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

Licentiate thesis from the Department of Immunology, Wenner-Gren Institute,
Stockholm University, Sweden
Dendritic cells and Plasmodium falciparum:
studies in vitro and in the human host.
Pablo Giusti
Stockholm 2009
“Seamos realistas y hagamos lo impossible”
Ernesto Guevara de la Serna
ii
Summary
Malaria is one of the world’s most threatening diseases. About half the world’s population is
at risk of infection and the infection claims a million lives each year. A vast majority of the
deaths occur in children below the age of 5 in sub-Saharan Africa. Survivors typically acquire
immunity only after long time of repeated exposure and immunity is rapidly lost. Immunity is
created by the activation of naive T cells and their differentiation into effector cells. The most
potent activators of naive T cells are dendritic cells (DCs). The life cycle of DCs is adapted to
find and process microbes in order to be able to present their antigens to T cells and thereby
activate them. Antigen presentation typically takes place in the lymph nodes and that is why
migration to these areas is an essential part of the DC life cycle. Various studies have shown
that DC function may be hampered by the malaria parasite or its components.
We have investigated activation and migratory capacities of DCs upon in vitro exposure of
the malarial pigment hemozoin and Plasmodium falciparum infected red blood cells.
Furthermore, we have assessed the activation status of blood DCs in the Fulani, a traditionally
nomadic population that respond better to malaria infection and exhibit less clinical symptoms
than other ethnicities living under similar conditions, and a neighbouring ethnic group, the
Dogon, in Mali.
Our results indicate that DCs are semi-activated upon malaria exposure in vitro, including
enhanced migratory capacity, partial up-regulation of co-stimulatory markers and no IL-12,
which may lead to inappropriate T-cell priming. We also observed that DCs from the Fulani
have a higher degree of activation than DCs from the Dogon upon malaria exposure in vivo.
We hypothesize that this increased DC activation may be the reason for the relatively
increased protection against malaria.
Taken together, our findings suggest that improper DC activation may contribute to poor
immunity in Malaria.
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List of Papers
This thesis is based on the following manuscripts which will be referred to by their roman
numerals:
I.
Giusti Pablo, Urban Britta, Frascaroli Giada, Tinti Anna, Troye-Blomberg Marita,
Varani Stefania. Synthetic hemozoin induces partial maturation of human
dendritic cells and increases their migration towards lymphoid chemokines.
Manuscript in preparation.
II.
Arama Charles *, Giusti Pablo *, Boström Stéphanie, Dara Victor, Traore Boubacar,
Dolo Amagana, Doumbo Ogobara, Varani Stefania and Troye-Blomberg Marita. Low
frequency of circulating dendritic cells is associated with higher cell activation,
raised specific antibody levels and increased pro-inflammatory responses in
Fulani, an ethnic group with low susceptibility to malaria. Manuscript in
preparation.
* These authors contributed equally to this work.
iv
Table of Contents
Summary .................................................................................................................................. iii
List of Papers ........................................................................................................................... iv
Table of Contents ..................................................................................................................... v
Abbreviations ........................................................................................................................... vi
Introduction .............................................................................................................................. 7
The immune system .............................................................................................................. 7
Innate Immunity ................................................................................................................. 10
Recognition strategies ...................................................................................................... 10
Innate immune cells ......................................................................................................... 13
Effector molecules ............................................................................................................ 18
Adaptive Immunity ............................................................................................................ 19
Recognition strategies ...................................................................................................... 19
Adaptive immune cells ..................................................................................................... 20
Effector molecules ............................................................................................................ 23
Coordinated Immune responses ....................................................................................... 24
Leukocyte movement ....................................................................................................... 24
Antigen presentation ........................................................................................................ 25
Co-stimulation .................................................................................................................. 27
Inflammation .................................................................................................................... 28
Intercellular communication ............................................................................................ 28
Malaria ................................................................................................................................ 31
Malaria burden ................................................................................................................. 31
Parasite life cycle ............................................................................................................. 32
Hemozoin ......................................................................................................................... 33
Project background ................................................................................................................ 34
Immune response to malaria ............................................................................................. 34
Innate responses ............................................................................................................... 34
Antigen-presenting cells in malaria .................................................................................. 34
Antibodies and protective immunity ................................................................................ 35
Cytokines and clinical implications in malaria ................................................................ 36
Fulani and Dogon in northern Mali .................................................................................. 36
Present Study .......................................................................................................................... 38
Aims ..................................................................................................................................... 38
First Study ........................................................................................................................ 38
Second study .................................................................................................................... 38
Methods ............................................................................................................................... 38
First Study ........................................................................................................................ 38
Second study .................................................................................................................... 39
Results and Discussion ....................................................................................................... 39
First Study ........................................................................................................................ 39
Second study .................................................................................................................... 42
General Conclusions .............................................................................................................. 45
Future work ............................................................................................................................ 46
Acknowledgements ................................................................................................................. 47
References ............................................................................................................................... 49
v
Abbreviations
APC
Antigen-presenting cells
MyD88
BCR
B-cell receptor
BDCA
Blood dendritic cell antigen
NK cells
Natural-killer cells
CC
Chemotactic cytokines
NF-kb
Nuclear factor kappa beta
CCR
Chemokine receptor
PAMPs
Pathogen-associated
CD
Cluster of differentiation
DC
Dendritic cell
GPI
Glycosylphosphatidylinositol
primary response gene 88
molecular patterns
PBMC
Peripheral blood
mononuclear cells
anchors
GM-CSF
Myeloid differentiation
PRRs
Granulocyte macrophage-
Pathogen-recognition
receptors
colony stimulating factor
RANTES
Regulated on Activation
Hz
Hemozoin
Normal T Expressed and
HLA
Human-leukocyte antigen
Secreted Protein
Ig
Immunoglobulins
Tc
Cytotoxic-T cell
IFN
Interferon
Th
Helper-T cell
IRAK
IL-1 receptor-associated
TIR
Toll/IL-1 receptor domain
kinase
TRAF
TNF receptor associated
IRF
IFN regulatory factor
IL
Interleukin
LPS
Lipopolysacharide
MCP
Monocytes-chemoattractant
Treg
Regulatory-T cell
protein
TCR
T-cell receptor
Macrophage-inflammatory
TLR
Toll-like receptor
protein
TNF
Tumour Necrosis factor
MIP
MoDC
factor
TRIF
adapter-inducing interferon-β
Monocyte-derived dendritic
cells
MØ
TIR-domain-containing
Macrophages
vi
Introduction
The immune system
The mammalian immune system is an evolutionary product of the co-existence of a wide
variety of microbes with multi-cellular organisms. Consequently, the human immune system
has evolved to become a very complex network, involving everything from peptides to
signalling molecules to specialized cells dispersed all throughout the body. The immune
system is commonly divided into two different branches that are called the innate and the
adaptive immunity.
On the one hand, one branch has evolved to detect danger signals and indiscriminately attack
microbes. This branch is called the innate immune system. Innate immunity is very similar in
all humans and does not, from a short time perspective, seem to adapt to changes in the
surrounding environment. The innate protection is achieved by creating obstacles at many
different levels to impair the pathogens from colonizing the human body. The first barriers are
physical, such as the skin and mucosal surfaces of the body. In this context it is important to
remember that everything from mouth, to stomach, to the end of the intestine can be regarded
as a tunnel through the body and is actually to be regarded as the body’s exterior surfaces.
These surfaces are colonized by enormous amounts of microorganisms that, for the most part,
are not pathogenic. To this end the immune system in the gut is somewhat specialized to
induce tolerance against harmless microorganisms. There are also physiological barriers like
the acidic environment in the stomach. Then there are the antibacterial peptides and
complement proteins in circulation that recognize pathogens and initiate a cascade of
proteolytic activity ultimately leading to lysis of the bacteria. In addition, there are the cells of
the innate immune system equipped with different kinds of receptors to recognize infectious
stimuli.
7
However, since living conditions in different social, cultural and geographical environments
can be very variable, there is a need for adaptation to different circumstances. The other
branch of the immune system has evolved to specifically recognize and remember the
pathogens that it has been exposed to. For that purpose some lymphocytes have evolved to
recognize pathogens specifically and to remember them. That is what is known as the
adaptive immune system and is, in contrast to the innate, unique in every human.
In summary, the innate immune system is unspecific, fast acting and lacks memory while the
adaptive is specific, slower to act and has a memory component. The outcome is an immune
system that can act swiftly against all pathogens and learn to respond more efficiently against
any pathogen common to a given individuals specific environment. These branches are,
however, not two entirely separate systems but the adaptive is dependent on the innate
components to function and the innate immune responses are much more efficient when aided
by adaptive components.
The cells of the immune system are found throughout the body, particularly in the circulation
and in the lymphatic tissues. They all originate from the bone marrow and, at certain stages of
development, in the liver. These cells are collectively called leukocytes and are of either
lymphoid or myeloid origin. The lymphoid are B- , T-, natural killer (NK) - cells and possibly
some subsets of dendritic cells (DCs). The granulocytes, monocytes and most of the DCs stem
from a myeloid progenitor. A vast majority of the circulating immune cells are granulocytes,
in round numbers, granulocytes comprise about 60% of leukocytes in blood, about 30% are
lymphocytes and the final 10% are monocytes (Figure 1).
8
Figure 1. Cells of the Immune system
The cells of the innate immune system are monocytes, macrophages (MØ), granulocytes, DCs
and NK cells. All of them, except for the NK cells, have endocytic properties enabling them
to constantly survey their surroundings.
The cells of both the adaptive and innate immunity communicate through the secretion of
cytokines. There is an important subgroup of cytokines that governs cell motility and they are
called chemotactic cytokines (CC). These substances are shared between all the cells of the
immune system and will therefore be discussed in a separate chapter after a closer look at the
two separate branches.
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Innate Immunity
Recognition strategies
Perhaps the most essential task for the immune system is to be able to distinguish what is to
be regarded as friendly and what is to be regarded as dangerous. To solve this task, an array of
receptors has evolved to recognize components that are essential and unique to microbes. An
example is the cell-wall component lipopolysaccharide (LPS), in gram negative bacteria, that
potently activates the innate immunity.
The mechanisms that have evolved to know what is dangerous and what is not have, perhaps
in an oversimplified manner, been referred to as self/non-self recognition. It is now known,
however, that these mechanisms recognize not only foreign antigens but also self derived
“danger signals” and even healthy self (1). Among the microbes there are recurring pathogen
associated molecular patterns (PAMPs) particular or essential to their function. The
components of the innate immunity have evolved to take advantage of that and initiate
adaptive immune responses (2). The cells of the innate immune system express receptors that
recognize these PAMPs that are called pattern-recognition receptors (PRR) (3). There is also a
family of receptors called C-type-lectin receptors. The C-type-lectin receptors recognize
carbohydrate structures on glycosylated surface proteins of pathogens. Some examples are the
mannose receptor, DC-SIGN and Dectin-1. Another important mechanism for recognizing
pathogens is Fc receptors which binds the constant region of antibodies. Because of this they
are dependent on adaptive immunity and should not be counted as a part of innate immunity.
Toll like receptors
The most investigated family belonging to the PRRs in humans are the toll-like receptors
(TLRs). The TLRs were first discovered in drosophila and it became clear that they were
related to the IL-1 receptor suggesting a role in immunity (4). This has been proven as toll
mutants were shown to be more susceptible to fungi infections (5). One after another, the
roles and ligands for the different receptors were discovered (6-9). Ten different TLRs have
10
so far been characterized in humans. They all have specific ligands and localizations (Table 2)
and are expressed on different subtypes of DCs which will be further discussed in the chapter
of DCs. TLR1, TLR2, TLR4, TLR5 and TLR6 recognize recurring PAMPs in bacteria, fungi,
parasites and some viruses and are found in the outer cell membrane. However, the TLRs that
are specialized in recognition of intracellular bacteria and viruses, such as TLR3, TLR7,
TLR8, and TLR9 are located intracellularly. The motifs that they recognize are not
necessarily unique to viruses. In fact, nucleic acids are also present in human cells, but since
TLR are localized in endolysosomes there should not be any nucleic acids from the host
present and reactions against self-derived nucleic acids can therefore be avoided.
Signalling
TLRs are membrane-spanning proteins with one amino terminal extracellular domain of
leucine-rich repeats, that is responsible for binding the ligand, and an intracellular signalling
domain. The TLRs can form either homodimers or heterodimers. For example, it has been
shown that TLR2 forms a homodimer or heterodimers with either TLR1 or TLR6 (10). In this
manner different dimer formations can recognize different ligands. The signalling domain of
the TLRs is homologous to the signalling domain of the IL-1R and is called Toll/IL-1R
domain (TIR). In general this domain signals through TIR-TIR interactions with an adaptor
protein, thus recruiting an IL-1 receptor-associated kinase (IRAK) family protein and the TNF
receptor associated factor 6 (TRAF6). This complex activates Map kinases, that eventually
leads to the activation of transcription factors (11,12).
All TLRs except TLR3 signal through the adaptor protein myeloid differentiation primary
response gene 88 (MyD88). TLR3 signals through the TIR-domain-containing adapterinducing interferon-β (TRIF) leading to IFN regulatory factor (IRF) 3 and IFN-α secretion
(13). TLR4 however, can use both of these pathways. TLR 1, 2, 4 and 6 can also signal
through toll-interleukin-1 receptor domain containing adaptor protein (TIRAP).
11
However, signalling through the same adaptor protein can have different outcomes. Both
TLR7 and TLR9 signal through MyD88 and engage IRF7 which leads to secretion of IFN-α
(14). When the TLR4 is activated through the MyD88- dependent pathway it leads to IL12p70 secretion (15) but when the alternative pathway via TRIF is activated this results in
IRF3 activation and secretion of type 1 IFNs (13,16). The impacts of different TLR activation
on adaptive immune responses have been summarised in a recent review (Figure 2) (15).
Figure 2. Reprinted from Manicassamy S, Pulendran B. Modulation of adaptive immunity with Toll-like
receptors. Semin Immunol (2009), doi:10.1016/j.smim.2009.05.005 with permission from Elsevier
Briefly, IL-12p70 is strongly induced by TLR4, TLR5 and TLR8 and only weakly induced by
TLR2 in its different dimer complexes that instead lead to IL-10 production. TLR4 ligation
can lead to either IL-12p70 or IFN-α release whereas viral recognition by TLR3, TLR7 and
TLR9 induces IFN-α secretion. When the different TLR complexes involving TLR2 are
activated that would result in T-helper (Th) 2 or Treg skewing of the immune responses.
12
Conversely, activation of TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 would lead to a Th1type of response. Thus, depending on which TLR is engaged, the outcome will be different
and thereby a “custom made” default response mechanism exists to deal with different types
of antigens.
Innate immune cells
Monocytes
Monocytes are characterized as mononuclear leukocytes expressing the LPS co-receptor
CD14. The circulating monocytes constitute a reservoir for tissue specific macrophages (MØ)
and DCs. They may also have a role in maintaining tissue homeostasis by cleaning up debris.
About 20 years ago a subpopulation of monocytes expressing CD16 (Fcγ Receptor III) was
described (17). The classical CD14++CD16- cell expresses higher levels of CD14 and no
CD16 (Fcγ Receptor III), while the inflammatory CD14+CD16+ cell type expresses CD16 and
lower levels of CD14. A more recent review has better defined the classical and inflammatory
monocytes based on studies of cytokine secretion and T-cell stimulation (18). It seems like the
inflammatory monocytes are more prone to trans-endothelial cell migration and
differentiation to DCs (19).
Macrophages
Macrophage (MØ) means big eater and tissue specific MØ subsets have been described in
various organs such as lung, liver and nervous tissues. Intracellular macrosialin or CD68 has
been widely used for identification of macrophages (20), although it has been proposed that it
is not a marker of macrophages (21) but rather of phagocytosis. In analogy with the Th1/Th2
activation of T cells a M1/M2 characterisation of MØ has been suggested. Classical activation
of MØs has been defined as the response to LPS, with IFN-γ secretion and induction of
cytotoxic-Th1 immune response; these are the ones called M1. The alternative activation
induced by IL-4, IL-10 and IL-13 of monocytes consequently leads to a M2 polarized MØ
thus promoting a Th2 type of response (22,23). However, there have been objections to the
13
oversimplification of this concept (24) where a more complex picture of MØ activation is
presented. Nevertheless, a subcategorizing of M2 polarized MØs has been suggested (25)
where the M2 MØs now include all MØs that are not M1. This leads to a concept where the
M1 is well defined but the M2 is still a heterogeneous group of MØs. Recently, a model that
suggests dividing MØs in host defence, wound healing and immune regulation has been
proposed (26) that would better account for the role of MØs in homeostasis.
