Characterization of antigen-presenting cell function in...
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Doctoral thesis from the Department of Immunology, Wenner-Gren Institute, Stockholm University, Sweden Characterization of antigen-presenting cell function in vitro and ex vivo Pablo Giusti Stockholm 2011
Transcript of Characterization of antigen-presenting cell function in...
Doctoral thesis from the Department of Immunology, Wenner-Gren Institute,
Stockholm University, Sweden
Characterization of antigen-presenting cell
function in vitro and ex vivo
Pablo Giusti
Stockholm 2011
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Imagination is more important than knowledge. For knowledge is limited to all we now know and understand, while imagination
embraces the entire world, and all there ever will be to know and understand.
Albert Einstein
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ABSTRACT Long-term protective immune responses depend on proper initiation of adaptive immunity by
professional antigen-presenting cells (APCs). Autoimmune disorders and certain infections
can cause disease through manipulating the immune system and modulation of APCs may be
important for the outcome of these conditions. This work aimed to investigate the behaviour
of different APC subsets during conditions known to cause improper immune responses.
In Paper I, the effects of an anti-inflammatory compound called Rabeximod, intended for
treatment of rheumatoid arthritis (RA) were investigated. The results showed that generation
of monocyte-derived DCs (MoDCs) and inflammatory macrophages (MΦs) was impaired
upon exposure to Rabeximod while differentiation of anti-inflammatory (Ai)-MΦs was
unaffected. When MoDCs and inflammatory MΦs were exposed to Rabeximod they exhibited
a reduced capacity to stimulate allogeneic T cells while Ai- MΦs remained unaffected. These
findings suggest that Rabeximod acts by inhibiting the functionality of inflammatory subsets
of APCs.
In Paper II, the effects of different Plasmodium falciparum-derived stimuli such as hemozoin
(Hz) and infected red blood cells (iRBCs) on MoDCs were investigated. Both stimuli
triggered increased expression of certain co-stimulatory molecules and chemokine receptors,
indicating increased activation and migration of MoDCs. These MoDCs secreted TNF-α, IL-6
and IL-10, but no IL-12. MoDCs exposed to iRBCs induced allogeneic T-cell proliferation
while those exposed to Hz did not. These results indicate that DCs may be improperly
activated during malaria and may therefore be unable to efficiently activate T cells.
In Paper III, innate aspects of immunity in children from the Fulani, that displays better
protection against malaria, were investigated and compared to the sympatric ethnic group the
Dogon. We observed that distinct subsets of APCs from Fulani children expressed increased
levels of activation markers and their toll-like receptor (TLR) responses were unaffected
while undergoing P.falciparum infection. Conversely, the Dogon exhibited decreased
activation of APCs and severely suppressed TLR responses when undergoing infection. These
differences in innate responses, including APC behaviour may indicate an important role for
TLR and APC activation in malarial immunity.
In conclusion, detailed knowledge of precise mechanisms of APC activation can be very
helpful in understanding the proper course of specific effector immune responses. Moreover,
this knowledge would allow for manipulation of APCs that could be used for treatment of
inflammatory disorders and in the generation of efficient vaccines against infectious diseases.
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LIST OF PAPERS
The thesis is based on the following papers which will be referred to by their roman numerals:
I. Giusti P, Frascaroli G, Tammik C, Gredmark-Russ S, Söderberg-Nauclér C,
Varani S. “The novel anti-rheumatic compound Rabeximod impairs
differentiation and function of human pro-inflammatory dendritic cells and
macrophages.” Immunobiology. 2011 Jan-Feb;216(1-2):243-50.
II. Giusti P, Urban B, Frascaroli G, Tinti A, Troye-Blomberg M, Varani S.
“Plasmodium falciparum-infected erythrocytes and β-hematin induce partial
maturation of human dendritic cells and increase their migratory ability in
response to lymphoid chemokines.” Infection & Immunity 2011 Jul;79(7):2727-36
III. Arama C, * Giusti P, * Boström S, Dara V, Traore B, Dolo A, Doumbo O, Varani
S, Troye-Blomberg M, "Interethnic Differences in Antigen-Presenting Cell
Activation and TLR Responses in Malian Children during Plasmodium
falciparum Malaria.” PLoS One. 2011 Mar 31;6(3):e18319 * These authors
contributed equally to this work.
