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

ii

“Seamos realistas y hagamos lo impossible”

Ernesto Guevara de la Serna

iii

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.

iv

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.

v

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

vi

Abbreviations

APC Antigen-presenting cells

BCR B-cell receptor

BDCA Blood dendritic cell antigen

CC Chemotactic cytokines

CCR Chemokine receptor

CD Cluster of differentiation

DC Dendritic cell

GPI Glycosylphosphatidylinositol

anchors

GM-CSF Granulocyte macrophage-

colony stimulating factor

Hz Hemozoin

HLA Human-leukocyte antigen

Ig Immunoglobulins

IFN Interferon

IRAK IL-1 receptor-associated

kinase

IRF IFN regulatory factor

IL Interleukin

LPS Lipopolysacharide

MCP Monocytes-chemoattractant

protein

MIP Macrophage-inflammatory

protein

MoDC Monocyte-derived dendritic

cells

MØ Macrophages

MyD88 Myeloid differentiation

primary response gene 88

NK cells Natural-killer cells

NF-kb Nuclear factor kappa beta

PAMPs Pathogen-associated

molecular patterns

PBMC Peripheral blood

mononuclear cells

PRRs Pathogen-recognition

receptors

RANTES Regulated on Activation

Normal T Expressed and

Secreted Protein

Tc Cytotoxic-T cell

Th Helper-T cell

TIR Toll/IL-1 receptor domain

TRAF TNF receptor associated

factor

TRIF TIR-domain-containing

adapter-inducing interferon-β

Treg Regulatory-T cell

TCR T-cell receptor

TLR Toll-like receptor

TNF Tumour Necrosis factor

7

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.

8

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).

9

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.

10

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

11

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 adapter-

inducing 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).

12

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 IL-

12p70 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.

13

Conversely, activation of TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 would lead to a Th1-

type 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

14

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.

15

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 CD1c CD11c CD123 CD2 CD16 CD32 CD64

BDCA1 + + - + - + +

BDCA2 - - + -/+ - - -

BDCA3 - -/+ - - - - -

CD16+DC - -/+ + + + Nd 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).

16

Receptor Ligand and origin location mDC pDC MoDC

TLR1 Triacyl LpP (bacteria) surface + + +

TLR2 Zymozan, PGN (fungi, G+ bacteria) surface + - +

TLR3 dsRNA (viruses) surface + - +

TLR4 LPS, proteins (G- bacteria, viruses) surface - - +

TLR5 Flagellin (bacteria) surface + - +/-

TLR6 Diacyl LpP, Zymosan (bacteria, fungi) surface +/- + +

TLR7 ssRNA (virus) internal +/- + -

TLR8 ssRNA (virus) internal + - +

TLR9 CpG-rich DNA, Hemozoin (virus, protozoa) internal - + -

TLR10 Nd surface + + 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 B-

cells (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).

17

Several protocols have been established to obtain purified mDCs from precursors in vitro (49-

51). 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 vitro-

generated 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

18

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.

19

Adaptive Immunity

The adaptive immune system has to be selective, in that it has to discriminate self from non-

self, 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

20

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 antigen-

binding 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 T-

helper (Th), T-cytotoxic (Tc) and T-regulatory cells (Treg).

21

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 T-

bet 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

22

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

23

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.

24

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.

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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 Ligand Location iDC mDC

CCR1 CCL3/MIP-1α, CCL5/RANTES Inflammation + -

CCR2 MCP1-4 Inflammation + -

CCR5 MIP-1α, CCL4/MIP-1β, CCL5/RANTES Inflammation + -

CCR7 CCL19/ELC/MIP-3β, CCL21/SLC Lymphatic tissue - +

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

26

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.

27

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 co-

stimulatory 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

28

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).

29

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

30

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

31

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

32

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)

33

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).

34

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

35

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 up-

regulation 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

36

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

37

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.

38

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

39

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-

40

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

41

induced a short lasting up regulation of CD83 on the surface of MoDCs as compared to TNF-

α/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 IL-

1β.

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

42

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 Fc-

receptors and thereby induce inflammatory cytokines (186).

43

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

44

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.

45

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.

46

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.

47

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.

48

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!

49

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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: marita.troye-blomberg@wgi.su.se

2

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.

3

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

4

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 high-

doses 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.

5

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 Ficoll-

Paque (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

6

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

7

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 co-

cultured 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

8

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).

9

Statistical analysis

Data was analyzed using the Wilcoxon’s rank-sum test. Comparisons were considered

statistically significant at p < 0.05.

10

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 HLA-

DR 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

11

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.

12

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, TNF-

α/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

13

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/SDF1-

α (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

14

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 IL-

1β (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.

15

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 sHz-

exposed 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

16

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 T-

cell 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

17

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 T-

cell 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)

18

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21

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.

22

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

23

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.

24

Figure 1

25

Figure 2

26

Figure 3

27

Figure 4

28

Figure 5

29

Figure 6

30

Figure 7a

31

Figure 7b

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 Dara

2, Boubacar Traore

2,

Amagana Dolo2, Ogobara Doumbo

2, Stefania Varani

1,3 and Marita Troye-Blomberg

1†

1Department of Immunology Wenner-Gren Institute Stockholm University, Sweden.

2Malaria Research & Training Centre, Faculty of Medicine, Pharmacology & Dentistry,

University of Bamako, Bamako, Mali.

3Department 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: marita.troye-blomberg@wgi.su.se

2

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.

3

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

4

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.

5

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.

6

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. Biotin-

conjugated 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) (Sigma-

Aldrich, 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

7

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), IL-

12(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%

8

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.

9

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).

10

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, IL-

10, 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 IL-

6 (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

11

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

12

ethnic group as compared to the Dogon (p=0.05) but not on the BDCA1 (p = 0.1578) or the

pDCs (p = 0.1224).

13

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).

14

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 IL-

12p70 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 IL-

12p70 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.

15

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

16

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.

17

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).

18

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21

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.

22

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.

23

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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