Dendritic Cells
DCs are a very heterogeneous group of cells both when it comes to their origins and their
distribution as specialized tissue specific cells in different organs (27). Unlike MØs, some DC
subsets may have a lymphoid origin. DCs are the most prominent professional APCs because
their life cycle is adapted to find, process and present antigens to naive T cells in lymphoid
tissues. The immature DCs (iDCs) travel the peripheral tissues expressing high amounts of
PRRs, particularly TLRs, and a repertoire of chemokine receptors (CCR) that are specialized
to detect inflammatory mediators. Upon encountering a pathogen, DCs become activated and
alter the expression of their surface molecules (28). The surface antigen CD83 is one of the
most important maturation markers on DCs and it has been shown that CD83 knockout mice
were blocked in the generation of CD4+ T cells (29). The DC-maturation process also
involves down regulation of PRRs and inflammatory CCRs, such as CCR1, CCR2 and CCR5.
Simultaneously, DCs up regulate co-stimulatory molecules and CCRs with ligands expressed
in lymphoid tissues such as CCR7 and CXCR4 (30). This CCR switch enables DCs to migrate
to T-cell rich areas in secondary lymphoid organs and encounter massive amounts of naive T
cells (31). When the proper combination of T-cell receptor (TCR) on a CD4+ T cell and
human-leukocyte antigen (HLA) class II complex expressed on a DC is found, co-stimulation
and cytokine secretion increases. This triggers an activation programme in the T cell that then
starts to grow and divide so that thousands of clones will be active in a matter of days.
14
There are many subpopulations of DCs in circulation and different models for the
development of DCs have been suggested (32). Most commonly blood DCs are described as
two distinct populations; the myeloid DCs (mDCs) and the plasmacytoid DCs (pDCs) with
complementary functions (33). However, it seems that there is certain plasticity even in these
two subtypes of DCs (34). A more careful characterisation has resulted in specific surface
marker for different DC subsets in circulation (Table 1) (35).
Subtype
BDCA1
BDCA2
BDCA3
CD16+DC
CD1c
+
-
CD11c
+
-/+
-/+
CD123
+
+
CD2
+
-/+
+
CD16
+
CD32
+
Nd
CD64
+
Nd
Table 1. Expression of surface molecules on distinct blood DC subsets.
Abbreviation: Nd- no data. References: (35-37).
The major mDC subpopulation in circulation is defined as lineage negative, CD1c+ CD11c+
CD123- cells. They are characterised by their expression of the blood dendritic cell antigen
(BDCA)-1 also called CD1c. Conversely, BDCA-2 (CD303) is strictly expressed on pDCs.
These cells are defined as lineage negative, CD123+ and CD11c- cells. In addition, BDCA-3
(CD141) is expressed on a subpopulation of mDCs that are also defined as lineage negative,
CD1c+ CD11c+, CD123- cells. Unlike the BDCA-1+ DCs, these cells lack the expression of
BDCA-1, CD2, CD32 and CD64. The monocytes and the different DC subsets differ in the
expression of TLRs. Different subtypes of DCs are directed towards different kinds of
pathogens and therefore express different TLRs (Table 2). The pDCs express TLR1, TLR6,
TLR7, TLR9 and TLR10, while mDCs express TLR1, TLR2, TLR3, TLR5, TLR6, TLR8 and
TLR10 (38).
15
Receptor
TLR1
TLR2
TLR3
TLR4
TLR5
TLR6
TLR7
TLR8
TLR9
TLR10
Ligand and origin
Triacyl LpP (bacteria)
Zymozan, PGN (fungi, G+ bacteria)
dsRNA (viruses)
LPS, proteins (G- bacteria, viruses)
Flagellin (bacteria)
Diacyl LpP, Zymosan (bacteria, fungi)
ssRNA (virus)
ssRNA (virus)
CpG-rich DNA, Hemozoin (virus, protozoa)
Nd
location
surface
surface
surface
surface
surface
surface
internal
internal
internal
surface
mDC
+
+
+
+
+/+/+
+
pDC
+
+
+
+
+
MoDC
+
+
+
+
+/+
+
Nd
Table 2. TLR receptors localization and ligands. Abbreviations: mDC-myeloid DC, pDC-plasmacytoid DC,
MoDC-monocyte derived DCs, LpP-Lipoprotein, PGN-Peptidoglycan, ds-double stranded, ss-single stranded,
Nd-No data. References: (38,39).
The pDCs primarily recognize and react to viral infections and respond by secreting high
amounts of IFN-α (40). On the other hand mDCs primarily respond to LPS and other
bacterial, viral and parasitic stimuli by secreting high levels of TNF-α, IL-6, IL-8, IL-12 and
up regulate the maturation markers HLA-DR and CD80 when exposed to LPS (41). This has
also been subject of more recent reviews and the different roles in pathogen recognition and
subsequent responses of pDCs and mDCs have been well characterized (42,43).
The BDCA-1+ mDC subset expresses higher levels of CD86 and HLA class II molecules than
the BDCA-3+ mDC subpopulation (35). The function of the rare BDCA-3+ mDC
subpopulation is still not clear, but a possible immunomodulatory role has been suggested for
this cell subset upon parasitic infection in humans (44).
Taken together, the DC specialisation in pathogen recognition, their transition from immature
to mature state and their high capacity to activate naive T cells, make them a crucial link
between the innate and adaptive immune responses. It is well established that the secretion of
pro-inflammatory Th1 cytokines like IL-12 and type 1 IFNs is essential in directing adaptive
responses. It has also been shown that DC-derived IL-12 induces differentiation of naive Bcells (45) and induces class switching to IgG and IgA, thus regulating humoral responses (46).
Moreover, the typical pDC type 1 IFN secretion discussed earlier can also induce plasma-cell
differentiation and Ig production (47,48).
16
Several protocols have been established to obtain purified mDCs from precursors in vitro (4951). DCs derived from CD34+ cord blood cells differentiate along two independent pathways
in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF-.
After 5-6 days, two subsets (one CD1a+CD14- and one CD1a-CD14+) can be observed; after
12 days in culture, all cells are CD1a+CD14-. The CD1a+ precursors differentiate into
Langerhans cells that contain Birbeck granules, whereas the CD14+ precursors lead to CD1a+
mDC that do not produce Birbeck granules and that possess the characteristics of interstitial
DCs (52). It is also believed that monocytes represent a pool of circulatory precursor cells that
are capable of differentiating into mDCs that resemble the features of interstitial DCs (28).
Monocyte-derived DCs (MoDCs) with the typical phenotype of CD1a+CD14- cells can be
obtained in vitro by stimulation with IL-4 and GM-CSF for 6 days (51) (256). In vitrogenerated monocyte-derived DCs are considered to have inflammatory properties (53). These
cells can further mature upon incubation with various stimuli, such as bacterial LPS,
inflammatory cytokines (TNF- and IL-1) and T-cell signals (e.g. CD40 ligand, CD40L). In
the first study of this thesis we have employed monocyte-derived DCs to resemble
inflammatory DCs that develop upon infection in vivo.
Granulocytes
Granulocytes are by far the most prevalent leukocytes in the circulation. Their morphology is
somewhat different from other leukocytes in that they have a segmented nucleus and their
cytoplasm contains granules. The granules contain highly reactive oxygen or nitrogen-derived
compounds, highly acidic contents or enzymes that digest bacterial components. The
granulocytes can be subdivided in three categories based on their staining characteristics. The
vast majority are neutrophils that quickly move to the site of infection and once there, they
release their granules and phagocytose as much bacteria as they can until they die. The pus
that is formed in wounds upon infection is remnants of neutrophils and their phagocytosed
bacteria. The other two subcategories of granulocytes, the eosinophils and the basophils, are
17
believed to be more adapted to clear parasite infections as they express IgE receptors (54,55).
Another class of densely granulated cells are the mast cells that contain histamine granules
and have been implicated in allergic reactions (56).
Natural Killer Cells
NK cells were named after their ability to find and kill tumour or virus infected cells without
prior activation (57). They express a variety of receptors that can bind to HLA class I
molecules. When NK cells meet a cell with no expression or an aberrant expression of HLA
class I, they release granules containing perforin and granzyme that will lyse the host cell.
This is known as the missing self hypothesis (58). NK cells express immunoglobulin-like
receptors that have either inhibitory or activating motif, as signalling sequences, on their
intraplasmic domain (59). The sum of the signals that the NK cell receives upon an encounter
with another cell will determine whether or not that cell should be killed (60). NK cells are
characterized by the expression of CD56 but not TCRs or CD3. It is also possible to subdivide
them according to the levels of CD56 and CD16 they express (61). The CD56high NK cells are
more prone to secrete high levels of cytokines while the CD56dim NK cells have a higher
cytotoxic activity.
Effector molecules
The complement system consists of about 30 serum proteins that attach directly to the surface
of pathogens or antibodies bound to the surface of pathogens (62). When activated they
recruit not only other complement proteins but also acts as chemotactic stimuli to leukocytes.
The final outcome, of the complement activating each other, is lysis of the pathogen they are
bound to and recruitment of cells to the site (63). In addition, there are some peptides in
circulation with bactericidal properties they were first found in plants but now various
antimicrobial peptides have been found in human as well.
18
Adaptive Immunity
The adaptive immune system has to be selective, in that it has to discriminate self from nonself, specific in order to recognize every particular pathogen and respond accordingly, flexible
in order to confront changes in the pathogen and have a memory to recognize previous
infectious agents. The destiny and developmental pathways of the lymphocytes make them
well suited to solve these tasks.
Recognition strategies
During maturation, B and T lymphocytes randomly rearrange the joining (J), diversity (D) and
variable (V) segments of their genome. These segments code for antigen-binding molecules
and are responsible for specifically recognising a particular antigen. In the case of T cells,
gene rearrangement occurs in the genes coding for the α, β, γ and δ chains of the T-cell
receptors (TCRs) while in B cells gene rearrangement occurs in the immunoglobulin (Ig)
genes coding for the heavy and light chains of the B-cell receptor (BCR).
Naive T cells gather in T-cell areas of the secondary lymphoid organs where they await an
activated APC that will present the right combination of peptide loaded HLA complex and
thereby activate them. A similar antigen from the same microbe may also bind a BCR thus
preparing the B cell for the encounter of an activated T cell. In this encounter the B cell will
become potently activated, expand clonally and produce vast amounts of antibody.
Most T cells recognize antigens only when presented to the TCR in the context of an HLA
molecule. The TCR cannot be expressed alone on the T-cell surface but co expression of TCR
and CD3 is mutually required for the successful expression of either (64). The TCR can be
composed of one α and one β chain and in that case it lacks an intracellular signalling domain.
It can also be composed of one γ and one δ chain this gives rise to the γδ-T cells. They
recognize antigens in a different manner than the αβ-T cells and are discussed further later in
the text. The intracellular signalling upon binding of the TCR goes through CD3 and triggers
19
the activation of various intracellular pathways leading to elevated Ca2+, actin remodelling
and activation of NF-κβ (65).
Adaptive immune cells
T Cells
T lymphocytes are a heterogenous group of cells that have undergone gene rearrangement and
express a TCR. The heterogeneity consists mainly in their manner of activation and
consequently their effector functions. They range from the most pro-inflammatory Th1 to the
most anti-inflammatory regulatory T cells (Tregs).
Development and Selection
The random rearrangement of the TCR genes creates a large enough variety of antigenbinding molecules so that for almost any given antigen, there will be a TCR to recognize it.
To avoid the release of potentially harmful T cells from the thymus to the periphery there is a
selection process in the thymus. During early thymic development, before the TCR genes are
rearranged, the cells express neither CD4 nor CD8 and are therefore termed double negative
(66). Since the gene recombination is random the specificity of the respective TCR is also
random. When the cells have rearranged their TCR, they express it on the surface along with
both co-receptors CD4 and CD8. At this stage they are termed double positive and a selection
process takes place where less than 5% of the cells survive (67). The T cells are presented to
self antigens in association with human-leukocyte antigen (HLA) molecules. During positive
selection the T cells that can bind self HLA molecules survive. If a T cell undergoing the
positive selection process is rescued by a cell expressing HLA class I it will maintain the
expression of CD8 but loose CD4 and vice versa. During the negative selection those who
bind too firmly to the HLA are potentially self reactive and therefore eliminated. The
objective is that the mature T cells released from the thymus all recognize self but should not
be self reactive. The T cells leaving the thymus can be subdivided into sub categories the Thelper (Th), T-cytotoxic (Tc) and T-regulatory cells (Treg).
20
T helper cells
The classical Th cells express CD4 and can be further subdivided in Th1 and Th2 cells
according to cytokine secretion and their functional properties (68,69). When the naive Th
cell is activated by an APC the co stimulation and the cytokines secreted are what will decide
what type of helper cells they become. The classical Th cells are the Th1 / Th2 cells. Th1 cells
are induced by high IL-12 that in turn leads to IFN-γ secretion by the Th1 cells. IFN-γ
secretion creates a positive feedback loop and causes up regulation of transcription factor Tbet that enhances Th1 response (70). Similarly IL-4 induces the transcription factor GATA3
that also maintains the expression of IL-4 and thus, a Th2 phenotype.
T-cytotoxic cells
The T-cytotoxic cells (Tc) express CD8 but not CD4 and therefore only recognizes antigens in
association with HLA class I. Consequently, the cells bind to all nucleated cells in the body
and when an aberrant expression of HLA-antigen complex is encountered, as it would be in a
cancer or virus infected cell, the Tc are activated. Upon activation the Tc cells kill the other
cell by induction of apoptosis or the release of granules with perforin and granulosin that lyse
the membrane of the infected cell (71).
Regulatory T cells
An additional CD4+ subtype expressing high levels of CD25 was found more than 10 years
ago exhibiting immunosuppressive properties (72). These T cells are released from the
thymus expressing CD4+CD25++ and the transcription factor FoxP3 and are called natural
regulatory T cells (nTreg). In addition there are Tregs that are induced in the periphery that
secrete high amounts of IL-10 and TGF-β these are referred to as inducible Tregs (iTregs).
The nTregs are believed to act in a contact dependent manner while the iTregs are cytokine
dependent (73).
Gamma delta T cells
About 1-10% of the T cells have TCR composed of one γ and one δ subunit instead of the αβ
subunits. It was suggested that a γδ clone could recognize a viral protein independently of
21
antigen presentation (74) and that recognition of non-peptidic antigens required cell contact
between T cells (75). The distribution of γδ-T cells differs from that of the αβ-T cells in that
they are mainly found in blood and peripheral tissues but rarely in the lymphoid organs. This
fact agrees well with the theory that these cells do not require APC to recognize antigens but
recognize them either alone or in a different context (76). The γδ-T cells are now considered
to work as a link between innate and adaptive immunity (77).
T helper 17
Recently, another type of Th cell has emerged because of its cytokine production it has been
called the Th17 cell. These cells secrete high amounts of IL-17, but not IL-4 or IL-12 (78).
Although it is not yet clear what drives Th17 differentiation it seems to be dependent on the
transcription factor Ror-γ that is induced by TGF-β and the transcription factor STAT-3 that is
regulated by IL-6, IL-21 and IL-23 (79). In humans it is not yet clear which cytokines induce
the Th17 while in mice this cell subset is induced by IL-6 and TGF-β (78).
Natural killer T cells
This subset of cells expresses the αβ-TCR and are therefore defined as T cells. They do
however display properties of NK cells and seem to be important for recognizing antigen
displayed on non-classical HLA molecules.
B cells
Much like the random rearrangement of the TCR in T cells, B cells in the bone marrow
rearrange their genome in the variable region coding for Ig. The Igs are expressed either as a
membrane bound form, i.e. the BCR, or secreted as antibodies and their production is
exclusive to B lymphocytes. The naive B cell expresses the BCR on the plasma membrane
and secretes low amount of IgM antibodies. When the BCR binds its specific antigen it is
internalised, processed and displayed on an HLA II molecule on the surface. At this stage, an
activated T cell whose TCR can bind the HLA-antigen complex on the B cell to form the
immunological synapse, can thereby potently activate the B cell. This leads to clonal
22
expansion of the B cell; the clones undergo somatic hypermutation in the epitope binding
region and as a result increase the affinity to the antigen. In this way the antibodies will have
higher affinity to the antigen the longer the infection persists. B cells can also be activated
without the aid of Th cells through for example TLR ligation. Upon activation the B cells
become either plasma cells that produce high amounts of antibodies or memory cells. After
activation, the B cells also undergo class switching where the Ig production is shifted from
mainly IgM to IgG or IgE subtype.