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ABBREVIATIONS APC Antigen-presenting cell
Ai-MΦ Anti-inflammatory MΦ
Allo-MΦ Allostimulated MΦ
BCR B-cell receptor
BDCA Blood-dendritic cell antigen
CARD Caspase recruitment domain
CC Chemotactic cytokines
CCR Chemokine receptor
CD Cluster of differentiation
CLIP Class II-associated invariant chain
peptides
CLR C-type lectin receptor
CR Complement receptor
DAMP Danger associated molecular
pattern
DC Dendritic cell
Flt3L FMS-like tyrosine kinase 3 ligand
GPI Glycosylphosphatidylinositol
anchor
GM-CSF Granulocyte macrophage-colony
stimulating factor
HLA Human-leukocyte antigen
HMGB1 High mobility group box 1
HSP Heat shock protein
Hz Hemozoin
Ig Immunoglobulin
IFN Interferon
iRBC Infected red blood cell
IRAK IL-1 receptor-associated kinase
IRF IFN regulatory factor
IL Interleukin
ISG IFN-stimulated genes
ITAM Immunoreceptor tyrosine-based
activation motif
LPS Lipopolysaccharide
M-CSF Monocyte-colony stimulating
factor
MΦ Macrophage
MCP Monocyte-chemoattractant protein
MDA Melanoma-differentiation
associated gene
mDC Myeloid DC
MIP Macrophage-inflammatory protein
MLR Mixed leukocyte reaction
MoDC Monocyte-derived dendritic cell
MMP Matrix metalloprotease
MyD88 Myeloid differentiation primary
response gene 88
NK cell Natural-killer cell
NF-kb Nuclear factor kappa beta
NLR Nod-like receptor
PAMP Pathogen-associated molecular
pattern
PBMC Peripheral blood mononuclear cell
pDC Plasmacytoid DC
PRR Pathogen-recognition receptor
RA Reumathoid arthritis
RANK Receptor activator of NFκB
RANTES Regulated on Activation Normal T
Expressed and Secreted Protein
RIG Retinoic acid-inducible gene
Tc T-cytotoxic cell
Th T-helper cell
TIR Toll/IL-1 receptor domain
TIRAP TIR- adaptor protein
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
TABLE OF CONTENTS ABSTRACT ........................................................................................................................................................ iii LIST OF PAPERS .............................................................................................................................................. iv ABBREVIATIONS ............................................................................................................................................. v TABLE OF CONTENTS ................................................................................................................................... vi INTRODUCTION ............................................................................................................................................... 7
THE IMMUNE SYSTEM ......................................................................................................... 7 T cells ....................................................................................................................................................... 10 Antigen-presenting cells of innate immunity........................................................................... 12 Recognition strategies of antigen-presenting cells ................................................................ 19
ANTIGEN PRESENTATION ................................................................................................. 24 Signal 1 .................................................................................................................................................... 24 Signal 2 .................................................................................................................................................... 29 Signal 3 .................................................................................................................................................... 30
INFLAMMATION ................................................................................................................. 35 Leukocyte movement ........................................................................................................................ 36
RELATED BACKGROUND .......................................................................................................................... 38 APCS IN INFLAMMATION AND THE LINK TO ADAPTIVE IMMUNITY ............................. 38
Monocytes .............................................................................................................................................. 38 Macrophages ......................................................................................................................................... 39 Dendritic cells ....................................................................................................................................... 40 Endogenous ligands ........................................................................................................................... 42 The manipulation of APCs by cytokines ..................................................................................... 43
MALARIA ............................................................................................................................. 44 REUMATHOID ARTHRITIS ................................................................................................. 49
THE STUDIES ................................................................................................................................................. 53 GENERAL AIM ..................................................................................................................... 53 SPECIFIC AIMS .................................................................................................................... 53
Paper I ..................................................................................................................................................... 53 Paper II .................................................................................................................................................... 53 Paper III .................................................................................................................................................. 53
METHODS ........................................................................................................................................................ 55 Paper I ..................................................................................................................................................... 55 Paper II .................................................................................................................................................... 55 Paper III .................................................................................................................................................. 56
RESULTS AND DISCUSSION ...................................................................................................................... 57 Paper I ..................................................................................................................................................... 57 Paper II .................................................................................................................................................... 59 Paper III .................................................................................................................................................. 63
GENERAL CONCLUSIONS .......................................................................................................................... 68 FUTURE WORK .............................................................................................................................................. 71 ACKNOWLEDGEMENTS ............................................................................................................................. 72 REFERENCES .................................................................................................................................................. 75
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INTRODUCTION
THE IMMUNE SYSTEM
Mammals have evolved in co-existence with a wide variety of viruses, parasites and bacteria.
Many of these microbes have adapted to niches in or outside the body and do not normally
cause disease, they are called commensals. However, these and other microbes can, under
certain conditions, sometimes invade tissues where they can cause disease. Therefore, the
body has developed defence mechanisms to keep tissues free from invading microbes and
maintain tissue homeostasis. Those defence mechanisms comprise antimicrobial peptides,
signalling molecules and a complex network of cells that have evolved to find and eliminate
pathogens and even remember the invading microbes. The immune system is commonly
divided into two, although intervening, different branches that we call the innate and the
adaptive immune system.
The innate immune system has evolved to detect danger signals and indiscriminately attack
pathogens. Innate immunity does not, from a short time perspective, seem to adapt to changes
in the surrounding environment and is very similar in all humans. In order to impede
microbial colonization of the human body, obstacles are created at many different levels.
