 11, 41-48 (1993).
111. John, C. C., Park, G. S., Sam-Agudu, N., Opoka, R. O. & Boivin, M. J. Elevated serum
levels of IL-1ra in children with Plasmodium falciparum malaria are associated with
increased severity of disease. Cytokine 41, 204-208 (2008).
112. Prakash, D. et al. Clusters of cytokines determine malaria severity in Plasmodium
falciparum-infected patients from endemic areas of Central India. J. Infect. Dis. 194, 198-207
(2006).
113. Clark, I. A., Cowden, W. B., Butcher, G. A. & Hunt, N. H. Possible roles of tumor
necrosis factor in the pathology of malaria. Am. J. Pathol. 129, 192-199 (1987).
114. Kurtzhals, J. A. et al. Low plasma concentrations of interleukin 10 in severe malarial
anaemia compared with cerebral and uncomplicated malaria. Lancet 351, 1768-1772 (1998).
115. May, J., Lell, B., Luty, A. J., Meyer, C. G. & Kremsner, P. G. Plasma interleukin10:Tumor necrosis factor (TNF)-alpha ratio is associated with TNF promoter variants and
predicts malarial complications. J. Infect. Dis. 182, 1570-1573 (2000).
51
116. Othoro, C. et al. A low interleukin-10 tumor necrosis factor-alpha ratio is associated with
malaria anemia in children residing in a holoendemic malaria region in western Kenya. J.
Infect. Dis. 179, 279-282 (1999).
117. Peyron, F. et al. High levels of circulating IL-10 in human malaria. Clin. Exp. Immunol.
95, 300-303 (1994).
118. de Waal Malefyt, R. et al. Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigenspecific human T cell proliferation by diminishing the antigen-presenting capacity of
monocytes via downregulation of class II major histocompatibility complex expression. J.
Exp. Med. 174, 915-924 (1991).
119. Charo, I. F. & Ransohoff, R. M. The many roles of chemokines and chemokine receptors
in inflammation. N. Engl. J. Med. 354, 610-621 (2006).
120. Burgmann, H. et al. Serum concentrations of MIP-1 alpha and interleukin-8 in patients
suffering from acute Plasmodium falciparum malaria. Clin. Immunol. Immunopathol. 76, 3236 (1995).
121. Abrams, E. T. et al. Host response to malaria during pregnancy: placental monocyte
recruitment is associated with elevated beta chemokine expression. J. Immunol. 170, 27592764 (2003).
122. Chaisavaneeyakorn, S. et al. Levels of macrophage inflammatory protein 1 alpha (MIP-1
alpha) and MIP-1 beta in intervillous blood plasma samples from women with placental
malaria and human immunodeficiency virus infection. Clin. Diagn. Lab. Immunol. 10, 631636 (2003).
123. Ochiel, D. O. et al. Differential regulation of beta-chemokines in children with
Plasmodium falciparum malaria. Infect. Immun. 73, 4190-4197 (2005).
124. Fahey, T. J.,3rd et al. Macrophage inflammatory protein 1 modulates macrophage
function. J. Immunol. 148, 2764-2769 (1992).
125. COHEN, S., McGREGOR, I. A. & CARRINGTON, S. Gamma-globulin and acquired
immunity to human malaria. Nature 192, 733-737 (1961).
52
126. Rotman, H. L., Daly, T. M., Clynes, R. & Long, C. A. Fc receptors are not required for
antibody-mediated protection against lethal malaria challenge in a mouse model. J. Immunol.
161, 1908-1912 (1998).
127. Bolad, A. & Berzins, K. Antigenic diversity of Plasmodium falciparum and antibodymediated parasite neutralization. Scand. J. Immunol. 52, 233-239 (2000).
128. Bouharoun-Tayoun, H., Attanath, P., Sabchareon, A., Chongsuphajaisiddhi, T. &
Druilhe, P. Antibodies that protect humans against Plasmodium falciparum blood stages do
not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with
monocytes. J. Exp. Med. 172, 1633-1641 (1990).
129. Bouharoun-Tayoun, H. & Druilhe, P. Plasmodium falciparum malaria: evidence for an
isotype imbalance which may be responsible for delayed acquisition of protective immunity.
Infect. Immun. 60, 1473-1481 (1992).