Effector molecules
The effector molecules of adaptive immunity are the antibodies. An antibody is composed of
two heavy and two light chains each consisting of one constant and one variable region. The
two heavy chains are linked to each other and to one light chain each by disulfide bridges.
After the rearrangement of the heavy and the light chain the B cells are released into
circulation. Each B cell expresses its BCR bound in the membrane as IgM or IgD isotype and
undergoes activation when BCR binds its specific antigen. An activated B cell undergoes a
recombination of the heavy-chain genes coding for the constant region but not the variable
region allowing it to keep its specificity but switching isotype (80). There are five different
isotypes or classes of antibodies; IgA, IgD, IgE, IgG and IgM, and the isotype is determined
by the constant region of the Ig heavy chain. It is this constant region that interacts with
complement proteins and FcRs thereby regulating the immune response (81). In addition, in
humans there are four subclasses of IgG antibodies the IgG1, IgG2, IgG3 and IgG4 and two
subclasses of IgA the IgA1 and IgA2.
23
Coordinated Immune responses
As already mentioned above the innate and adaptive branches of the immune system are not
two separate systems. Therefore it is impossible to generally write about immune responses
under two separate headings. The connecting factor between innate and adaptive immunity is
antigen presentation. Finally, molecules governing cell motility and signalling are shared
between the two branches of immunity and are therefore discussed here with regards to
coordinated immune response.
Leukocyte movement
In order to efficiently mount a proper immune response the cells need to travel to the site of
infection. Cell movement is guided by a class of small proteins called chemotactic cytokines
(CC) or more commonly, chemokines. They were named because of their ability to induce
chemotaxis i.e cell movement towards a gradient of a certain substance. The chemokines are
divided according to structural properties and the C symbolises the location of highly
conserved cystein residues in the peptide chain of chemokines. The CC are divided into two
major subgroups; the CC and the CXC (82). and there has been an addition of two smaller
subgroups; i.e. the C and the CX3C subgroups to this major families (83). The chemokine
receptors (CCRs) have a 7 transmembrane domain and are coupled to a G protein for
intracellular signalling (84).
Inflammatory CCRs
CCR1, CCR2 and CCR5 have various ligands and share many of them (Table 3) (85). They
typically bind inflammatory stimuli such as the macrophage inflammatory protein (MIP)-1
and MIP-1, the Monocytes-Chemoattractant Protein (MCP)1-4 and the Regulated on
Activation Normal T Expressed and Secreted Protein (RANTES) (85). In this manner cells
that express these CCRs will sense inflammation and start to move towards the site of
inflammation.
24
Lymphoid CCRs
Contrary to the inflammatory CCRs, CXCR4 and CCR7 do not have many known ligands.
CXCR4 is expressed on T and B lymphocytes and dramatically affects the development of B
cells and cerebral tissues (86). CCR7 is expressed on naive T cells and and it has been shown
that lack of this receptor severely impairs primary immune responses (87). The CXCR4
ligand stromal-derived factor (SDF)1/CXCL12 and the two ligands for CCR7, CCL19 and
CCL21 are expressed mainly in lymphoid tissues (Table 3).
Receptor
CCR1
CCR2
CCR5
CCR7
Ligand
CCL3/MIP-1α, CCL5/RANTES
MCP1-4
MIP-1α, CCL4/MIP-1β, CCL5/RANTES
CCL19/ELC/MIP-3β, CCL21/SLC
Location
Inflammation
Inflammation
Inflammation
Lymphatic tissue
iDC
+
+
+
-
mDC
+
CXCR4
CXCL12/SDF-1
Lymphatic tissue
+
+
Table 3. A few examples of CCRs expressed on immature and mature DCs and some of their ligands.
Abbreviations: CCL-Chemokine ligand, iDC-imature DC, mDC-mature DC, MCP-monocytes-chemoattractant
protein, MIP-Macrophage inflammatory protein. References: (83,85)
Antigen presentation
It is well established that two signals are required in order to activate T cells (88) and it was
suggested that a third signal also is needed (89) which has now been widely accepted.
The three signals required to properly activate a CD4+ T-cell are:
1. Binding of the T-cell receptor to a HLA class II antigen bound complex
2. Binding of co-stimulatory molecules
3. Cytokine signalling
Human Leukocyte Antigen
The HLA molecules are what define an individual’s immunological “self”. They are the
reason for organ rejection upon transplant. If the transplant has a different HLA expression it
will not be considered self but instead be treated as foreign and attacked. T cells can only bind
an antigen when presented to them as a complex with HLA molecules. The classical HLA
25
proteins and are subdivided into class I and II. The HLA class I molecules are expressed on
all nucleated cells of the body and bind to the distal part of the CD8 molecule present on T
cells. The expression of HLA class II molecules is restricted only to professional APCs they
bind the distal part of the CD4 receptor present on T cells.
Antigen uptake
APCs are the cells that activate the adaptive branch of immunity. For this purpose it is
necessary for APCs to internalize the pathogen, and this is done by a mechanism called
endocytosis. Endocytosis is a process by which the cell takes up extracellular contents by
budding of inwards forming an endosome of the plasma membrane and internalizing it to the
cytoplasm. There are different forms of endocytosis. There is pinocytosis or cell drinking and
phagocytosis or cell eating. Pinocytosis is formation of vesicles seemingly at random that
serves to sample the environment for soluble antigen in a non-specific manner. Phagocytosis
is receptor-mediated uptake of an antigen bound to receptors on the surface such as the C-type
lectin family of receptors or the Fc receptors (90). This leads to reorganisation of the
cytoskeleton, thus, surrounding the complex and internalising the antigen. Depending on the
nature of the antigen and the receptor there may be different inflammatory responses (91).
Antigen processing
All nucleated cells present endogenous peptide fragments on HLA class I molecules. This is
done through a mechanism called the cytosolic pathway. The professional antigen-presenting
cells (APCs) can take up particles from the extracellular environment, process them and
present peptide fragments on HLA class II molecules. That is called the endocytic pathway.
The endocytic pathway
Typically, the endosome is fused with lysosomes containing digestive enzymes in an acidic
environment to form an endolysosome where the pathogen is degraded. The peptide
fragments are loaded on HLA molecules intracellularly and transported to the surface of the
cell.
26
The cytosolic pathway
Instead of loading foreign peptide fragments on the HLA class II molecules the HLA class I
molecules are loaded with endogenous fragments of cytosolic proteins. The cytosolic proteins
are degraded by the proteasome and transported to the endoplasmatic reticulum. In the
endoplasmatic reticulum they are loaded on HLA class I molecules and put on the surface.
Cross-priming
The protein production in a tumor or infected cell is different from a healthy cell. The display
of these products will be recognized as aberrant and killed by NK cells (92). In order to be
able to activate CD8+ T cells antigens must be presented on HLA class I molecules.
Professional APCs have an alternative pathway that enables them to present exogenous
antigens on HLA class I molecules, thus inducing antigen-specific CD8+ T cells (93). This
phenomenon is called cross priming and is restricted to DCs and to lesser extent macrophages.
Cross priming is induced by viral recognition receptors in particular TLR9 (94,95).
Co-stimulation
As a fundamental part in the antigen-presentation process, APCs express a wide variety of costimulatory molecules. These will aid in the binding between the cells and create what is
called the immunological synapse. Two very important co-stimulatory molecules are the
CD80/B7.1 and the CD86/B7.2 that bind to CD28 and CD152 expressed on T cells. Ligation
of CD28 will lead to T-cell activation while ligation of CD152 will suppress activating
signalling. Co-stimulatory molecules can also skew T-cell responses in different directions.
The ligands specific for the TNFR family members CD134/OX40 and CD137/4-IBB are two
other molecules that are known to have an impact on T-cell responses. For example, if an
APC express high amounts of the co-stimulatory factor OX40L that binds OX40 on the T
cells they will be more prone to differentiate into CD4+ cells (96), while if 4-1BBL, expressed
on the APC, binds CD137 or 4-1BB that are present on the T cells, the T cells will be more
27
prone to become CD8+ cells (97). Recently it has been proven that both the ligands OX40 and
4-1BBL can support proliferation of naturally occurring regulatory T cells (Tregs) while
OX40 can also antagonise the induction of peripherally induced Tregs (98). Finally, evidence
shows that T-cell priming in the absence of proper co-stimulation (99,100) or of activating
cytokines will instead lead to anergic T cells or to differentiation of Tregs (101).
Inflammation
At the site of inflammation complement proteins and other inflammatory mediators act as
signal substances that call the attention of inflammatory cells. As soon as the cells reach the
site of inflammation cytokines such as IL-1, IL-6 and TNF- as well as inflammatory
chemokines such as IL-8 are secreted. This will lead to an increased permeability of the
vessels increasing the blood flow at the inflammation site. Simultaneously, the vessel walls
express adhesion molecules like selectins which contribute to adhesion of circulating cells to
the vessel wall, followed by transendothelial migration. The granulocytes are early at the site
of inflammation and kill the pathogen by release of granules containing toxic compounds and
by phagocytosis. Monocytes will also phagocytose the pathogen and secrete pro inflammatory
factors until the infection is resolved. Finally tissue specific APCs will also be activated,
internalize the pathogen and prepare for encountering naive T cells. If the pathogen is
removed the inflammation is resolved and tissue balance is restored. If the pathogen persists
there is increased risk for a permanent inflammation leading to tissue damage.
Intercellular communication
For coordinating an attack against a pathogen there are many types of signalling molecules
that the cells use for intercellular communication. Cytokines have traditionally been given
names according to whatever function they were discovered for, but as they are sequenced
they become renamed to an interleukin (IL).
28
Cytokines are a means of communication between leukocytes and other cells. They are
typically small proteins induced by activated immune cells to act on other immune cells.
There is not a very clear distinction between hormones and cytokines. For example, it has
been suggested that IL-6 acts in a hormone-like manner as it is secreted by muscle cells
during exercise and has major metabolic effects (102).
Interferons
These cytokines were discovered over 50 years ago and named interferons (IFNs) because
they interfered with viral replication (103). Nowadays, it is known that there are many
subgroups of IFNs and that they are found in all vertebrates where they have been
investigated (104). In this thesis I will only discuss the type 1 and the type 2 IFNs.
Type I IFN
The classical antiviral type 1 IFNs are the IFN-α and IFN-β. The NK cells and the pDCs are
very potent producers of IFN-α in response to activation by viral antigens. IFN-α acts on other
immune cells to elicit an anti viral response and also functions as an analgesic.
Type II IFN
IFN-γ is the hallmark cytokine of Th1 responses and is secreted by activated T cells and NK
cells as a response to IL-12 (105). IFN-γ acts in response to virus or intracellular bacteria and
tumor cells. IL-12 is secreted by mDCs, MØs and B cells in response to inflammatory stimuli
(106). It acts on NK and T cells to induce the production of IFNs and induces differentiation
of naive T cells to activated effector-T cells.
IL-1β
IL-1β does not seem to play a role in homeostasis but is a potent pro-inflammatory cytokine
that induces fever. IL-1β is produced by monocytes, MØs and DCs in response to
inflammation. It is implicated in inflammation through induction of cyclo-oxygenase 2 and
inducible nitrous oxide synthase. It is an inflammatory cytokine that helps to increase
29
expression of adhesion molecules on the endothelium at a site of inflammation and acts as a
bone marrow stimulant increasing the number of neutrophils in circulation (107).
IL-4
IL-4 was originally known as the B-cell differentiation factor and is the hallmark cytokine for
Th2 responses (108). It is secreted by various subsets of T cells and granulocytes (109) and
acts on two different receptors. The type 1 receptor binds only IL-4 while the type 2 receptor
can also bind IL-13 although the response is not as strong as compared to IL-4 (110).
IL-6
IL-6 was initially named B-cell differentiation factor upon its discovery (111) but was later
renamed to IL-6. IL-6 is induced by inflammatory cytokines such as TNF- and IL-1β but is
inhibited by IL-4 and IL-13. Nevertheless, this cytokine has been shown to skew CD4+ T cells
to a Th2 phenotype (112). More recently it has been suggested that IL-6 inhibits
differentiation of Tregs but promotes Th17 by increasing expression of RORγt while
inhibiting Foxp3 (113).
IL-8
IL-8 is secreted by almost all cells of the innate immune system during an inflammatory
response upon activation through TLRs. It is a chemokine and its major role is to recruit T
cells to the site of inflammation (114).
IL-10
IL-10 was first described as the substance secreted by Th2 cells inhibiting Th1 actions (115)
under the name of cytokine-synthesis inhibitory factor. Later, this cytokine was also shown to
inhibit the expression of co-stimulatory molecules as well as MHC class II molecules
(116,117). IL-10 is now widely recognized as an anti inflammatory cytokine secreted by
monocytes, MØs, DCs and some T cells to counteract inflammatory stimuli. It inhibits the
30
production of a great number of cytokines, even its own production, but the inhibition of IL-1
and TNF- is crucial to its anti inflammatory effects (118).
IL-12 family
IL-12 was the first member of the IL-12 family of interleukins initially discovered as NK-cell
stimulatory factor (119). More recently, IL-23 (120) and IL-27 (121) were added to the IL-12
family. IL-12 is the classical Th1-inducing cytokine secreted by neutrophils, monocytes, MØs
and DCs and induces IFN-γ release in NK cells and T cells. Although similar in many ways,
the members of the IL-12 family have varying structures and their actions and are
differentially regulated depending on the nature of the microbial stimuli. This is evidenced by
the fact that LPS induces IL-12 and IL-23 in DCs whereas peptidoglycan induces IL-23 but
not IL-12 (122).
Tumour Necrosis Factor-
TNF-α is a member of the TNF super family of proteins and is a pro inflammatory cytokine
initially discovered for killing of tumour cells. In infectious diseases, TNF promotes
inflammation and helps to combat the pathogen. However, too high levels of this cytokine are
responsible for tissue damage and pathogenicity (123). TNF-α can trigger apoptosis via the
activation of pro caspases of the TNF receptor and thus induce cell death.
Malaria
Malaria burden
There are as many as 3.3 billion people that live at risk of infection and 247 million cases
where reported in 2006. The vast majority of the victims are children that live in Sub-Saharan
Africa. About 98% of the cases in Africa are caused by Plasmodium falciparum and 85% of
the deaths are children below the age of 5 (124). Another group at risk are pregnant women
that are susceptible to pregnancy associated malaria (125). Malaria is not only one of the most
31
devastating infectious diseases in the world it is also tightly connected to poverty. Not only is
the most severe impact of malaria on the poorest populations in the poorest countries but these
populations are also kept in poverty as an effect of infection (126,127).
Parasite life cycle
The word malaria is Italian and means “bad air” because the disease was initially thought to
be airborne. The infection is caused by a protozoan parasite of the genus Plasmodium and is
probably one of the oldest diseases of mankind (128). The definitive host is the mosquito of
the genus Anopheles and the parasite is transmitted from mosquitoes to intermediate hosts
such as reptiles, birds and mammals. There are five species infecting humans and the four
“classical” are Plasmodium falciparum, vivax, malariae, ovale. Recently, several cases of
human Plasmodium knowlesi that was formally believed to only infect macaques have been
reported from Southeast Asia (129-135).
P. falciparum remains, however, by far the most lethal species of the genus Plasmodium. The
sexual stage of the parasite lifecycle takes place in the mosquito gut and thousands of
sporozoites are released from there to travel to the salivary glands (Figure 3). The sporozoites
are then transmitted to humans by the bite of an infected mosquito. The human stage of
infection is asexual. The sporozoites enter the blood stream and immediately infect the liver
where the parasites develop into merozoites. The length of the liver stage depends on the
species of Plasmodium. When the merozoites are released from the liver they infect red blood
cells (RBCs) in circulation and undergo replication. This stage is known as the blood stage
and RBCs go from ring stage infected RBCs to trophozoites and finally schizonts. At the
rupture of the schizont, thousands of new merozoites are released into the blood stream. A
small portion of the released parasites are gametocytes, which are responsible for sexual
stages in the mosquito gut. It has been shown that, when the parasitemia is reduced during
anti-malarial treatment, the amount of gametocytes increases (136)
32
Figure 3. Ménard, Robert, 2005: Knockout malaria vaccine? (Nature 433, 113-114).