Physical barriers, such as the skin and mucosal surfaces of the body are the first ones.
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 considered as the body’s exterior surfaces. These
surfaces are colonized by enormous amounts of microorganisms that do not normally cause
disease. The acidic environment in the stomach is an additional physical barrier. Inside the
body, in circulation, there are antibacterial peptides and complement proteins that recognise
pathogens and initiate a cascade of proteolytic activities ultimately leading to lysis of
microbes.
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Finally, there are the cells of the innate immune system. They are equipped with different
kinds of receptors to recognize disruptions of the normal homeostasis and infectious stimuli.
These receptors have evolved to recognize danger signals and recurrent patterns that are
essential to, and therefore conserved among, pathogens. These pathogen associated molecular
patterns (PAMPs) and danger associated molecular patterns (DAMPs) are recognized by
pattern recognition receptors (PRRs). The best studied PRRs are the toll-like receptors (TLRs)
but there are many different PRRs and some debate as to what their function really is,
therefore, they will be further discussed in a separate chapter.
However, since living conditions in different social, cultural and geographical environments
can be variable there is a need for adaptation. In order to be able to adapt to these variable
conditions, thus responding as quickly and efficiently as possible, a distinct branch of the
immune system has evolved to specifically recognize and remember pathogens that it has
been exposed to. This is the adaptive branch of the immune system and is, in contrast to the
innate, unique in every human being.
In summary, the innate immune system is fast acting, unspecific and lacks memory while the
adaptive immunity is slower to act but is specific and has a memory component. The
interaction of these two branches results in an immune system that can act swiftly against all
pathogens and learn to respond more efficiently against pathogens that are common to a given
individual´s specific environment. These branches are dependent on each other for optimal
performance. The innate components are necessary for the adaptive branch to function and the
innate immune responses are much more efficient when aided by adaptive components.
All cells of the immune system originate in the bone marrow and, at certain stages of
development, in the liver. They are found throughout the body, particularly in the lymphatic
tissues and in circulation. These cells are collectively called leukocytes and they all stem from
hematopoietic stem cells. The pluripotent stem cells differentiate into common lymphoid
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progenitors or common myeloid progenitors. These then further differentiate into their
respective immune cells. B- , T- and natural killer (NK)- cells and possibly some subsets of
dendritic cells (DCs) stem from a lymphoid progenitor, while granulocytes, monocytes and
most 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).
Figure 1. Cells of the immune system
Monocytes, macrophages (MΦs), granulocytes, DCs and NK cells belong to the innate branch
of the immune system. With the exception of NK cells these cells are able to constantly
survey their surroundings through endocytosis.
Leukocytes can communicate through the secretion of cytokines that are sometimes called the
hormones of the immune system. Since they are a means of communication between
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leukocytes they are called interleukins (IL). An important subgroup of cytokines that governs
cell motility are the chemotactic cytokines (CCs). These substances are shared among all cells
of the immune system and will be discussed in a separate chapter.
T cells
T lymphocytes are defined by expression of a T-cell receptor (TCR), through which they
recognize antigens. These cells are not able to recognize antigens directly, but the pathogen
needs to be degraded into peptide fragments that in association with human leukocyte antigen
(HLA) molecules on antigen-presenting cells (APCs) are presented to T cells. The details of
how this activation is orchestrated, the cells responsible and its consequences will be
discussed in more detail below.
The TCR lacks an intracellular signalling domain instead it is expressed together with a
molecule called cluster of differentiation (CD) 3. The CD3 molecule contains an
immunoreceptor tyrosine-based activation motif (ITAM) that can elicit intracellular
signalling. Intracellular signalling via CD3 is initiated upon binding of the TCR to an antigen-
HLA complex. The genes coding for the TCR are randomly rearranged during T-cell
development thus providing a unique specificity for each TCR. Because this rearrangement is
at random, T cells must be prevented from recognizing and attacking ones healthy tissues. A
selection process occurs in the thymus where less than 5% of the cells survive [1]. This
process is carried out in order to avoid the release of potentially harmful T cells. The final
outcome of this process is the release of T cells expressing a TCR repertoire with unique
specificities and harmless to healthy self. T cells leaving the thymus can be subdivided
according to the expression of their co-receptors, CD4+ cells are called T- helper (Th) cells,
CD8+ cells are called T-cytotoxic (Tc) cells.
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T helper cells
Th cells are defined by the expression of the co-receptor CD4. The activation of naïve CD4+ T
cells can lead to skewing of the T cell in different directions thus creating a heterogeneous
population of Th cells. Depending on the cytokines secreted by the activating APC the naïve
T cell will commit to a given lineage (Figure 2).