130. Groux, H. & Gysin, J. Opsonization as an effector mechanism in human protection
against asexual blood stages of Plasmodium falciparum: functional role of IgG subclasses.
Res. Immunol. 141, 529-542 (1990).
131. Sarthou, J. L. et al. Prognostic value of anti-Plasmodium falciparum-specific
immunoglobulin G3, cytokines, and their soluble receptors in West African patients with
severe malaria. Infect. Immun. 65, 3271-3276 (1997).
132. Bolad, A. et al. Distinct interethnic differences in immunoglobulin G class/subclass and
immunoglobulin M antibody responses to malaria antigens but not in immunoglobulin G
responses to nonmalarial antigens in sympatric tribes living in West Africa. Scand. J.
Immunol. 61, 380-386 (2005).
133. Israelsson, E. et al. Differences in Fcgamma receptor IIa genotypes and IgG subclass
pattern of anti-malarial antibodies between sympatric ethnic groups in Mali. Malar J. 7, 175
(2008).
134. Vafa, M. et al. Relationship between immunoglobulin isotype response to Plasmodium
falciparum blood stage antigens and parasitological indexes as well as splenomegaly in
sympatric ethnic groups living in Mali. Acta Trop. 109, 12-16 (2009).
53
135. Aucan, C. et al. High immunoglobulin G2 (IgG2) and low IgG4 levels are associated
with human resistance to Plasmodium falciparum malaria. Infect. Immun. 68, 1252-1258
(2000).
136. Schantz-Dunn, J. & Nour, N. M. Malaria and pregnancy: a global health perspective.
Rev. Obstet. Gynecol. 2, 186-192 (2009).
137. McGregor, I. A. Epidemiology, malaria and pregnancy. Am. J. Trop. Med. Hyg. 33, 517525 (1984).
138. Flick, K. & Chen, Q. var genes, PfEMP1 and the human host. Mol. Biochem. Parasitol.
134, 3-9 (2004).
139. Beeson, J. G., Amin, N., Kanjala, M. & Rogerson, S. J. Selective accumulation of mature
asexual stages of Plasmodium falciparum-infected erythrocytes in the placenta. Infect. Immun.
70, 5412-5415 (2002).
140. Fried, M. & Duffy, P. E. Adherence of Plasmodium falciparum to chondroitin sulfate A
in the human placenta. Science 272, 1502-1504 (1996).
141. Menendez, C. et al. The impact of placental malaria on gestational age and birth weight.
J. Infect. Dis. 181, 1740-1745 (2000).
142. Ordi, J. et al. Massive chronic intervillositis of the placenta associated with malaria
infection. Am. J. Surg. Pathol. 22, 1006-1011 (1998).
143. Rogerson, S. J. et al. Placental monocyte infiltrates in response to Plasmodium
falciparum malaria infection and their association with adverse pregnancy outcomes. Am. J.
Trop. Med. Hyg. 68, 115-119 (2003).
144. Ismail, M. R. et al. Placental pathology in malaria: a histological, immunohistochemical,
and quantitative study. Hum. Pathol. 31, 85-93 (2000).
145. Le Hesran, J. Y. et al. Maternal placental infection with Plasmodium falciparum and
malaria morbidity during the first 2 years of life. Am. J. Epidemiol. 146, 826-831 (1997).
54
146. Guyatt, H. L. & Snow, R. W. Malaria in pregnancy as an indirect cause of infant
mortality in sub-Saharan Africa. Trans. R. Soc. Trop. Med. Hyg. 95, 569-576 (2001).
147. Dorman, E. K. et al. Impaired uteroplacental blood flow in pregnancies complicated by
falciparum malaria. Ultrasound Obstet. Gynecol. 19, 165-170 (2002).
148. Suguitan, A. L.,Jr et al. Changes in the levels of chemokines and cytokines in the
placentas of women with Plasmodium falciparum malaria. J. Infect. Dis. 188, 1074-1082
(2003).
149. Chaisavaneeyakorn, S. et al. Immunity to placental malaria. IV. Placental malaria is
associated with up-regulation of macrophage migration inhibitory factor in intervillous blood.
J. Infect. Dis. 186, 1371-1375 (2002).
150. Fievet, N. et al. Plasmodium falciparum induces a Th1/Th2 disequilibrium, favoring the
Th1-type pathway, in the human placenta. J. Infect. Dis. 183, 1530-1534 (2001).