Hemozoin
When the parasites degrade hemoglobin as a source for amino acids, the free heme group that
is left is toxic to the parasites. It is therefore transformed into innocuous crystals called
malarial pigment or hemozoin (Hz) as described in (137). Hz is released into the circulation
and taken up by APCs. The detection of circulating leukocytes containing Hz has been
suggested as a good method of diagnosing malaria because it can be correlated to total
parasite burden. In fact, one study has shown that this better predicts the prognosis then the
peripheral parasite count (138).
Methods have been developed to form hemozoin crystals synthetically from hemin (137) and
the product is usually referred to as synthetic (s)Hz or β-hematin. Except for the effect in
humans that will be discussed in the next section, several studies in vivo in mice have assessed
the immunogenicity of Hz. It was shown that sHz induces potent pro inflammatory responses
and recruitment of leukocytes, mainly monocytes and neutrophil populations (139-141).
33
Project background
Immune response to malaria
Innate responses
Different parts of the parasites have been shown to bind the TLRs and elicit innate immune
responses and it has been suggested that MyD88 plays an important role in malarial
pathogenesis (142). Glycosylphosphatidylinositol anchors (GPIs) of protozoan parasites have
been implicated as ligands for TLR2 (143). TLR4 has been shown to induce pro inflammatory
responses to GPI from another apicomplexan parasite Trypanosoma cruzi (144). Even one of
the intracellular nucleic acid recognising TLRs, TLR9, has been suggested to be the receptor
for Hz and binding of Hz to TLR9 would induce cell activation (145). However, a recent
study suggests that malarial DNA attached to Hz is responsible for TLR9 activation and Hz
would only function as a carrier of malarial DNA to the endolysosomes (146). Recent studies
indicate that children with a polymorphism in the TLR4 gene are predisposed to severe
malaria (147) and that TLR4 and TLR9 polymorphisms in pregnant women infected with
malaria are associated with increased risk of low birth weight in the offspring (148). These
studies suggest a role for innate immunity and in particular TLRs in the responses against
malaria. In addition, it has been shown that whole blood stimulation with malaria antigens
increase the secretion of pro- and anti- inflammatory cytokines upon subsequent specific TLR
stimulation (149). This clearly indicates that P. falciparum modulates TLR activity.
Antigen-presenting cells in malaria
As crucial cells of innate immunity and by expressing several TLRs, APCs may play a
fundamental role in the host response against the parasites. It has been suggested that T cells
are hampered in immune responses to malaria and that their suppressed function may be
34
mediated by DCs (150,151). A lower expression of HLA-DR on peripheral blood DCs in
children with acute malaria as compared to healthy children has been observed in vivo (152).
In vitro observations have shown increased pro as well as anti-inflammatory mediators when
exposing APCs to Hz as reviewed in (153). Hz mediates activation of pDCs via TLR9
although it is unclear whether the ligand is Hz itself, parasite DNA or both in a complex
(145,146,154). Monocytes loaded with Hz secrete high TNF-α and IL-1β (155) but lower
levels of IL-12p70 by an IL-10 dependent mechanism (156). They also exhibit impaired upregulation of MHC class II (157) and inhibited differentiation to DCs (158). As for human
MoDCs, it has been shown that maturation in response to LPS was impaired when the
immature DCs were exposed to P.falciparum infected red blood cells (iRBCs) prior to the
LPS challenge (159). On the other hand, enhanced maturation was observed when immature
DCs were exposed to parasite derived Hz (160). The heterogeneity of DCs, the different
parasite strains, the different type of Hz employed and the time of exposure are all factors that
could possibly explain these discrepancies (161,162). Taken together, these studies indicate
that P.falciparum can influence the function of various APC subsets and suggest that immune
responses to malaria may be modulated by hampering APC function, in particular DCs.
Antibodies and protective immunity
Immunity to malaria is both stage and species specific. It is acquired gradually and is lost if
the individual leaves the endemic area for a prolonged time period. T cells are essential to
acquire and regulate immunity to malaria (163). In mice, immunity to Plasmodium chaubaudi
is mainly cell mediated while immunity to Plasmodium yoeeli is mainly antibody mediated
thus proving that both T and B-cell mediated immunity are important for protection (164). It
has been known since the early sixties that Ig play an important role in malarial immunity.
Already over 40 years ago it was shown that transferring γ-globulins from adults living in
endemic areas to children, both from the Gambia (165) and from east Africa (166) exerted a
35
therapeutic function. It was also shown that monocytes could inhibit parasite growth when
incubated with sera from immune individuals (167). More recently it has been suggested that
the more cytophilic subclasses such as IgG1 and IgG3 (168) are more important for protection
against the infection (169).
Cytokines and clinical implications in malaria
The clinical stage of malaria is the blood stage of infection and the characteristic fever
returning at each rupture of the schizonts is a hallmark for malaria. Pro-inflammatory
cytokines seem to play an important role in the development of severe malaria. In fact, high
levels of TNF-α and low levels of IL-10 have been associated with disease severity and IL-12
was found to be lower in the severe cases as compared to the mild cases in Gabonese children
(170). More recently, high plasma levels of IL-1β have been associated with the development
of cerebral malaria (171) and high levels of both IL-6 and IL-10 were associated with severe
malaria as compared to uncomplicated malaria controls (172).
Fulani and Dogon in northern Mali
The Fulani are traditionally a nomadic pastoral people that have settled in various African
countries. The inhabitants of the Fulani and the Dogon village in this area do not intermarry.
The Fulani still have cattle but are now sedentary in northern Mali and have lived in the study
area for at least 200 years. The Dogon are farmers and migrated to this particular area about
50 years ago. Many studies have been performed showing that the Fulani are less susceptible
to malaria than their sympatric counterparts i.e. other ethnicities living under similar
geographical economic and social conditions. It has been shown that the Fulani respond more
potently to the infection than other populations under similar conditions. The Fulani exhibit
higher malaria-specific antibody titres, increased spleen rate, and have higher proportions of
36
malaria specific IL-4 and IFN-γ producing cells as compared to their sympatric neighbours
(173-176). In addition, it has been shown recently that a population of Fulani in Burkina Faso
have lower Treg activity than a neighbouring sympatric group (177). The underlying reasons
for the different susceptibility to malaria infection of the Fulani have not yet been completely
clarified.
37
Present Study
Aims
First Study
The goal of the first study was to characterize the behavior of MoDCs in malaria by
challenging these cells with iRBCs or Hz and analyze their phenotype, migratory behaviour
and cytokine secretion. We hypothesize that an impaired activation of DCs by the parasite or
by its derived products may contribute to the poor development of immunity to malaria.
Second study
It is well established that the Fulani are less susceptible and mount a relatively stronger
immune response to malaria than their sympatric neighbouring populations. The underlying
mechanisms for that are still not well understood. We hypothesize that the prevalence or
activation status of different DC subsets in circulation of the Fulani children could be related
to the relative protection against malaria seen in the Fulani.
Methods
Here follows only a brief description of the methods used in the different projects. A more
detailed description of the methods is included in each manuscript.
First Study
Monocytes were purified from peripheral blood mononuclear cells (PBMCs) from healthy
blood donors using Ficoll separation. The monocytes were then cultured in the presence of
GM-CSF and IL-4 to produce MoDCs. The MoDCs were then challenged with sHz, crude
malaria antigen or iRBCs. The phenotype of the cells was analyzed using FACS, their
38
migratory properties were assessed using a Boyden chamber and their culture supernatant was
analysed for cytokine secretion.
Second study
Study area
The study area is in northern Mali in the area of Sahel just south of the Sahara desert. Malaria
transmission is seasonal and the rainy season starts in July and lasts until October. The rate of
infected mosquitoes is similar between the Fulani and Dogon villages but the Dogon have had
more clinical episodes of malaria infection (175).
Methods
A total of 40 Dogon children were enrolled of whom 20 were slide positive for P. falciparum
and 20 were negative. In the Fulani there were a total of 37 children enrolled, 14 were
infected and 23 were uninfected. Venous blood from infected and uninfected children of the
Fulani and Dogon ethnicities was collected. The plasma was frozen in order to analyze levels
of cytokines and antibodies in circulation. PBMCs were isolated and stained for DC subtypes
and activation markers and analysed by FACS.
Results and Discussion
First Study
Various studies have investigated the effect of Hz or iRBCs on monocytes and MoDCs as
recently reviewed in (178,179). It is now known that MoDCs that have been exposed to high
doses of iRBCs do not mature properly in response to LPS (159). It has also been shown that
Hz has a similar effect on MoDCs (158). In this study, we further analyzed the effect of sHz
and iRBCs on MoDC activation, CCR expression and migratory capacity. As a positive
control we used a combination of TNF-α and prostaglandin E2 (hereafter referred to as TNF-
39
α/PGE2). We choose this combination because of the enhanced migratory capacity of MoDCs
exposed to these stimuli (180).
Our data show that upon incubation with sHz, MoDCs significantly increased the expression
of CD80 and CXCR4. We also observed a slight up regulation of CD83, HLA-DR and CCR7.
Nevertheless, upregulation of these maturation markers upon sHz stimulation was
significantly lower than the one observed upon TNF-α/PGE2 stimulation. This may indicate
that MoDCs exposed to sHz are poor inducers of adaptive immune responses. A similar
pattern of activation was found when MoDCs were incubated with iRBCs. These results are in
line with other findings that reported only partial maturation of DCs upon incubation with
malaria derived products (160,181).
The increased expression of the lymphoid CCRs on DCs in response to malaria stimuli led us
to test the migratory ability of these cells in vitro towards the ligands for CCR7 and CXCR4,
CCL19/MIP-3β and CXCL12/SDF-1α, respectively. The results revealed that immature
MoDCs exposed to 20 µg sHz increased their migration towards both ligands as compared to
the cells that where incubated with medium alone. The increased migration towards lymphoid
chemotactic ligands homing to lymph nodes that we observed in immature MoDCs exposed in
vitro to malaria derived products may indicate that immature, circulating DCs are induced to
travel to the lymph nodes during a natural P. falciparum infection.
Conversely, when MoDCs were exposed to sHz before TNF-α/PGE2 was added to the
culture, they failed to properly up regulate CCR7. Other than just inducing chemotaxis
towards lymphoid organs, this receptor has been reported to enhance maturation, survival and
migratory speed of DCs (182). Thus, we hypothesize that malaria derived products could
negatively affect DC responses in the presence of other maturation stimuli.
The kinetics of sHz effect on MoDCs was also evaluated and cells were phenotypically
analyzed at 12, 36 and 60 hours upon incubation with sHz. The results indicated that sHz
40
induced a short lasting up regulation of CD83 on the surface of MoDCs as compared to TNFα/PGE2, thus further indicating that MoDCs are only partially activated during malaria
infection.
To better characterize the response of MoDCs to malaria derived products, we analyzed the
cytokine release by DCs in response to sHz and iRBCs. An increased release of IL-6, IL-10
and TNF- was seen when MoDCs were incubated with 20 µg sHz. Similarly, incubation of
DCs with iRBCs increased the secretion levels of the same cytokines with the addition of IL1β.
It has been shown that IL-10 secreted by monocytes-MØs upon contact with Hz decreases the
levels of IL-2, IL-12 and IFN-γ in PBMC cultures (183), thus down-regulating Th type 1
responses. In addition, increased levels of TNF-α, IL-1β and IL-6 were reported to be
implicated in severe malaria. Recently, the serum levels of IL-1β, IL-6, IL-10 and TNF-α
have been associated to severe complications during malaria infection in various studies
(171,172). Furthermore, it has been shown that rapid induction of IL-1β may help to control
malaria infection, but persistence of this cytokine can lead to anemia (184).
Secretion of pro-inflammatory cytokines is generally important in the initial phases of malaria
infection but maintained high levels are associated with inflammatory induced tissue injury.
We suggest that mDCs can be crucial cells in inducing the large inflammatory response that is
often observed during acute P. falciparum infection.
Taken together, our results show partially increased expression of maturation markers,
increased migration towards lymphoid chemokines and increased secretion of IL-10 but not
IL-12 by MoDCs upon contact with malaria derived products. These in vitro observations
suggest that DC responses in malaria are potent enough for DCs to make contact with naive T
cells in the lymph nodes. However, proper induction of adaptive immunity requires high
expression of co-stimulatory molecules and IL-12 release by DCs. It is therefore possible that
41
the actions of Hz on mDCs, with induction of partial DC maturation and increased IL-10
secretion, may lead to insufficient activation of T cells or even induction of T cells with
regulatory properties upon infection in the natural host. The poor activation of T cells may in
turn lead to impaired adaptive immune responses and therefore insufficient clearance of the
parasites. We therefore hypothesize that an impaired activation of DCs by the parasite or by
its derived products may contribute to the poor development of immunity to Malaria.
Second study
Many studies have been conducted in different African countries comparing different aspects
of the immune responses to malaria of the Fulani and their sympatric ethnic groups (175,185).
It has been confirmed that the Fulani show less clinical symptoms of infection than sympatric
ethnic groups although they are exposed to the same parasite pressure (173). The Fulani
exhibit higher antibody titres, enlarged spleen rates and higher levels of cytokines in
circulation. Our study was conducted to investigate some aspects of the innate immune
responses in children belonging to the Fulani and Dogon ethnic groups in Mali. In particular,
the study aim was to investigate whether the properties of circulating DCs of Fulani and
Dogon children could account for their different response to malaria infection.
We confirmed the higher levels in circulation of Malaria specific antibodies in Fulani as
compared to Dogon. In fact, our findings showed significantly higher levels of malaria
specific IgG, IgM and higher levels of IgG1, IgG2 and IgG3 but not IgG4 subclasses in the
Fulani children as compared to Dogon children. For the IgG3 subclass of antibodies,
decreased levels were found in the Fulani children undergoing P. falciparum infection as
compared to uninfected Fulani. This result may suggest that the IgG3 antibodies have bound
the parasite-derived antigen, formed immune complexes and thus, could not act in an
antibody-cell-mediated way (168), although the immune complexes could act through Fcreceptors and thereby induce inflammatory cytokines (186).
42
The level of IFN-α, IFN-γ, IL-1β, IL-6, IL-8, IL-10, IL-12(p70) and TNF- were measured in
serum of 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 exhibited values that were
below the detection limit of our assay.
When comparing the Fulani and the Dogon, regardless of infectious status, the levels of IL-6,
IL-8, IL-12, IFN-α and IFN-γ were higher in the Fulani children. Thus, similarly to what was
observed for the antibodies, the Fulani had higher levels of several inflammatory cytokines in
plasma as compared to the Dogon children.
The infected Dogon children had significantly higher levels of IL-6 and IL-12 while increased
levels of IL-6 and IL-12 were not observed when comparing uninfected and infected Fulani.
This may suggest a different skewing of immune responses of the Dogon as compared to the
Fulani. When comparing only infected children from the two ethnicities, differences were
only found for IFN-γ but not the other cytokines, and higher IFN-γ levels were observed in
the Fulani ethnic group as compared to the Dogon.
Taken together, cytokine analysis of plasma samples in the two ethnic groups revealed that
upon P falciparum infection an IFN- mediated response seems to be increased in the Fulani,
while a different pro-inflammatory pattern lacking IFN- up-regulation would be activated in
the Dogon.
We also analyzed three subtypes of blood DCs as well as their activation status in the children
involved in the study. We observed increased frequency of circulating pDCs and BDCA-3+
mDCs in the infected Dogon as compared to the uninfected Dogon. Conversely, the infected
Fulani exhibited lower frequency of the same subsets than the infected counterpart.
The different levels of DCs in the blood of the two ethnic groups undergoing malaria might be
explained by the different activation status of these cells. In fact, with regard to the expression
of HLA class II molecules, the DC activation status was consistently higher in pDCs of the
43
infected Fulani as compared to the uninfected children belonging to the same ethnic group,
while HLA class II expression on BDCA-1+ and BDCA-3+ mDCs and on pDCs was
significantly lower in infected Dogon than in the uninfected peers. The increased activation
status of circulating pDCs in infected Fulani children may indicate that these cells have
migrated to lymphoid organs to trigger T and B-cell responses and are therefore no longer
found in circulation, thus possibly explaining the reduced frequency of these cells subsets in
the blood of infected Fulani children. Conversely, our findings suggest that malaria infection
impairs activation of circulating DCs in the Dogon ethnic group, thus possibly affecting
downstream cellular and humoral immune responses.