Figure 2. Th-cell differentiation and their consequences for immunity
Reprinted from Medzhitov, Nature 449, 819-826 (18 October 2007), doi:10.1038/nature06246 with permission
from Nature Publishing Group.
This lineage is established and sustained by promoting the expression of a specific
transcription factor which then suppresses transcription factors that direct the other lineages.
This was first discovered as the first two lineages defined were shown to be reciprocally
inhibitory and defined as Th1 and Th2 cells [2,3]. Later, additional subsets have been defined
that are now known as regulatory T cells (Tregs) and Th17 [4]. Tregs can either be induced in
the periphery and are then called Tr1 or Th3 cells or they can be released from the thymus in
which case they are referred to as natural (n) Tregs [5]. The transcription factors involved in
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establishing the distinct Th lineages are T-bet, GATA-3, RORγt and FoxP3 for Th1, Th2,
Th17 and Tregs respectively [6].
The functions of the different subsets of effector T cells are also different. Th1 responses have
traditionally been linked to the elimination of intracellular bacteria or viruses while Th2
confers protection against parasites and Th17 clears extracellular bacteria and fungi. Finally,
Tregs are necessary to avoid excessive tissue damage upon inflammation. This model
explains how adaptive immunity has evolved to produce diverse responses to different kinds
of pathogens. These directed responses can in turn enhance innate immunity.
T cytotoxic cells
Tc cells are defined by the fact that they express CD8 as co-receptor. Upon activation, Tc
cells kill other cells by induction of apoptosis or release of granules containing perforin or
granulosin that can lyse the infected cell [7]. Much like Th cells, some of the Tc effector cells
become memory cells, in order to sustain long term immunity, while the remaining cells
undergo apoptosis as soon as the primary infection is cleared [8].
Antigen-presenting cells of innate immunity
Professional APCs act as sentinels of the immune system. These cells play a pivotal role in
the induction of adaptive immune responses to antigenic stimulation by presenting antigens to
T cells in the context of HLA molecules expressed on their cell surface. Innate APCs
comprise two types of DCs, i.e. plasmacytoid DCs (pDCs) and myeloid DCs (mDCs),
monocytes and tissue-specific macrophages (MΦs). Importantly, B cells are also capable of
presenting antigens but are not considered as part of innate immunity and will not be dealt
with in this thesis.
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Monocytes
Monocytes develop from a myeloid progenitor in the bone marrow and are then released into
the bloodstream. Monocytes constitute about 10% of the blood leucocytes in humans and are
characterized as mononuclear leucocytes expressing the lipopolysaccharide (LPS) co-receptor
CD14. These cells play an important role in tissue homeostasis by patrolling vessels and
tissues [9] and constitute a reservoir for tissue-MΦs and DCs [10].
Monocyte subsets
About 20 years ago, a subpopulation of monocytes expressing Fcγ receptor (FcγR) III/CD16
was described [11]. The classical CD14++
CD16- cell expresses high levels of CD14 and no
CD16, while the non-classical CD14+CD16
++ cell type expresses CD16 and lower levels of
CD14. The different patterns of cytokine secretion of the two subsets have led to the terms
“inflammatory” and “classical” monocytes. However, since the function of the more recently
discovered monocyte subset is still not clearly defined the term “inflammatory” can be
unfortunate and the term “non-classical” is preferred. More recently another monocyte
population, that is present at a low frequency but exhibit unique features and expand in
response to cytokine treatment and inflammation, has been found. The immunophenotype of
this subset is somewhere in between classical and non-classical (CD14++
CD16+) which has
led to the definition of “intermediate” monocytes [12]. The role of monocytes when they enter
tissues or become activated in response to inflammatory stimuli will be further discussed
when discussing the role of APCs in inflammation.
Macrophages
Over 100 years ago, in 1908, Ilya Ilyich Mechnikov and Paul Ehrlich were awarded the Nobel
prise in Medicine for their work on phagocytosis. Much of their work was performed in MΦs
that Mechnikov described as mononuclear phagocytes playing an important role in pathogen
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elimination and housekeeping [13]. The term macrophage is derived from Greek and means
“big eater” in contrast to the granulocytes that were called “small eaters”. Phagocytosis by
MΦs can be induced by different types of receptors such as FcRs and will be further discussed
in the chapter of antigen presentation. MΦs have since then been described in various organs
such as lung, liver and nervous tissues as tissue-specific subsets. For the identification of MΦs
intracellular macrosialin, also called CD68, has been widely used [14] although it has been
proposed to be a marker for phagocytosis rather than a specific marker of MΦ subsets [15].
These cells also express the monocyte marker CD14 and, as professional APCs, they typically
express HLA class II, henceforth referred to as HLA-II, molecules on their surface.