151. Rogerson, S. J. et al. Placental tumor necrosis factor alpha but not gamma interferon is
associated with placental malaria and low birth weight in Malawian women. Infect. Immun.
71, 267-270 (2003).
152. Dolo, A. et al. Difference in susceptibility to malaria between two sympatric ethnic
groups in Mali. Am. J. Trop. Med. Hyg. 72, 243-248 (2005).
153. Modiano, D. et al. Different response to Plasmodium falciparum malaria in west African
sympatric ethnic groups. Proc. Natl. Acad. Sci. U. S. A. 93, 13206-13211 (1996).
154. Farouk, S. E. et al. Different antibody- and cytokine-mediated responses to Plasmodium
falciparum parasite in two sympatric ethnic tribes living in Mali. Microbes Infect. 7, 110-117
(2005).
155. Bereczky, S. et al. Spleen enlargement and genetic diversity of Plasmodium falciparum
infection in two ethnic groups with different malaria susceptibility in Mali, West Africa.
Trans. R. Soc. Trop. Med. Hyg. 100, 248-257 (2006).
55
156. Torcia, M. G. et al. Functional deficit of T regulatory cells in Fulani, an ethnic group
with low susceptibility to Plasmodium falciparum malaria. Proc. Natl. Acad. Sci. U. S. A.
105, 646-651 (2008).
157. Arama, C. et al. Interethnic differences in antigen-presenting cell activation and TLR
responses in Malian children during Plasmodium falciparum malaria. PLoS One 6, e18319
(2011).
158. Kassam, S. N. et al. Pregnancy outcomes in women with or without placental malaria
infection. Int. J. Gynaecol. Obstet. 93, 225-232 (2006).
159. Opsjln, S. L. et al. Tumor necrosis factor, interleukin-1, and interleukin-6 in normal
human pregnancy. Am. J. Obstet. Gynecol. 169, 397-404 (1993).
160. Fried, M., Muga, R. O., Misore, A. O. & Duffy, P. E. Malaria elicits type 1 cytokines in
the human placenta: IFN-gamma and TNF-alpha associated with pregnancy outcomes. J.
Immunol. 160, 2523-2530 (1998).
161. Moormann, A. M. et al. Malaria and pregnancy: placental cytokine expression and its
relationship to intrauterine growth retardation. J. Infect. Dis. 180, 1987-1993 (1999).
162. Were, T. et al. Suppression of RANTES in children with Plasmodium falciparum
malaria. Haematologica 91, 1396-1399 (2006).
163. John, C. C., Opika-Opoka, R., Byarugaba, J., Idro, R. & Boivin, M. J. Low levels of
RANTES are associated with mortality in children with cerebral malaria. J. Infect. Dis. 194,
837-845 (2006).
164. Crawford, A., Angelosanto, J. M., Nadwodny, K. L., Blackburn, S. D. & Wherry, E. J. A
role for the chemokine RANTES in regulating CD8 T cell responses during chronic viral
infection. PLoS Pathog. 7, e1002098 (2011).
165. Kabyemela, E. R. et al. Maternal peripheral blood level of IL-10 as a marker for
inflammatory placental malaria. Malar J. 7, 26 (2008).
166. Armah, H. B. et al. Cerebrospinal fluid and serum biomarkers of cerebral malaria
mortality in Ghanaian children. Malar J. 6, 147 (2007).
56
167. Jain, V. et al. Plasma IP-10, apoptotic and angiogenic factors associated with fatal
cerebral malaria in India. Malar J. 7, 83 (2008).
168. Vacca, P., Moretta, L., Moretta, A. & Mingari, M. C. Origin, phenotype and function of
human natural killer cells in pregnancy. Trends Immunol. 32, 517-523 (2011).
169. Duffy, P. E. Plasmodium in the placenta: parasites, parity, protection, prevention and
possibly preeclampsia. Parasitology 134, 1877-1881 (2007).
170. Hartman, T. K., Rogerson, S. J. & Fischer, P. R. The impact of maternal malaria on
newborns. Ann. Trop. Paediatr. 30, 271-282 (2010).
171. McCall, M. B. et al. Early interferon-gamma response against Plasmodium falciparum
correlates with interethnic differences in susceptibility to parasitemia between sympatric
Fulani and Dogon in Mali. J. Infect. Dis. 201, 142-152 (2010).
57