Our findings suggest that there is a difference in the prevalence and activation status of blood
DCs, cytokine secretion and Ig levels between the Fulani and the Dogon in response to
malaria infection. Such differences are likely to be important for the relatively stronger
protection against malaria seen in the Fulani as compared to the Dogon.
44
General Conclusions
We have shown that malaria derived products can activate MoDCs in vitro leading to
migration of DCs towards lymphoid ligands and to secretion of both pro and anti
inflammatory cytokines. However, such activation is partial, and may lead to an impaired DC
functionality. Additionally, we have shown that blood DCs in the Fulani express higher levels
of activation markers during malaria infection, while blood DCs in the infected Dogon exhibit
significant lower levels of activation markers than the uninfected counterpart. Moreover, the
Fulani had higher levels of IFN- in plasma as compared to the Dogon children, indicating a
more efficient response to this parasitic infection (187). We also know that any malaria naive
adult that becomes infected typically becomes severely ill and unable to efficiently combat the
infection.
Impaired DC activation upon malaria infection, as indicated in the Dogon population in our
study, may result in a deficient and delayed adaptive immune response as commonly seen in
malaria. This may not be the case in the Fulani, where we observed increased DC activation
and a more Th-1 skewed response upon Malaria infection. We know that DCs play a pivotal
role in initiating immune responses and directing adaptive immunity. The early IFN-skewed
response seen in the Fulani is the most beneficial response for the host and we suggest that
such a response may well depend on the manner of DC activation upon infection.
45
Future work
The immunogenic effects of Hz and in particular its impact on APCs are still not clarified. In
relation to the first study an interesting aspect to study is whether the increased migration
towards lymphoid ligands can be reproduced upon stimulation of DCs with iRBCs instead of
Hz. Another interesting follow up would be to test the capacity of Hz-induced “partially
matured” DCs to activate T cells.
In relation to the second study a follow up during the dry season would yield very important
information about the innate immune system of the children included in the study when not
exposed to the malaria parasite. In addition, to test the response of APCs obtained from these
children in vitro to Hz and iRBCs, similar to the experimental setup used in the first study
presented here, might yield important information of the function of individual cell types in
the different ethnicities upon exposure to malaria. This may even lead to proof that the initial
response to P. falciparum infection is indeed decisive in the outcome of the infection and thus
may in the future lead to focus efforts of preventive strategies to the initial, innate response to
the parasite.
46
Acknowledgements
I would like to thank everybody at the department of immunology as well as other
departments, institutes and universities that have in any way contributed to my education and
development within the fields of malaria and immunology.
First and foremost my gratitude goes to my supervisor Marita Troye-Blomberg for giving me
the opportunity to work with you and always taking your time to help me with answers to all
my questions and suggest solutions to my problems.
I am also very grateful to my co-supervisor Stefania Varani for support and endless
comments, sometimes difficult but I really am grateful and I know that in the end it is all for
the best, that has greatly improved the quality of this work and hopefully my own abilities as
an author. Grazie mille!
Another great support in writing this thesis was Carmen Fernandez muchísimas gracias por
toda la ayuda no solo con esta tesis pero también con todo lo demás.
I am also grateful to Klavz Bersins, Eva Sverremark-Ekström and Eva Severinson for aiding
in all kinds of worries from cross-words to C-course problems.
For all things practical Gelana, Maggan, Ann och Anna-lena tack, tack och tack för kaffet,
korsorden medmera men först och främst att ni orkar med all röra och kaos jag sprider
runtomkring mig.
All the other students that I have, or have had, the pleasure to work with, “Allergy girls”, “TB
boys”, “Hispanic crew” and prominent Malaria student investigators. To those that are now
roaming about in the great big world outside these walls, in particular Lisa
(Vemskajanuskyllapå) Isralesson. And of course those whose company I still have the
pleasure to enjoy.
47
For the time spent in and outside the lab, country and continent, it has been great fun and I am
sure it will stay that way for years to come. I especially want to mention Charles “Malian
genius” Arama, Ebba “duvetvem” Sohlberg and Stéphanie “TanzanianElisaOracle” Boström.
I am very glad to have crossed paths with you and for being able to share your experiences be
they good, bad or even everyday ones.
To all the former students at the department not mentioned here, the corridor in front or at all
the different departments where important issues are being resolved, be it by Chen girls or by
Portuguese mafia.
Last but not least, all my family past, present or future, by blood or picked up along the way I
will keep you forever with me.
Para toda mi familia, pasada, presente y porvenir, de sangre o recuparada por los caminos los
llevare por siempre conmigo. Mamma, Pappa, Erre och Marre vad hade jag varit utan er?
Hasta la Victoria siempre!
48
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Synthetic hemozoin induces partial maturation of human dendritic cells and increases
their migration towards lymphoid chemokines.
Giusti Pablo (1), Urban Britta (2)(6), Frascaroli Giada (3), Tinti Anna (4), Troye-Blomberg
Marita (1) †, Varani Stefania. (1)(5)
(1)Department of Immunology, Wenner-Gren Center, Stockholm University, Stockholm,
Sweden
(2) Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK
(3)Institute for Virology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm,
Germany
(4)Dept. of Biochemistry G. Moruzzi, University of Bologna, Bologna, Italy
(5)Dept. of Laboratory Medicine, Division of Clinical Microbiology, Karolinska University
Hospital, Huddinge, Sweden.
(6) KEMRI-Wellcome Trust Collaborative Research Programme, Centre for Geographical
Medicine, Coast, PO Box 230, Kilifi, Kenya
†
Corresponding author:
Prof. Marita Troye-Blomberg, PhD
Department of Immunology
Wenner-Gren Institute
Svante Arrheniusväg 16
Stockholm University
S-106 91 Stockholm
Sweden
Tel +46 (0)8164164
Fax + 47 (0)8 157356
E-mail:
[email protected]
Abstract
Acute and chronic Plasmodium falciparum infections alter the immune competence of the
host possibly through changes in dendritic cell (DC) functionality. DCs are the most potent
activators of T cells and migration is integral to their function. Mature DCs express lymphoid
chemokine receptors (CCRs) which enables them to migrate to the lymph nodes where they
encounter naïve T cells. The present study aimed to investigate the impact of the malaria
pigment hemozoin (Hz) or infected erythrocytes (iRBCs) on the expression of CCR.
Monocyte derived DCs partially matured upon incubation with synthetic Hz or iRBCs as
indicated by a moderately increased expression of CD80, CD83 and HLA-DR. Both stimuli
also provoked the release of pro-inflammatory and anti-inflammatory cytokines such as IL-6,
IL-10 and TNF-α and induced up-regulation of the lymphoid chemokine receptor CXCR4,
which was coupled to an increased migration to lymphoid ligands.
Taken together, these results suggest that the partial maturation of DCs upon stimulation with
malaria-derived products and the increased IL-10 secretion may lead to insufficient activation
of T cells or even induction of T cells with regulatory properties. The poor activation of T
cells may in turn lead to impaired adaptive immune responses and therefore insufficient
clearance of the parasites.
2
Introduction
Plasmodium falciparum (Pf)-malaria is one of the most severe infectious diseases in the world
where as many as 3.3 billion people live at risk of infection and 247 million cases where
reported in 2006. The vast majority of victims are children below 5 years of age (1).
Immunity to malaria is developed only after repeated exposure and is not long lasting (2).
Several studies have demonstrated impairment of immune responses in acute and chronic Pf
malaria (3).
Previous studies indicate that an early pro-inflammatory cytokine-mediated mechanism is
crucial to control parasitemia and for the clearance of parasites. Excessive pro-inflammatory
responses, however, can cause severe disease (4). The rapid production of cytokines implies
release either from pre-existing memory T cells or from cells of the innate immune system,
such as antigen presenting cells (APCs) or natural killer (NK) cells. Among APCs, dendritic
cells (DCs) are the most potent in initiating, controlling and regulating functionally distinct T
cell responses (5,6). Three signals are required from a DC to potently activate naive T cells
and they consist of high expression of peptide-loaded human leukocyte antigen (HLA) class II
molecules, co-stimulatory molecules and cytokine secretion. For proper stimulation of T cells,
upon encountering a pathogen in the periphery DCs need to migrate to secondary lymphoid
organs in order to encounter them (7). The migratory capacities of DCs are guided by the
expression of chemokine receptors (CCRs), which in turn is regulated by DC maturation. In
the periphery, the expression of CCR1 and CCR5 enable DCs to detect inflammation and find
the causative agent. Upon maturation, those inflammatory CCR are down regulated and
lymphoid CCRs, like CXCR4 and CCR7, whose ligands are expressed in lymphatic tissues
are instead up regulated (5).
The Pf parasite has been shown to affect DC responses through the action of the malaria
pigment hemozoin (Hz) or through adhesion of Pf infected red blood cells (iRBCs) to these
3
cells. However, the data obtained so far are somewhat contradictory. Coban et al. reported
activation of monocyte-derived DCs (MoDCs) upon Hz stimulation, as indicated by elevated
expression of maturation markers and IL-12 secretion (8). On the other hand, Hz-loaded
monocytes do not differentiate properly into MoDCs, and Hz-loaded MoDCs exhibit an
impaired maturation in response to lipopolysaccharide (LPS), suggesting that Hz could have
an inhibitory role in the development of inflammatory DCs (9). It has been shown that highdoses of iRBCs inhibit LPS-induced maturation of MoDCs and alter their immunostimulatory
abilities (10). It was later suggested that this effect could have been induced by apoptosis of
DCs upon contact with high doses of iRBCs (11). Yet another study has recently
demonstrated that low doses of iRBCs induce semi-maturation of MoDCs and that these cells
release TNF-α, IL-6 and IL-10 but not IL-12 (12).
In vivo, the proportion of circulating activated DCs is reduced in children with Pf malaria
(13). Recent evidence shows that a minor blood myeloid DC population, the blood dendritic
cell antigen (BDCA)-3+ DCs, is expanded in children with severe malaria as compared to
healthy controls, which was associated with augmented IL-10 plasma levels and impaired DC
allostimulatory ability, indicating an immunomodulatory function for this APC subset (14).
Therefore, despite contradictory in vitro results, analysis of DCs during natural malaria
infection suggests that these cells are activated during infection, and importantly exhibit an
immunomodulatory phenotype as compared to classical type-1 DC activation which leads to a
Th1 skewed T cell response.
In the present study, the phenotype and function of MoDCs were evaluated upon exposure to
synthetic Hz (sHz), parasite lysates and iRBCs. As a measure of function the expression of
CCRs and their capacity to migrate towards chemoattractants were investigated.
4
Materials and Methods
Dendritic cell cultures
Peripheral blood mononuclear cells (PBMCs) from anonymous blood donors (Karolinska
Hospital, Stockholm, Sweden) were isolated on a density gradient centrifugation using FicollPaque (GE Healthcare, Uppsala, Sweden) and used for further cell purification. MoDCs were
generated by modification of a described method (15). Briefly, monocytes were isolated by
negative selection using MACS microbeads according to the manufacturer's instructions
(Monocyte Isolation Kit II, Miltenyi Biotech, Bergisch Gladbach, Germany). Purified
monocytes were resuspended in complete RPMI 1640 supplemented with 10% fetal calf
serum (FCS), 35 ng/ml IL-4, and 50 ng/ml GM-CSF (R&D Systems, Minneapolis, MN,
USA). Cell differentiation was monitored by light microscopy and flow cytometry. At day 5,
cells were harvested and 89 % ± 1 % of the cells exhibited a typical MoDC phenotype (CD14 CD1a+). Parasite lysate, sHz or iRBCs were then added to the differentiated MoDC cultures.
In order to induce maturation of the MoDC, 1 µM of prostaglandin E2 (PGE2) (R&D
Systems) and 50 ng/ml of TNF- (R&D Systems) were added to the culture medium at day 5
if nothing else is stated.
Preparation of synthetic hemozoin
Synthetic Hz, also called -hematin, is structurally similar to Hz formed naturally by parasites
(16,17) and was prepared from hemin chloride as previously described (18) with some
modifications (19). Briefly, 75 mg of porcine hemin (Sigma-Aldrich, Steinheim, Netherlands)
were dissolved in 14.5 ml NaOH 0.1 M and incubated at 60°C. Within 10 min, 1.45 ml HCl
(1M) and 8.825 ml sodium acetate (12,8 M) were added and the solution was incubated for
two hours at 60°C constantly stirring at a rate of ~150 rpm. The solution was then centrifuged
at 15000 g for 13 min, and the pellet was resuspended in Tris-HCl buffer (100 mM, pH=8)
containing 2.5% SDS (KEBO Lab, Stockholm, Sweden) and incubated for 30 min 37°C to
5
dissolve heme aggregates. The solution was then centrifuged at 15000 g for 13 min and the
pellet resuspended in pre-warmed NaHCO3 (100 mM, pH=9.2). After washing twice, Hz was
dried at 37°C. The resulting pellet was weighed and re-suspended to 10 mg/ml in sterile
water. In agreement with previous findings (20), the spectrum of Hz exhibited the presence of
intense bands at 1710, 1660, 1299, 1279, 1209 cm-1 (Figure 1). Two intense bands at 1660 and
1209 cm-1 were present in our preparation indicating a high grade of purity of the sample (18).
The endotoxin level in the preparation was below 0.05 endotoxin units (EU) EU/ml
(Bacteriology Lab, Sahlgrenska Hospital, Göteborg, Sweden) in our highest working
concentration.
Parasite cultures
Pf parasite strains were cultured using human O+ RBCs (Karolinska Hospital, Stockholm,
Sweden) in RPMI 1640 medium containing 25mM Hepes, 2mM L-glutamine, 50 μg/ml
gentamycin, and 0.2% sodium bicarbonate and supplemented with 0,5% Albumax (Gibco™,
Paisley, UK). The Tanzanian laboratory strain F32 was maintained in continuous cultures in
candle jars as previously described (21). Parasite cultures were regularly tested for
Mycoplasma contamination by the mycoplasma detection kit Myocoalert® (Lonza, Rockland
ME, USA). The parasite cultures were kept synchronous by treatment with 5% D-sorbitol in
distilled water as previously described (22). In order to produce parasite lysates, late stage
parasites of the F32 strain were purified on 60% Percoll gradient centrifugation, sonicated as
previously described (23) and stored at -80° C until used.
Intact iRBCs harbouring late parasite stages (schizonts and late trophozoites) were harvested
when the parasitemia reached approximately 10% by using magnetic separation as previously
described (24).
Briefly, parasite cultures were pelleted and re-suspended in phosphate-buffered saline (PBS)
supplemented with 2% bovine serum albumin (BSA) and 2 mM EDTA. Parasites were added
6
to CS columns (Miltenyi Biotech) and incubated 10 min before washing the column. The
column was then removed from the magnet and the parasites were rinsed out with PBS.
Percentage of late stage iRBCs was determined by staining with acridine orange and counting
under UV-microscope. Uninfected RBCs run in parallel under the same conditions were used
as negative controls. iRBCs and uninfected RBCs were derived from the same donor in each
experiment.
MoDCs-parasites co-cultures
In order to prevent burst of the infected erythrocytes during co-culture, iRBCs were incubated
in aphidicolin which arrests the parasite-cell cycle in the trophozoit stage (25). Briefly,
synchronous parasite cultures at ring stage (8-10% parasitemia) were cultured in 15 µg/ml of
aphidicolin (Sigma-Aldrich, St. Louis, USA) for maximum 24 hours. When the parasites had
reached trophozoit stage the cultures were purified using magnetic separation as previously
described (24). Immature MoDCs were harvested and resuspended at 106 cells/ml in 24-well
plates. Purified trophozoites (90 % ± 1 % purity) were added at the ratios of iRBCs/DCs 10:1,
and 100:1 or uninfected RBCs at the high ratio. MoDCs and uninfected or iRBCs were cocultured for 10 hours and TNF-/PGE2 was used as a positive maturation control.