Macrophage subsets
More than 25 years ago, interferon (IFN) -γ was identified as an outstanding factor that could
induce production of superoxides in MΦs and activate them to become potent immune cells
[16,17]. It was later described that IL-4 and IL-13 activation of MΦs inhibited the respiratory
burst and secretion of proinflammatory cytokines [18,19]. It was then proposed that these
factors could lead to a different kind of activation, which led to the concept of alternatively
activated MΦs [20].
In analogy with the Th1/Th2 activation of T cells, an M1/M2 characterisation of MΦs has
been suggested. Since the classical Th1 cytokine IFN-γ was the first activating factor
described, stimulation of MΦs induced by this mediator has been defined as “classical”
activation or more recently M1. In contrast, “alternative” activation of monocytes induced by
IL-4, IL-10 or IL-13 leads to M2 polarized MΦs, thus promoting a Th2 type of response
[21,22]. A subcategorizing of M2 polarized MΦs, where the M2 MΦs now include all MΦs
that are not M1, has been proposed [23]. This leads to a concept where the M1 is well defined
but the M2 would then comprise a heterogenous group of cells that have different roles in
infection and wound healing. Therefore this concept has been accused of oversimplification
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and a more complex picture of MΦ activation has been suggested that separates MΦ subtypes
according to their functions [24]. This classification defines classically activated M1 as MΦs
devoted to host defence, while the M2 are separated into wound healing MΦs and regulatory
MΦs [25]. This definition was suggested to better account for the role of MΦs in homeostasis.
For simplicity, we will refer to the different MΦ subsets as M1 for classically activated MΦs
and M2 for all other subsets.
Macrophage differentiation in vitro
Two important factors for macrophage differentiation are macrophage colony-stimulating
factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF). M-CSF is
constitutively produced by many different cell types and is readily detectable under steady
state conditions while GM-CSF is produced upon stimulation with inflammatory mediators.
Both factors induce MΦ differentiation and they have been suggested to represent the
extremes of the full spectrum of MΦ activation [26].
MΦs can be differentiated in vitro from monocytes using M-CSF or GM-CSF. Both GM-CSF
and M-CSF are individually important in the development of MΦs as lack of either lead to
altered MΦ homeostasis or pathology [27]. Cells obtained by these stimulations are
phenotypically similar. Nevertheless M-CSF-derived MΦs express higher levels of CD16 and
CD163 than GM-CSF derived MΦs. Both cell types are capable of antibody-dependent
cytotoxicity although the M2 MΦs seem slightly more efficient in this function, possibly as a
consequence of the increased CD16 expression [28]. Upon exposure to Mycobacterium, in
contrast to monocyte-derived DCs (Mo)DCs, MΦs do not secrete IL-12 but M1 secrete IL-23
which leads to more efficient T-cell priming, while M2 secrete IL-10 and are poor activators
of T cells [29].
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Dendritic Cells
DCs were first described in 1973 by Ralph Steinman and Zanvil Cohn [30,31]. Four years
later the authors concluded that DCs are at least 100 times more effective in stimulating
allogeneic T cells than any other major APC subset [32]. We now know that DCs are
specialized professional APCs and that their life cycle is adapted to find, process and present
antigens to naive T cells in lymphoid tissues. DCs are a very heterogeneous group of cells
both when it comes to their distribution as specialized cells in different tissues and to their
origin [33].
Immature DCs (iDCs) express high levels of PRRs to detect PAMPs and a repertoire of
chemokine receptors (CCRs) that are specialized to detect inflammatory mediators while
these cells patrol peripheral tissues. When DCs encounter inflammatory signals and/or
microbial components, they become activated and the expression of their surface molecules is
modified [34]. One of the most important maturation markers on DCs is the surface antigen
CD83, which is crucial for the induction of activated CD4+ T cells [35].
Down-regulation of PRRs and inflammatory CCRs, such as CCR1, CCR2 and CCR5 is also
part of the maturation process of DCs. Simultaneously, DCs up-regulate HLA molecules, co-
stimulatory molecules and CCRs specific for ligands expressed in lymphoid tissues such as
CCR7 and CXCR4 [36]. This change in CCR expression enables migration of DCs to T-cell
rich areas in secondary lymphoid organs where lymphoid chemokines are produced and
where DCs encounter massive amounts of naive T cells [37]. When DCs and T cells find their
perfect match, that is, an antigen-HLA-II complex on the DC and the TCR of a CD4+ T cell,
co-stimulation and cytokine secretion are increased. This T-cell activation triggers its
proliferation and thousands of T-cell clones will be produced in a matter of days.
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DC subsets
In humans, two complementary subsets of DCs called mDCs and pDCs have been described
in peripheral blood [38]. More recently, subgroups of mDCs according to specific surface
markers have been suggested (Table 1). Careful characterisation of these subsets has resulted
in surface markers specific for different DC subsets in circulation [39].
The major circulating mDC subpopulation is defined as lineage negative, CD1c+ CD11c
+
CD123- cells and characterised by blood dendritic cell antigen (BDCA)-1/CD1c expression.
Conversely, pDCs are defined as lineage negative, CD123+ and CD11c
- cells and express
BDCA-2 (CD303). BDCA-3 (CD141) is expressed on a subpopulation of mDCs that is also
defined as lineage negative, CD1c+, CD11c
+, CD123
-. These cells lack the expression of
BDCA-1, CD2, CD32 and CD64.
Blood DCs lack dendrites and markers of mature DCs such as CD83 and thus do not have
typical characteristics of DCs in tissue. These immature DCs appear to be in transit when
circulating in the blood, and they mature into functional DCs only after entering the tissue
[12].
Subtype CD1c CD11c CD123 CD303 CD2 CD16 CD32 CD64
BDCA-1 + + - - + - + +
BDCA-2 - - + + -/+ - - -
BDCA-3 - -/+ - - - - - -
CD16+DC - -/+ + - + + Nd Nd
Table 1. Expression of surface molecules on distinct blood DC subsets.
Abbreviation: Nd- no data. References: [39-41].
Dendritic cell differentiation in vitro
In order to obtain in vitro cultures of DCs, several protocols have been established to obtain
purified mDCs [42-44]. DCs derived from CD34+ cord blood cells differentiate along two
independent pathways in response to GM-CSF and TNF-α. After 5-6 days, two subsets
(CD1a+CD14
- and CD1a
-CD14
+ cells) can be observed; after 12 days in culture, all cells are
CD1a+CD14
-. The CD1a
+ precursors differentiate into Langerhans cells that contain Birbeck
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granules, whereas the CD14+ precursors lead to CD1a
+ mDCs that do not produce Birbeck
granules and that possess the characteristics of interstitial DCs [45]. MoDCs with the typical
phenotype of CD1a+CD14
- cells can be obtained in vitro by stimulation of purified monocytes
with IL-4 and GM-CSF for 5-6 days [44]. In vitro-generated MoDCs are considered to have
inflammatory properties [46]. 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. CD40L). A protocol for in vitro generation of the BDCA-3+ DC subset has been recently
reported that will be helpful for comparative studies on different DC populations and enable
manipulation of these cells to be used in different therapeutic settings [47].
Origin of DCs
The origin of DCs remains an unresolved issue. There are many subpopulations of DCs in
peripheral blood and different models for the development of DCs have been suggested [48].
It is believed that monocytes represent a pool of circulatory precursor cells that are capable of
differentiating into mDCs that resemble the features of interstitial DCs [34]. In support of this
hypothesis, it has been shown that monocytes can differentiate into DCs and migrate to the
lymph nodes upon ingestion of latex beads [10]. Similarly, Langerhans DCs in the skin can
arise from monocytes that infiltrate tissues [49], but under steady-state conditions they can
renew themselves to maintain the resident population [50]. Recently, it has been shown that
LPS can induce monocyte differentiation to DCs and that these differentiated cells can
migrate to lymph nodes and activate both CD4+ and CD8
+ T cells [51].
The origin of pDCs is particularly intriguing since they bare gene rearrangement previously
thought to be unique for lymphocytes and it seems that they can originate from both lymphoid
and myeloid precursors [52]. Even though pDCs and mDCs have been suggested to stem from
precursors of different origins it seems that the system allows some plasticity since pDCs
from bone marrow were able to differentiate to mDCs upon viral infection [53].
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Recognition strategies of antigen-presenting cells
It has now been more than 20 years since Charles Janeway proposed a link between innate
and adaptive immune responses [54]. He hypothesised that there are recurring PAMPs that are
essential to the function of microbes which are recognized by receptors of the host innate
immunity. These receptors are called PRRs. This was in line with the prevailing idea that
these receptors had evolved to distinguish self from non-self.
Five years later, Polly Matzinger suggested an alternative theory called “the danger model”
[55]. The “danger model” suggested that signals sent by stressed or injured tissues are the key
triggers to APCs. We now know that PRRs expressed on APCs have, in addition to
exogenous microbial derived ligands i.e. PAMPs, many endogenous ligands. Most of these
ligands can be related to stress, injury or necrotic cell death i.e. DAMPs [56-59]. The most
investigated family belonging to the PRRs in humans are the TLRs.
Toll-like receptors
Toll was first discovered as a protein involved in the development of drosophila but its role in
immunity was first shown by Bruno Lemaitre and Jules Hoffman as they showed that toll-
deleted mutants of drosophila were susceptible to lethal fungal infections [60]. The tolls were
later shown to be structurally related to the IL-1 receptor, suggesting an important role in
immunity [61].