Immunophenotype
All antibodies used were mouse monoclonal antibodies either unconjugated or conjugated
with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Conjugated antibodies used for
cell-surface staining included those recognizing CD1a, CD14, CD80, CD83, CD86, HLA-DR,
(Pharmingen, San Diego, CA, USA). Unconjugated antibodies were CCR7, CXCR4 (R&D
Systems) that were made detectable using a secondary polyclonal goat anti-mouse PE
conjugated antibody (Dako Cytomation, DAKO A/S, Denmark). Data acquisition and analysis
were performed on a FACSCalibur flow cytometer (Beckton Dickinson, Mountain View, CA)
using BD CellQuest™Pro version 5.2.1 software. FACS data are presented here as mean
7
fluorescence intensity (MFI) indicating the level of expression of a certain marker on an
individual cell.
Assessment of migratory properties of MoDCs
The human recombinant chemokines used were the chemokine (C-X-C motif) ligand,
(CXCL)-12/stromal cell-derived factor (SDF)-1α and the CCL19/MIP-3β (PeproTech Inc.,
Rocky Hill, NJ, USA). Both chemokines were used at a final concentration of 100 ng/ml in
RPMI 1640 medium containing 1% FCS. Cell migration was evaluated using a 48-well
Boyden chamber (Neuroprobe, Pleasanton, CA, USA) with 5-µm-pore-size polycarbonate
filters. Medium without FCS was used as control of background migration. Cells were
allowed to migrate for 1.5 hours after which MoDCs on the membrane were stained and
counted. All samples were assayed in duplicates, and the number of cells that migrated in five
visual fields (original magnification, x100) was determined for each well, as previously
described (26). The net number of cells that migrated was calculated by subtracting the
number of cells that migrated in response to medium alone from the number of cells that
migrated in response to chemokines.
Cytokine measurement in the supernatant of cell cultures
Supernatants from MoDC cultures were used to analyse cytokine production in the presence
or absence of malaria-derived material. Supernatants were collected when the cells where
harvested and stored at -70°C. Supernatants were analyzed for the levels of interferon (IFN)γ, IL-1β, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF- and TNF-β using a Human Th1/Th2
11plex bead assay (Bender MedSystems, Vienna, Austria) according to manufacturer’s
instructions. Data acquisition and analysis were performed by FACSCalibur and the FCAP
Array software (Soft Flow, Hungary Ltd. Hungary).
8
Statistical analysis
Data was analyzed using the Wilcoxon’s rank-sum test. Comparisons were considered
statistically significant at p < 0.05.
9
Results
sHz induces partial maturation of MoDCs and increases lymphoid CCR expression
In order to evaluate the effect of sHz on the phenotype of MoDCs, cells were stimulated with
sHz or TNF-α/PGE2 overnight. MoDCs cultured in medium alone were used as a negative
control. As expected, cells that were stimulated with TNF-α/PGE2 showed a statistically
significant increased expression of CD80 and CD83 (Figure 2). After stimulation with sHz, an
upregulation of CD80 was observed (p=0.046 and p=0.018, for 10 or 20 µg/ml, respectively).
Non-stimulated cells expressed low levels of CD83 and there was a statistically significant up
regulation of this maturation marker when the cells were stimulated with 10 µg/ml sHz
(p=0.043), while the highest concentration of sHz (20 µg/ml) did not induce a significant
increase of CD83 on MoDCs. TNF-α/PGE2 induced a strongly increased expression of HLADR and CCR7 molecules, while the expression levels of these markers were only slightly, not
significantly, increased upon sHz stimulation (Fig 2c and d). Conversely, similarly to the
effect caused on MoDCs by TNF-α/PGE2, sHz induced significant increased expression of
CXCR4 as compared with unstimulated MoDCs when added at the highest concentration (20
µg/ml) (p=0.028) and a similar increase was observed using10 µg/ml although this was not
significant (p=0.0797). Thus, sHz induces partial maturation of MoDCs and increased
lymphoid CXCR4 expression on the cell surface.
To evaluate the kinetics of MoDC activation upon sHz or TNF-α/PGE2 stimulation, a
phenotypic analysis at different time points after incubation with these stimuli was performed.
Given the results obtained in the previous section the kinetics of activation markers was
analyzed only after stimulating cells with 20 µg/ml of sHz. At first, data was evaluated
without taking into consideration the time points to confirm the partial activation. The
spontaneous MFI expression of CD80 was 56 +/- 3 (mean–value +/- standard error) and
increased to 85+/- 4 when stimulated with 20 µg/ml sHz and 96 +/- 4 with TNF-α/PGE2
10
stimuli. For CD83 the background expression was 21 +/- 4 and increased to 35 +/- 6 upon sHz
stimuli and to 68 +/- 6 when stimulated with TNF-α/PGE2. HLA-DR expression increased
from 75+/- 3 to 91 +/- 2 upon sHz stimulation and to 100 +/- 1 using TNF-α/PGE2.
The data were then evaluated by considering different time intervals, and increased expression
of CD80 and HLA-DR was maintained after 60 hours of stimulation regardless of the type of
stimulus (Figure 3a and c). For CD83, there was a tendency of continuously decreased
expression when stimulated with sHz. The statistical analysis showed that the decrease
between the 12 and 60 hour time points was significant for unstimulated cells (p=0.021) or
cells stimulated with sHz (p=0.043) (Figure 3b). When the cells were stimulated with
TNFα/PGE2 however, no statistically significant differences were seen (p=0.19). Thus, the
partial activation that was observed in MoDCs stimulated with sHz was not fully maintained
at 60 hours of incubation.
sHz impairs the up regulation of CCR7 induced by maturation stimuli on MoDCs
In addition to the stimulatory effect of sHz seen on unstimulated MoDCs we investigated
whether sHz could interfere with the maturation induced by TNFα/PGE2. As shown in Figure
4, addition of sHz to the cultures prior to TNF-α/PGE2 stimulation significantly impaired the
up-regulation of CCR7 (p= 0.042 and p=0.017, for 10 µg and 20 µg of sHz, respectively). No
differences were observed for the expression levels of CD80, CD83, HLA-DR and CXCR4 in
MoDCs matured with TNF-α/PGE2 in the presence or absence of sHz (data not shown). Thus,
sHz selectively blocks the CCR7 up-regulation induced by TNF-α/PGE2 on MoDCs.
11
Contact with iRBCs induces partial maturation of MoDCs
Next, we examined whether parasite lysates or intact iRBCs could induce activation of
MoDCs. MoDCs from four donors were incubated for 18 hours with 15 or 75 µg/ml of
parasite lysate. The cells were subsequently harvested and stained for surface expression of
CD80, CD83, HLA-DR, CXCR4 and CCR7 and analyzed by FACS. No significant
differences were observed in the investigated markers upon contact with parasite lysates (data
not shown).
To evaluate the effect of iRBCs on MoDCs, cells were incubated with aphidicolin-treated
iRBCs, (F32 laboratory strain) or uninfected RBCs, for 12 hours and then stained for CD83,
HLA-DR, CXCR4 and CCR7 before FACS acquisition. The expression levels of the different
markers did not differ significantly between MoDCs cultured in medium alone or with
uninfected RBCs . Similarly, low doses of iRBCs (10:1, iRBCs:MoDCs) did not significantly
alter the expression of the investigated markers (data not shown). Conversely, the high iRBC
to MoDCs ratio revealed a strong tendency, similar to that seen for TNF-α/PGE2, for an up
regulation of MFI values from 82 +/- 4 (mean–value +/- standard error), 26 +/- 6 and 58 +/14 in the unstimulated cells to 90 +/- 11, 39 +/- 1 and 89 +/- 12 for HLA-DR, CCR7, and
CXCR4, respectively. The levels of HLA-DR, CCR7 and CXCR4 were 99 +/- 8, 51 +/- 9 and
96 +/- 12, respectively, in cells cultured with TNF-α/PGE2 (Figure 5). In addition, TNFα/PGE2 stimuli lead to an increased CD83 expression from 20 +/- 15 to 44 +/- 16. No
difference was seen in cells stimulated with high ratio of iRBCs as compared to uninfected
RBCs (data not shown). Thus, high doses of iRBCs seem to induce an incomplete maturation
12
of MoDCs. Importantly, intact parasitized erythrocytes are required to induce this effect, since
parasite lysates could not induce any maturation/activation of DCs.
sHz-induced maturation of MoDCs is coupled to increased migratory capacity
in
response to lymphoid chemokines.
Upon maturation, MoDCs acquire the ability to migrate in response to lymphoid chemokines
by up regulating the CXCR4 and CCR7 molecules (27), Since we found that sHz induced a
partial maturation of MoDCs with an up regulation of CXCR4, the capacity of sHz-treated
MoDCs to migrate in response to inflammatory and lymphoid ligands was assessed. MoDCs
treated with TNF-α/PGE2 exhibited a significantly increased migratory capacity, as compared
to untreated cells, in response to the CXCR4 (n=6) and CCR7 (n=5) ligands, CXCL12/SDF1α (p=0.03) and CCL19/MIP3-β (p=0.04), respectively (Figure 6). An increased migration
towards CXCL12/SDF1-α (p=0.03) as well as for CCL19/MIP3-β (p=0.04) was also
observed in sHz-treated MoDCs as compared to untreated cells.
MoDCs exhibit increased secretion of pro- and anti- inflammatory cytokines after
stimulation with sHz or high concentrations of iRBCs.
To investigate whether malaria-derived stimuli could induce the production of soluble
mediators in MoDCs, the levels of selected cytokines were measured in the cell-culture
supernatant obtained from sHz-, iRBC- and un- treated MoDCs. Baseline production of all
inflammatory cytokines in unstimulated MoDCs was negligible except for IL-8, as shown in
13
Figure 7a and b. In agreement with published data (28), the levels of all examined cytokines
were unaffected upon stimulation with TNF-α/PGE2 (Figure 7a and b). MoDCs stimulated
with 20 µg of sHz released significantly higher levels of IL-6 (p=0.009) and IL-10 (p=0.021)
as compared to unstimulated cells (Figure 7a). The secretion of IL-6 was also increased when
stimulated with 10 µg of sHz (p=0.05). There was also a slight increase in TNF-α secretion
in sHz-stimulated cultures as compared to unstimulated cultures, but this did not reach
statistical significance. No differences were found in IL-1β, IL-8 or IL-12p70 secretion.
Next, we analyzed cytokines release by MoDCs after stimulation with iRBCs. At the low
(10:1) iRBCs:MoDCs ratio no significant increment in the release of IFN-γ, IL-1β, IL-2, IL-5,
IL-6, IL-8, IL-10, IL-12p70, TNF- or TNF-β was observed (data not shown). However,
using a high (100:1) iRBCs:MoDCs ratio there was a significantly increased secretion of IL1β (p=0.021), IL-6 (p=0.021), IL-10 (p=0.021) and TNF-α (p=0.043) by MoDCs. Of note, the
secretion of IL-12p70 was very low upon iRBCs stimulation in all donors examined.
When comparing the levels of cytokines secreted by DCs upon stimulation with sHz and
iRBCs the same cytokines were elevated with both stimuli except for IL-1β that was not
increased upon sHz stimulation. The levels of IL-6, IL-10 and TNF- were higher when
MoDCs were stimulated with iRBCs as compared to sHz stimulation. Thus, these results
indicate that both sHz and iRBCs induce secretion of a number of pro-inflammatory cytokines
in MoDCs.
14
Discussion
Previous studies evaluating the effect of malaria-derived products have found that immune
cells exposed to malaria-derived antigens are impaired in their activation and subsequent
function. In this study a partial activation of MoDCs upon exposure to both iRBCs and sHz
was observed, as demonstrated by an increased, albeit moderate, expression of maturation
markers, as well as lymphoid CCRs on the surface of DCs as compared to the TNF-/PGE2
induced maturation. The up-regulation of CXCR4 and the slight up regulation of CCR7 on the
surface of MoDCs was coupled to a significantly increased migration towards their respective
ligands CXCL12 /SDF1-α and CCL19/MIP3-β. A kinetic study revealed that MoDCs,
unstimulated or activated by sHz could not maintain their expression of CD83 for a 60 hour
period. When the cells where stimulated with TNF-α/PGE2, however, they did maintain
elevated levels of CD83 expression. These data strengthen the results indicating that malaria
derived products activate MoDCs, but not as potently as TNF-α/PGE2.
It is known that the quality of the interaction between T cells and DCs affects
the outcome of the immune response (5,6) and that co-stimulation and cytokine release are
crucial factors in antigen presentation and the modulation of T-cell responses (5,6). In our
experiments, iRBC- and sHz- exposed MoDCs were not fully matured with regard to proper
up-regulation of maturation markers nor did these cells release IL-12, which is crucial to
activate a type 1 response including IFN-γ secretion (29). In addition, our iRBC- and sHzexposed MoDCs released IL-10.
We therefore propose that the partial activation of myeloid DCs in combination
with the release of IL-10 might lead to impaired T-cell activation, in line with what has been
observed in vitro (10) and during natural infections (14). Alternatively, it is possible that this
DC phenotype may lead to the induction of a regulatory-T cell (Treg) phenotype. Tregs have
been reported to contribute to immune evasion during malaria in mice and humans, indicating
15
that this is one mechanism whereby the malaria parasites could subvert the host immune
system (30). In support of this, evidence shows expansion of Treg population in a controlled
model of experimental malaria in humans (31), which was related to a more rapid parasite
growth (32). In addition, a field study in Kenya reported that expanded CD4+CD25high cell
population, potentially Tregs, was associated with a higher risk of clinical malaria (33).
We also observed that sHz selectively impaired the up-regulation of CCR7
induced by TNF-/PGE2 stimulation. Recent evidence indicates that, apart from chemotaxis,
CCR7 controls the cytoarchitecture, the migratory speed, and the maturation rate of DCs (34).
Therefore, blocking the CCR7 up-regulation on maturing MoDCs may be an effective tactic
for the malaria parasite to block DC functionality, thus impairing the initiation of the T-cell
responses, as seen in other parasitic infections (35). In support of this, it has been observed
that Hz-containing DCs reside in the T-cell areas of the spleen, but fail to induce a proper Tcell effector function in a murine model of malaria infection (3).
Increased secretion of a number of pro-inflammatory cytokines such as IL1-β,
IL-6, and TNF-α by MoDCs upon contact with Pf-derived stimuli was observed in our study.
Malaria creates a large inflammatory response during acute infection and a correlation has
been found between high levels of serum TNF-α and IL-10, severe malaria and high
parasitemia in African children (36). Recently, the serum levels of IL1-β, IL-6, IL-10 and
TNF-α have been associated with severe complications during malaria infection in various
studies (37,38).
Our results indicate that myeloid DCs may be crucial cells in inducing the large
inflammatory response that is often observed during acute Pf infection in the natural host.
In conclusion, contact of MoDCs with sHz or iRBCs induced partial maturation of these cells,
provoked the release of some inflammatory cytokines and up-regulated lymphoid CCR on the
cell surface. It was also observed that the up regulated expression of CXCR4 was coupled to
16
increased migration to the related ligand. This might suggest that Pf induces MoDCs to
migrate to lymphoid organs. On the other hand, it appears that MoDCs activated by malaria
stimuli might not be fully equipped to potently activate T cells or that they may skew the Tcell differentiation towards a regulatory phenotype. This phenomenon may contribute to
altered Pf specific immunity that is often observed in malaria-infected patients.
Acknowledgements
This work was supported by grants from the Swedish Agency for Research and Development
with Developing Countries (Sida/SAREC), as well as a grant within the BioMalPar European
Network of Excellence (LSHP-CT-2004-503578)
17
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20
Figure legends
Figure 1. Characteristics of sHz preparation.
The spectrogram exhibits characteristic peaks of hemozoin. The scale represents the sHz as
prepared according to the Tripathi’s modification of the Egan’s protocol. The spectrum
exhibited the presence of intense bands at 1710, 1660, 1299, 1279, 1209 cm-1 characteristic
of hemozoin.
Figure 2. sHz induces partial maturation of MoDCs and causes upregulation of
lymphoid CCR expression.
MoDCs were incubated with or without sHz (10 or 20 µg/ml), TNF-α and PGE2 were added
or not as maturation stimuli in designated wells. After 18 hours of incubation, the cells were
harvested, stained for surface markers and analyzed by FACS. Y-axis reports mean
fluorescence intensity (MFI) for CD80, CD83, HLA-DR, CXCR4 and CCR7. The boxplots
illustrate the medians and the 25th and 75th quartile and the whiskers represent the 10% and
90% percentiles for 5 donors. Data were analyzed using Wilcoxon’s rank sum test * = P<0.05.