The ligands and roles for the different receptors were discovered one after another [62-65]. So
far, ten different TLRs have been characterized in humans. They are all expressed by different
subtypes of APCs and have specific localizations and ligands (Table 2). TLR1, TLR2, TLR4,
TLR5 and TLR6 are found in the outer cell membrane and recognize recurring PAMPs in
bacteria, fungi, parasites and some viruses. Conversely, TLR3, TLR7, TLR8, and TLR9 are
located intracellularly and are specialized in recognition of intracellular bacterial and viral
nucleic acids. The motifs that TLRs recognize are not necessarily unique to pathogens. For
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example, it has been shown that TLR7 does not distinguish between influenza- and self-
derived single stranded RNA [66]. However, TLR7 is localized in endolysosomes where host
ssRNA is not normally present, thus avoiding any reaction against self-derived nucleic acids
under steady-state condition.
The expression of TLRs is different in monocytes, MΦs and different DC subsets as
previously mentioned. Different subtypes of DCs are devoted to defend against different kinds
of pathogens and therefore express different TLRs (Table 1). The pDCs express TLR1, TLR6,
TLR7, TLR9 and TLR10, while mDCs express TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,
TLR8 and TLR10 [67].
Receptor Ligand and origin Location mDC pDC MoDC MΦ Monocyte
TLR1 Triacyl LpP (bacteria) Surface + + + + +
TLR2 Zymozan, PGN (fungi, G+ bacteria) Surface + - + + +
TLR3 dsRNA (viruses) Internal + - + - -
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,(virus, protozoa) Internal - + - - +
TLR10 Nd Surface + + Nd - -
Table 2 TLR localization and ligands.
Abbreviations: mDC-myeloid DC, pDC-plasmacytoid DC, MoDC-monocyte-derived DCs, LpP-Lipoprotein,
PGN-Peptidoglycan, LPS-lipopolysaccharide, ds-double stranded, ss-single stranded, Nd-No data. References:
[67,68].
Signalling through TLRs
TLRs are membrane-spanning proteins with one intracellular signalling domain and one
amino terminal extracellular domain containing leucine-rich repeats, which are responsible for
binding the ligand. The signalling domain signals as a dimer and TLRs can form either
heterodimers or homodimers. For example, it has been shown that TLR2 can form
heterodimers with either TLR1 or TLR6 or form a homodimer and that the ligands for each
conformation can vary [69].
21
The TLR signalling domain is homologous to the IL-1R signalling domain and is called
Toll/IL-1R (TIR) domain. In general TIR-TIR interaction with an adaptor protein initiates
signalling thus recruiting an IL-1-receptor-associated kinase (IRAK) family protein and the
TNF-receptor associated factor 6 (TRAF6). This complex activates Map kinases, which
eventually lead to the activation of transcription factors [70,71].
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 IFN-β (TRIF) and TNF-receptor associated factor 3 (TRAF3) leading to activation
of the transcription factor IFN regulatory factor (IRF) 3 and secretion of type I IFNs [72]. In
addition to MyD88, TLR1, TLR2, and TLR6 can also signal through toll-interleukin-1
receptor domain containing adaptor protein (TIRAP). TLR4 is unique in that it can signal via
MyD88, TIRAP and TRIF.
Interestingly, 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 type I
IFNs [73]. Conversely, when the MyD88 - dependent pathway is activated through TLR4 it
leads to IL-12p70 secretion [74] while when TLR4 engages the alternative pathway via TRIF,
it results in IRF3 activation and secretion of type I IFNs [72,75].
In addition, the production of type I IFNs can be induced by two different pathways; either
through IRF3 via TRAF3 or through IRF7 via TRAF6 [76]. TRAF3 was shown to be essential
for the induction of type I IFNs and IL-10. In addition, deficiency of TRAF3 augments
production of IL-12 because of defective IL-10 production [77]. Therefore, this may be an
additional way to create diverse responses. The impact of different TLR activation on
adaptive immune responses has been summarised in a review [74] (Figure 3).
22
Figure 3. TLRs in the regulation of adaptive immunity.
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 only weakly induced by TLR2 but strongly induced by TLR4, TLR5 and
TLR8. Conversely, TLR2 activation leads to IL-10 production. TLR4 ligation can lead to
either IL-12p70 or the release of type I IFNs [74]. Viral recognition by TLR3, TLR7 and
TLR9 induces the release type I IFNs by different pathways. Thus, activation of different
TLR2 complexes results in T-helper (Th) 2 or Treg skewing of the immune responses, while
activation of TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 can lead to a Th1-type of
response [74].
The outcome will be different depending on which TLR is engaged, and thereby a “custom-
made” default response mechanism is put in place to deal with different types of antigens.
This activation process is further complicated by the fact that these signalling pathways
23
interact with the pathways of other PRRs. This interaction can skew the signalling pathways
in different directions as we will see in the next section.
C-type lectin receptors
Another group of PRRs are the C-type-lectin receptors (CLRs), which belong to a superfamily
of proteins recognising carbohydrate structures on glycosylated surface proteins of pathogens.