** = P<0.01.
Figure 3 Kinetics of expression levels for different activation markers on MoDCs upon
sHz stimulation.
MoDCs (1x106 cells/ml) were left unstimulated or were stimulated with sHz (20 µg/ml) or the
maturation stimuli (TNF-α and PGE2) and harvested after 12, 36 or 60 hours of incubation.
21
Cells were stained for surface markers and analyzed by FACS. The graphs illustrate the result
of one representative donor out of four independent experiments.
Figure 4. sHz impairs the upregulation of CCR7 upon DC maturation.
MoDCs were incubated with or without sHz (10 (n=5) or 20 (n=8) µg/ml) for 4 hours, when
the maturation stimuli (TNF-α and PGE2) were added. Cells were incubated for additional 18
hours, harvested, stained for surface markers and analyzed by FACS. Y-axis indicates mean
fluorescence intensity (MFI) for CCR7. The boxplots illustrate the medians and the 25th and
75th quartiles and the whiskers represent the 10% and 90% percentiles. Data were analyzed
using Wilcoxon’s rank sum test *; P<0.05.
Figure 5. Incubation with iRBCs induces partial maturation of MoDCs including up
regulation of lymphoid CCR expression.
MoDCs were co-cultured with uninfected RBCs (ctrl RBC) or iRBCs at the ratio of 100:1
(RBCs/DCs). Maturation stimuli (TNF-α and PGE2) were added in separate wells as a
positive control for maturation. Cells were incubated for 12 hours, harvested, stained for
surface markers and analysed by FACS. The mean fluorescence intensity (MFI) of HLA-DR,
CD83, CXCR4 and CCR7 is illustrated in the diagram as mean values and standard error of
the mean. Data were analyzed using Wilcoxon’s rank-sum test.
Figure 6. Partial maturation of MoDCs induced by sHz is coupled to increased
migration in response to lymphoid chemokines.
MoDCs were stimulated with sHz (20µg/ml) or the maturation stimuli (TNF-α/PGE2) and
incubated for 18 hours. Cells were then harvested and seeded in the upper compartments of a
Boyden chamber while 100 ng/ml of CXCL12/SDF1-α or CCL19/MIP3-β were added to the
22
lower compartments. Medium without FCS was used as control of background migration.
Cells were incubated for 1.5 hours to allow migration after which the cells that had migrated
to the lower compartment were counted in 5 representative fields for each well. The net
number of cells that migrated was calculated by subtracting the number of cells that migrated
in response to medium alone from the number of cells that migrated in response to
chemokines. The diagram represents mean values ± standard error of the mean of 6 donors for
CXCL12/SDF1-α and 5 donors for CCL19/MIP3-β. Data were analyzed using Wilcoxon’s
rank sum test *; P<0.05.
Figure 7. Increased secretion of pro and anti- inflammatory cytokines by MoDCs upon
stimulation with high concentrations of sHz and iRBCs.
(a) MoDC were incubated with 10 and 20 µg/ml of sHz and cell culture supernatants were
subsequently analyzed for cytokines. Each graph illustrates the mean values and SEM of 4
donors and the concentration in pg/ml of each cytokine. (b) MoDCs were incubated with a
high and a low concentration of iRBCs and cell culture supernatants were subsequently
analyzed for cytokines. Each graph illustrates the mean values and standard error of the mean
of 4 donors and the concentration in pg/ml of each cytokine.
23
Figure 1
24
Figure 2
25
Figure 3
26
Figure 4
27
Figure 5
28
Figure 6
29
Figure 7a
30
Figure 7b
31
Low frequency of circulating dendritic cells is associated with higher cell activation,
raised plasma levels of anti-malarial antibodies and increased pro-inflammatory
responses during Plasmodium falciparum infection in Fulani, an ethnic group with low
susceptibility to malaria.
Charles Arama*1,2, Pablo Giusti*1, Stéphanie Boström 1, Victor Dara2, Boubacar Traore2,
Amagana Dolo2, Ogobara Doumbo2, Stefania Varani 1,3 and Marita Troye-Blomberg1†
1
Department of Immunology Wenner-Gren Institute Stockholm University, Sweden.
2
Malaria Research & Training Centre, Faculty of Medicine, Pharmacology & Dentistry,
University of Bamako, Bamako, Mali.
3
Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska
University Hospital, Huddinge, Sweden.
* These authors contributed equally to this work.
†
Corresponding author:
Prof. Marita Troye-Blomberg, PhD
Department of Immunology
Wenner-Gren Institute
Svante Arrheniusväg 16
Stockholm University
S-106 91 Stockholm
Sweden
Tel +46 (0)8164164
Fax + 47 (0)8 157356
E-mail:
[email protected]
Abstract
The Fulani ethnic group from West Africa is relatively better protected against malaria
infection as compared to other sympatric ethnic groups, but little is known about the
mechanisms behind this lower susceptibility to Plasmodium falciparum (Pf) infection.
Dendritic cells (DCs) are the most potent activators of naive T cells, and thereby play an
important role in regulating the adaptive immune responses. It has been shown that the DC
subset expressing the blood-dendritic-cell antigen (BDCA)-3 is more prevalent in the
circulation of children with severe malaria. In this regard, we aimed to investigate whether
two ethnic groups exposed to similar malaria pressure but differing in their response to
malaria exhibit differences in terms of number and activation status of circulating DCs,
plasma levels of cytokines and anti-malaria specific antibodies.
The present study has been conducted in children during the malaria season of 2008. All
children belonged to either the Fulani or the Dogon ethnic groups, and they were divided into
two groups i.e. one with active Pf infection and one with no active infection.
In agreement with previous studies, the Fulani exhibited higher plasma levels of anti-malarial
antibodies as compared to the Dogon. In addition, plasma levels of IFN-, IFN- and a
number of pro-inflammatory cytokines were higher in the Fulani children as compared to the
Dogon, regardless of infection. During Pf infection, serum levels of IFN- were increased in
the Fulani, while a different pro-inflammatory pattern that lacked IFN- was seen in the
Dogon. Interestingly, when analysing the expression of activation markers on different DC
subsets, our results showed that malaria infection induced activation of pDCs in Fulani but
impaired activation in the Dogon. Taken together, this suggests a marked difference in innate
responses that could clearly affect adaptive immune responses already early in life. In
conclusion, the findings presented here suggest are likely to be important for the relative
protection against malaria seen in the Fulani as compared to the Dogon.
2
Introduction
Malaria is one of the most important infectious diseases in the world and has a severe impact
on child mortality. Mali in West Africa has seasonal malaria, with infections peaking during
the late rain period from September to November. Previous studies performed in a rural area
in Mali, conducted during the malaria transmission season, have shown different
susceptibility to Plasmodium falciparum (Pf) infection between two different ethnic groups;
the Fulani and the Dogon. These populations live under similar social, cultural and geographic
conditions (1) and are exposed to identical malaria pressure. However, the Fulani show less
clinical symptoms of malaria, and parasites are less frequently detected in their blood (2). In
addition they exhibit higher titres of Pf-specific IgG subclasses (3), and IgM antibodies (4,5).
It has been shown that individuals undergoing malaria infection respond less to
other infections as well, indicating that at least some functions of the immune system are
somehow impaired by the parasite (6). Immunity to malaria is achieved only after multiple
infections and is not long lasting. This may indicate a failure in the adaptive branch of the
immune system to achieve proper memory responses. The initiators of adaptive immune
responses are the antigen-presenting cells (APCs) and the most potent APCs are dendritic
cells (DCs). Two main populations of DCs have been described in peripheral blood; the
plasmacytoid DCs (pDCs) and the myeloid DCs (mDCs), and the latter can be further divided
into three subtypes; the blood dendritic cell antigen-1 (BDCA-1+) DCs, the BDCA3+ DCs and
the CD16+ DCs. It has been shown that mDC differentiation and function are impaired in vitro
by the malaria parasite (7) or by products derived from infection (8). In addition, HLA-DR
levels are decreased on circulating DCs of Pf-infected children (9) as compared to healthy
controls and higher numbers of circulating BDCA-3 DCs have been detected in blood of
severely infected children combined with reduced stimulatory ability, indicating that malaria
3
may impair DC functionality and disclosing a modulatory role for the BDCA-3+ DC subset in
immunity (10).
During viral infections pDCs are essential components of the innate immunity
(11,12). In addition, pDCs have been shown to play a crucial role in B-cell activation and in
the induction of antigen-specific and polyclonal IgG production in response to viruses
(12,13). The role of this DC subset during malaria infection is still under investigation. It is
known that the function of pDCs is affected by Pf and that this cell subset is activated by
asexual blood stage parasites in vitro with increased interferon (IFN)-α secretion and
enhanced ability to stimulate γδ T cells (14). In addition, higher IFN-α levels in plasma have
been observed in patients suffering of mild as compared to severe malaria (15,16), suggesting
a possible role of pDCs in protection against severe malaria in humans.
Using flow cytometry, we evaluated the prevalence of different DC subpopulations in
peripheral blood mononuclear cells (PBMCs) obtained from Fulani and Dogon children with
or without malaria infection in a rural area of Mali. In addition, the plasma samples collected
from the corresponding individuals were analysed for the levels of anti-malarial IgM, IgG
subclasses as well as a number of pro-inflammatory and anti-inflammatory cytokines.
4
Materials and Methods
Study population
The study was conducted in a rural area of the Dogon valley of Mali where malaria is
mesoendemic with intense transmission during the rainy season. The study area and the study
population have been described in detail elsewhere (1). Children between 2 and 10 years of
age belonging to either the Fulani or the Dogon ethnic groups were included in the study.
Forty children from the Dogon population and thirty seven from Fulani were recruited. The
study included healthy children and patients that were classified as uncomplicated malaria. A
thick blood smear was made from each volunteer. The slides were stained in 3% Giemsa and
examined for the presence of Pf parasites. Presence of malaria infection was defined as a
positive thick smear with or without any malaria symptom. Axillary temperature was
measured in all patients and symptomatic malaria was defined as fever (≥37.5°C), or history
of fever, plus the presence of any density of parasites in the blood. Among the Dogon, 20
children were undergoing malaria infection and 20 children were healthy, while among Fulani
14 children were suffering of malaria infection and 23 were healthy. Ethical approval was
obtained from the institutional review boards of the University of Bamako Mali
(N°08_64/FMPOS). For each volunteer, both community and an individual consent form
from the parents were obtained, before the inclusion of the children in the study.
Blood collection and cell preparation
Venous whole blood samples were collected from healthy and infected children of both
ethnicities. From each volunteer, 6 ml of peripheral blood were collected in heparin tubes (BD
Vacutainer® Plasma Tube, 143 USP Units of Sodium Heparin freeze dried) and were
transported to the laboratory of Bandiagara for further experiments.
5
PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare,
Uppsala, Sweden) and used for APC immunophenotyping and expression of activation
markers. Plasma was separated by centrifugation; plasma samples were stored at -80°C and
used for cytokine and antibody studies.
Determination of circulating levels of anti-malaria antibodies
Determination of malaria-specific antibodies was done using a standard sandwich enzyme
linked immunosorbent assay (ELISA) using a crude malaria antigen preparation (17) and
performed as previously described (3)(18). Briefly, 96-well ELISA plates (Costar, Corning,
NY, USA) were coated with crude malaria antigen (10 µg/ml) over night at 4oC. Biotinconjugated mouse anti-human monoclonal antibodies and alkaline phosphatase conjugated
streptavidine (Strep-ALP) (Mabtech AB, Nacka, Sweden) were used for each subclass. For
detection of malaria-specific IgG and IgM, goat-anti-human IgG-ALP (Mabtech AB) and
goat-anti-human IgM-ALP (Jackson ImmunoResearch Laboratories, PA, USA) were used.
The assays were developed with p-nitrophenyl phosphate disodium salt (pNPP) (SigmaAldrich, St Louis, MO, USA) as substrate, and the optical densities were measured at 405nm
in an ELISA plate reader (VmaxTM Kinetic Microplate Reader, Menlo Park, CA, USA). The
concentrations of anti-malarial antibodies were calculated from standard curves obtained from
sandwich ELISA with 6 dilutions of myeloma proteins of the IgG1-4 isotypes (Biogenesis,
Poole, England) or for total anti-malarial IgG and IgM antibodies, with highly purified IgG or
IgM antibodies, respectively (Jackson ImmunoResearch Laboratories).
Determination of cytokine levels in serum using ELISA
The levels of IFN-α and IFN-γ were measured using commercial ELISA kits (Mabtech). All
samples were assayed in duplicates according to manufacturer’s recommendation. The
6
cytokine concentrations were calculated from a standard curve obtained in a sandwich ELISA
with eight dilutions of lyophilized native human IFN-α standard (range 10,000-3 pg/ml) and
recombinant human IFN-γ standard (range 3.000-1.0 pg/ml). The assays sensitivity was 2
pg/ml for IFN-α and 7 pg/ml for IFN-γ, respectively.
Determination of cytokine levels in serum using cytometric bead array
Plasma levels of IL-1β, IL-6, IL-8, IL-10, IL-12(p70) and TNF- were determined by using
cytometric bead array (CBA) technology (BD Biosciences, San Diego, CA) according to
manufacturers’ recommendation. Briefly, 24 µl of bead populations with distinct fluorescent
intensities, coated with cytokine-specific capture antibodies were added to 24 µl of serum
sample, and 24 µl of phycoerythrin (PE)-conjugated anti-human inflammatory cytokine
antibodies. Simultaneously, standards for each cytokine (range 0-10000 pg/ml) were also
mixed with cytokine capture beads and PE-conjugated reagent. The lower limit of detection
for the various cytokines were as followed; IL-1β (7.2), IL-6 (2.5), IL-8 (3.6), IL-10 (3.3), IL12(p70) (1.9) and TNF-α (3.7) pg/ml. All data were acquired using a FACSCalibur flow
cytometer (Becton Dickinson, Mountain View, CA, USA) and CellQuest software. The
software used for the analysis was the FCAP Array software (Soft Flow, Hungary Ltd.
Hungary).
Immunophenotype
Purified PBMCs were fixed and frozen immediately after purification as previously described
(19,20). Briefly, cells were resuspended in PBS to a concentration of 2 million cells/ml and
treated with DNAse (Sigma-Aldrich) for 5 minutes at 37°C at a concentration of 666 units/ml.
Cells were then washed and resuspended in 3 ml of prewarmed (37°C) 4% paraformaldehyde
(PFA) (Sigma-Aldrich), incubated for 5 minutes at RT and then washed in cold PBS +1%
7
foetal bovine serum (FBS, Gibco, Paisley, UK) Finally, cells were resuspended in 1 ml PBS
+10% DMSO and frozen at -70C. For staining, the cells were thawed and resuspended in PBS
5 mM EDTA 2% FBS. FcR blocking reagent and mouse monoclonal antibodies conjugated
with fluorescein isothiocyanate (FITC) or PE, peridinin chlorophyll protein (PerCP) or
allophycocyanin were added. Antibodies used for cell-surface staining included those
recognizing BDCA-1, BDCA-2, BDCA-3, CD3, CD14, CD19, and mouse isotype controls
IgG1 and IgG2a (Miltenyi Biotec, GmbH, Germany) and CD16, CD56, CD86, HLA-DR
(Pharmingen, San Diego, CA, USA). The cells were finally resuspended in PBS 1% PFA,
acquired and analysed using a FACSCalibur (Beckton Dickinson) and the BD CellQuest™Pro
version 5.2.1 software.
Statistical analysis
Statistical significance was determined using non-parametric tests. A Kruskal-Wallis test was
employed to compare when more than two groups were involved, if the test exposed a
significant difference the Mann-Whitney U-test for significance between two unpaired groups
was employed. For all tests p≤0.05 was considered significant and no correction was made for
using multiple tests. The data were analyzed using the StatView software.
8
Results
Specific antibody responses to Pf are higher in Fulani children than in the Dogon
children
Plasma levels of total antimalarial IgG, IgM as well as IgG1, IgG2, IgG3 and IgG4 were
measured in plasma of both uninfected and infected Fulani and Dogon. When considering
both uninfected and infected subjects, higher titres of anti-malarial IgG and IgM were found
in the Fulani population as compared to the Dogon (p<0.001, Figure 1A and B). No
differences were observed between the infected and uninfected children within the two ethnic
groups except for IgM that was higher in the infected Dogon compared to the uninfected
Dogon (p=0.05, Figure 1B), while this was not the case in the Fulani (p=0.17). However, the
IgM levels in the infected Dogon were significantly lower as compared to the infected Fulani
(p<0.01). Similarly, when analyzing the plasma levels of antimalarial IgG subclasses in the
Fulani as compared to the Dogon, regardless of infection, we observed that IgG1 (p<0.0001),
IgG2 (p<0.0001), IgG3 (p<0.0001) but not IgG4 (p=0.1957) were higher among the Fulani
than the Dogon. The same differences were observed when comparing only the uninfected
children from the two groups. When comparing the infected children of both ethnicities, the
Fulani had higher levels of IgG1 (p<0.030) and IgG3 (p<0.001) than the Dogon. However, no
intraethnical differences were observed for IgG1, IgG2 and IgG4 between the infected and
uninfected children. Nevertheless, the Fulani children undergoing Pf infection had
significantly lower levels of IgG3 as compared to their uninfected peers (p=0.045), while the
Dogon children undergoing Pf infection exhibited higher levels of IgG3 in plasma than the
uninfected Dogon children (p=0.034; Figure 2C).
9
Intraethnic and interethnic difference in cytokine plasma levels
The plasma of the children participating in the study were analysed for IL-1β, IL-6, IL-8, IL10, IL-12p70 and TNF-. When comparing the Fulani to the Dogon, regardless of infectious
status, we found higher levels of IL-6 (p = 0.0003; Figure 3A), IL-8 (p < 0.0001; Figure 3B),
IL-12p70 (p = 0.0102; Figure 3C) , IFN-(p = 0.0027; Figure 3D) and IFN-γ (p = 0.0044;
Figure 3E) in the Fulani population as compared to the Dogon. When comparing only the
uninfected children, plasma levels of IL-6 (p < 0.0001), IL-8 (p < 0.0002), IL-12 (p = 0.0010)
and IFN-(p < 0.0025) were significantly higher in the Fulani than in the Dogon children.
Differences were only found for IFN-γ (p = 0.0175), but not the other cytokines, when
comparing infected children from the two ethnicities, and higher IFN-γ levels were observed
in the Fulani as compared to the Dogon.
Finally, differences were also observed within the same ethnic group depending on the
malaria status. The Dogon children undergoing infection had significantly higher levels of IL6 (p < 0.0001) and IL-12 (p < 0.0001) in the plasma than the uninfected Dogon subjects. In
the Fulani, the only significant difference observed, was that the infected children had lower
levels of IL-8 (p = 0.0485) in plasma than the uninfected children belonging to the same
ethnic group. The plasma levels of IL-1β were below detection limit for all samples (not
listed). Similarly, IL-10 detectable plasma levels were found only in four samples out of 76
totals (not listed).
Interethnic and intraethnic differences in circulating dendritic cell frequency and
activation
The percentage or absolute numbers of circulating BDCA-1+ DCs did not show any
interethnic or intraethnic difference (Figure 4A and B). However, the infected Dogon
10
exhibited significantly lower levels of HLA-DR expression on BDCA-1+ cells as compared to
uninfected Dogon (p<0.001), and infected Fulani (p=0.002, Figure 5A). When analyzing
BDCA-2+ pDCs and BDCA-3+ mDCs distribution, no interethnic differences were observed
between the uninfected Fulani and Dogon. However, differences were observed within the
same ethnic groups upon malaria infection. In fact, the infected Dogon exhibited significantly
higher frequency of circulating pDCs+ and BDCA-3+ mDCs than uninfected children of the
same ethnic group (p=0.034 and p=0.016, respectively), while the infected Fulani had a
reduced frequency of pDCs and BDCA-3+ mDCs in the circulation as compared to uninfected
Fulani (p=0.060 and p=0.038, respectively, Figure 4A). No difference in absolute numbers of
circulating BDCA-3+ mDCs was seen in any of the examined groups (Figure 4B) while there
was a significant increase of blood pDCs in infected Dogon children as compared to the
uninfected Dogon (p=0.020, Figure 4B). Additionally, pDCs of infected Fulani exhibited
higher expression levels of HLA-DR as compared to the uninfected Fulani (p=0.009), while
the expression of HLA-DR on pDCs was lower in the infected Dogon as compared to their
uninfected peers (p=0.035; Figure 5A). Expression levels of the activation marker CD86 was
also slightly increased on pDCs in infected Fulani as compared to uninfected children, while
the expression of CD86 was significantly lower in the infected Dogon as compared to
uninfected Dogon (p<0.001; Figure 5B). When analyzing BDCA-3+ mDCs, the infected
Fulani had slightly higher levels of HLA-DR on this cell subset than the uninfected subjects
belonging to the same ethnic group (p=0.057), while the BDCA3+ mDCs of infected Dogon
had significantly lower levels of HLA-DR than the uninfected Dogon (p=0.027; Figure 5A).
Finally, when analyzing CD86 expression on pDCs (p < 0.0129) and BDCA-1+
(p < 0.0061) and BDCA-3+ (p = 0.0002) mDCs of the Fulani as compared to the Dogon
regardless of infection, the levels were significantly higher in Fulani than in Dogon for all DC
subsets. In addition, expression of HLA-DR was higher on the BDCA-3+ mDCs in the Fulani
11
ethnic group as compared to the Dogon (p=0.05) but not on the BDCA1 (p = 0.1578) or the
pDCs (p = 0.1224).
12
Discussion
In this study, we investigated some immunological aspects of Pf malaria in terms of
prevalence of different subsets of DCs as well as certain activation markers, plasma levels of
cytokines, and specific anti-malaria antibodies in children from two different ethnic groups
living in a malaria endemic area in Mali. These populations, the Fulani and the Dogon, have
differences in the susceptibility to Pf infection.
Previous studies have shown higher levels of antimalarial antibodies in
asymptomatic adults belonging to the Fulani as compared to other sympatric ethnic groups
(3,5,21,22). In line with these findings our data show that Fulani children also have higher
plasma levels of anti-malarial IgG subclasses except IgG4 as compared to the Dogon children.
Although this has been established previously in adults, we here show that the higher humoral
specific immune response of the Fulani is evident already early in life.
During Pf infections, the presence of anti-malarial IgG3 is associated with protection against
malaria in humans, possibly by an antibody-dependent mechanism inhibiting the growth of
parasites (23,24). Interestingly, we observed that the plasma levels of anti-malarial IgG3 were
higher in the uninfected Fulani than in the uninfected Dogon, suggesting a protective role of
these antibodies in the Fulani. However, the levels of antimalarial IgG3 were found to be
lower in the infected Fulani children than in the uninfected subjects of the same ethnic group.
The lower IgG3 levels seen in the infected Fulani could be due to the rapid formation of Pf
specific IgG3 antibody-antigen complexes, thereby making the antigen-biding part of the
antibody incapable of interacting with infected erythrocytes. Thus, antibodies in the form of
immune complexes cannot function in antibody-dependent cellular inhibition assays (ADCI)
an important suggested killing mechanism of anti-malaria IgG3 antibodies (25). However,
immune complexes can act through Fc-receptors and thus play an important role in enhancing
inflammatory responses as seen in the Fulani (2).
13
Cytokines play a central role in the pathogenesis of malaria. The balance
between pro- and anti- inflammatory cytokines may be important in the clinical outcome of
malaria (26-28).
When analyzing uninfected subjects, the levels of IFN-α, IL-6, IL-8 and IL12p70 in plasma were higher in the Fulani than in the Dogon children. This may indicate that
the Fulani children exhibit higher levels of basal activation of their innate immune response as
compared to the Dogon.
We found significantly lower level of IL-8 in the infected Fulani children as
compared to the uninfected subjects belonging to the same ethnic group. These low levels
were similar to those seen in both infected and uninfected Dogon. The clinical significance of
reduced IL-8 levels in infected Fulani as compared to uninfected peers is presently unknown.
The levels of IL-6 and IL-12 were higher in infected Dogon than uninfected
children of the same ethnic group. Earlier studies have shown that acute-phase levels of IL12p70 in plasma were inversely correlated with parasitemia (15). Thus, the high level of this
cytokine observed in infected Dogon could be the consequence of the ongoing acute Pf
infection which is exacerbated in Dogon children as compared to the Fulani.
Conversely, we found higher plasma levels of IFN-γ in the infected Fulani
children as compared to the infected Dogon. This is in line with recent findings indicating that
mononuclear leukocytes from the Fulani produce a markedly stronger IFN-γ response to in
vitro stimulation with Pf than leukocytes from Dogon (McCall, Unpublished manuscript).
Studies in both humans and mice indicate that the magnitude of early IFN- response is a
crucial determinant of the outcome of malaria infection (29-31). Our findings suggest that the
elevated IFN- responses in Fulani undergoing Pf infection may play an important role in the
differences that we observe in the immune response of the Fulani as compared to the Dogon.
14
Taken together, these results may indicate that one of the underlying causes for
the relative protection against malaria, of the Fulani, may be related to increased IFN-γ
secretion that seems to be absent in the Dogon.
DCs initiate adaptive immune responses by providing antigenic stimulation to T
and B cells. Thus, DCs provide a crucial link between the innate and adaptive immune
response and are potent in the uptake, processing and presentation of antigens to T cells (32).
We found lower frequency of pDCs and BDCA-3+mDCs in the circulation of infected Fulani
as compared to uninfected children belonging to the same ethnic group, while the same DC
subsets were relatively increased in numbers in infected Dogon as compared to uninfected
Dogon. Interestingly, analysis of HLA-DR expression revealed that circulating pDCs and
BDCA-3+ mDCs in the Fulani were more activated in the infected children. DC activation
usually results in up-regulation of lymphoid chemokine receptors on the cell surfaces which
will result in an increased cellular migration to lymph nodes. DC migration may therefore
account for the lower frequency of pDCs and BDCA-3+ mDCs that we observed in peripheral
blood of infected Fulani. However, absolute numbers of circulating pDCs and mDCs were not
affected by malaria infection in Fulani, and reduced frequency of these DC subsets in infected
subjects may also be explained by a relative increase in some other leukocyte population. It is
known that the Fulani have a higher spleen rate than other sympatric ethnic groups (33,34)
that could be coupled to an increase in both T- and B-cell subsets.
In the Dogon children the malaria infection caused a strongly reduced
expression of HLA-DR in circulating pDCs and BDCA-1+ and BDCA-3+ mDCs. These
results are in accordance with previous findings in Kenyan children where it has been shown
that children with acute malaria exhibited reduced expression levels of HLA-DR on mDCs
(9). HLA class II expression on DCs is fundamental for presenting antigens to T cells and
inducing their activation (32,35). It has been previously shown that PBMCs from children
15
with severe malaria induced less T-cell proliferation than PBMCs from healthy children
(36,36). Thus, the reduced expression of HLA-DR on the surface of all DC subsets in Dogon
children undergoing malaria infection may indicate impaired DC functionality, which in turn,
may contribute to impaired cell-mediated responses against the parasite.
Taken together, our results show that malaria infection induces activation of
pDCs in Fulani, while the same infection impairs HLA-DR expression in all examined DC
subsets in Dogon. Since no differences were observed when comparing only the uninfected
children from the two ethnicities. These different responses observed in the two ethnic groups
may be the reason that we see a higher expression of activation markers when comparing the
two groups without considering the infectious status.
Since pDCs play a crucial role in triggering humoral immunity (12,13) increased
pDC responses in Fulani may support antibody production which could explain the higher Ig
levels observed in Fulani as compared to other ethnic groups living in the same areas
(3,5,21,33) On the other hand, impaired DC activation upon malaria infection, as reflected
here in the Dogon population, may result in a deficient immune response as commonly seen
in malaria (37). To further evaluate the innate response in the Fulani we are presently
analysing the cytokine secretion of the PBMCs after stimulation with specific TLR ligands.
In conclusion, the findings presented here suggest that there is a difference in the prevalence
and activation status of blood DCs, cytokine secretion and Ig levels between the Fulani and
the Dogon in response to malaria infection already in early life. Such differences are likely to
be important for the relative stronger protection against malaria seen in the Fulani as
compared to the Dogon.
16
Acknowledgements
This work was supported by grants from the Swedish Agency for Research and Development
with Developing Countries (Sida/SAREC), as well as a grant within the BioMalPar European
Network of Excellence (LSHP-CT-2004-503578) and from the European & Developing
Countries Clinical Trials Partnership (EDCTP).
17
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Figure legends
Figure 1. Specific antibody responses to Pf are higher in Fulani children than in the
Dogon children
Blood plasma samples from 77 children were analysed for antimalarial IgG (A) and IgM (B).
The samples were subdivided according to ethnicity and slide positivity to malaria; i.e.
uninfected Dogon (n=20), infected Dogon (n=20), uninfected Fulani (n=23) and infected
Fulani (n=14). The y-axis depicts the concentration of the respective antibody/ml of plasma .
The boxplots illustrate the medians and the 25th and 75th quartile and the whiskers represent
the 10% and 90% percentiles. Data were analyzed using Mann-Whitney rank sum test. *;
p≤0.05. **;p<0.01. ***; p<0.001.
Figure 2. Malaria specific IgG subclass responses to Pf in Fulani and Dogon children.
Blood plasma samples from 77 children were analysed for antimalarial IgG1 (A), IgG2 (B),
IgG3 (C) and IgG4 (D). The samples were subdivided according to ethnicity and slide
positivity to malaria; i.e. uninfected Dogon (n=20), infected Dogon (n=20), uninfected Fulani
(n=23) and infected Fulani (n=14). The y-axis represent µg of the respective antibody/ml of
plasma. The boxplots illustrate the medians and the 25th and 75th quartile and the whiskers
represent the 10% and 90% percentiles.. Data were analysed using Mann-Whitney rank sum
test. *; p≤0.05. **;p<0.01. ***; p<0.001.
21
Figure 3. Intraethnic and interethnic difference in cytokine plasma levels.
Blood plasma samples from 77 children were analyzed for cytokines. The levels of IL-6 (A),
IL-8 (B) and IL-12p70 (C) were measured using cytometric bead array. The levels of IFN- 
(D) and IFN- (E) were analyzed using commercial ELISA kits. The samples were subdivided
according to ethnicity and slide positivity to malaria; i.e. uninfected Dogon (n=20), infected
Dogon (n=20), uninfected Fulani (n=23) and infected Fulani (n=14).The y-axis depicts
picograms (pg) of the respective cytokine/ml of plasma. The boxplots illustrate the medians
and the 25th and 75th quartile and the whiskers represent the 10% and 90% percentiles. Data
were analyzed using Mann-Whitney rank sum test. *; p≤0.05.***; p<0.001.
Figure 4. Interethnic and intraethnic differences in the frequency and absolute numbers
of circulating DC.
PBMCs from 63 children were isolated using Ficoll and subsequently fixed and frozen. The
cells were harvested and stained using monoclonal antibodies against known subtypes of
blood DCs and analyzed using FACS Calibur. The samples were subdivided according to
ethnicity and slide positivity to malaria, i.e. uninfected Dogon (n=20), infected Dogon (n=20),
uninfected Fulani (n=13) and infected Fulani (n=10). The y-axis in (A) depicts the percentage
of circulating DC subsets while the y-axis in (B) depicts the absolute number of circulating
DCs/ml of blood. The boxplots illustrate the medians and the 25th and 75th quartile and the
whiskers represent the 10% and 90% percentiles. Data were analyzed by Mann-Whitney rank
sum test. *; p≤0.05.
22
Figure 5. Interethnic and intraethnic differences in blood DCs activation markers
PBMCs from 63 children were isolated using Ficoll and subsequently fixed and frozen. The
cells were harvested and stained using monoclonal antibodies against known subtypes of
blood DCs and activation markers, such as HLA-DR (A) and CD86 (B). Cells were
subsequently analyzed using FACS Calibur. The samples were subdivided according to
ethnicity and slide positivity to malaria, i.e. uninfected Dogon (n=20), infected Dogon (n=20),
uninfected Fulani (n=13) and infected Fulani (n=10). The y-axis depicts mean fluorescence
intensity (MFI) of activation markers. The boxplots illustrate the medians and the 25th and
75th quartile and the whiskers represent the 10% and 90% percentiles. Data were analyzed by
Mann-Whitney rank sum test. *; p≤0.05. **;p<0.01. ***; p<0.001.
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