Some examples of CLRs are the mannose receptors, DC-SIGN, CLEC9A/DNGR1, Dectin-1
and, importantly, a specific marker of the pDC subset, the BDCA-2 molecule. One of the
functions of these receptors is to internalize antigens for processing and presentation to T
cells, as shown for DC-SIGN [78]. It has been proposed that ligation of CLRs alone might
lead to immunosuppression while ligation of the same CLR during a danger situation would
induce activation [79]. Although many CLRs can induce signalling on their own, some CLRs
act by modulating TLR signalling [80].
Nod-like receptors
A more recently discovered family that senses inflammation includes the Nod-like receptors
(NLRs). These receptors assemble upon activation into large cytoplasmic multiprotein
complexes called inflammasomes [81] that recruit inflammatory caspases and trigger their
activation [82]. In contrast to TLRs, NLRs exhibit the ability to activate caspase-1 that is
necessary for IL-1β production [83,84]. Inflammasome activation may also have a role in
initiating adaptive immunity [85]. It has also been suggested that another NLR, the NLRX1
would have a regulatory function [86] thus indicating additional possibilities of regulating
immune responses.
RIG-I-like receptors
A family of cytoplasmic receptors specific for viral nucleic acids is known as the retinoic
acid-inducible gene I (RIG-I)-like helicases. These receptors have been shown to induce type
24
I IFNs by phosphorylation of IRF3 [87] through the action of a caspase recruitment domain
(CARD) [88]. RIG-I and the melanoma-differentiation-associated gene (MDA)5, a receptor
belonging to the same family, recognize single stranded RNA and double stranded RNA.
RIG-I responds to influenza-, paramyxoviruses and flaviviruses while MDA5 respond to
picorna viruses [89,90].
In summary, there is a wide variety of receptors that are able to recognize both exogenous
PAMPs and endogenous DAMPs. Some of them are specifically expressed on a given cell
type while expression of other receptors is less restricted. Thus, a myriad of possibilities to
fine tune the responses of APCs according to the specific stimulus are presented.
ANTIGEN PRESENTATION
It is well established that APCs need to transmit two signals in order to activate T cells [91]
and it was later suggested that a third signal is needed [92] 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-II-antigen complex
2) Binding of co-stimulatory molecules
3) Cytokine signalling
Signal 1
The immunological definition of an individual’s “self” lies in the HLA molecules. These
molecules are the main determinants for the rejection of allogeneic grafts upon
transplantation. The cells of a transplant have an aberrant HLA expression, as compared to the
host, and the host will not consider it as self but treat it as foreign and attack. T cells can only
recognize an antigen when it is presented as peptide fragments in association with HLA
molecules.
25
The classical HLA proteins are subdivided into class I and II. The HLA-I molecules are
expressed on all nucleated cells of the body. They have a peptide-binding groove that holds
fragments of about 8-10 amino acids. The distal part of the HLA molecule then binds to the
CD8 molecule present on T cells [93]. The expression of HLA-II molecules is restricted only
to professional APCs. The HLA-II molecules have a larger groove than the HLA-I and can
therefore hold larger fragments. The distal part of the HLA-II molecules bind CD4 present on
T cells [93].
In the steady state condition HLA-antigen complexes are constantly internalized by immature
APCs, but upon maturation the internalization stops and consequently the cell surface
becomes full of HLA-antigen complexes. This process can be regulated by the actions of
ubiquitin ligase that marks the β-chain of HLA molecules with ubiquitin. This mark induces
internalization of the complexes while ubiquitin ligase is down regulated upon activation of
DCs [94-97].
Antigen uptake
Professional APCs activate the adaptive branch of immunity. The first step in antigen
presentation is to internalize the pathogen, and this is performed by 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. Pinocytosis is a
form of endocytosis that is also referred to as “cell drinking” or internalization of soluble
products. Another form of endocytosis is “cell eating” or phagocytosis. Pinocytosis is
formation of vesicles seemingly at random that serves to sample the environment for soluble
antigen in a non-specific manner. Phagocytosis is a process that is defined as an actin-
dependent receptor-mediated uptake of particles larger than 0,5µm in diameter [98].
The receptors involved are cell-surface receptors, such as Fc receptors (FcR), complement
receptors (CR) or CLRs [99]. FcR and CR are called opsonic receptors because they
26
recognize the particles to be phagocytosed only when opsonised i.e when the particles are
bound to antibodies or complement proteins. The zipper model suggests that FcγR binding of
the particle leads to receptor clustering that are sequentially engaged to the surface of the
particle, leading to local actin polymerisation. The polymerisation creates outwards extension
of the cytoskeleton called phagocytic cup [100].
FcR ligation leads to activation of tyrosine kinases, belonging to the Src and Syk families
[101]. They in turn lead to activation of small GTPases that are important for remodelling of
the cytoskeleton [102]. CR3-mediated phagocytosis is not dependent on tyrosine kinases and
seem to be mediated by a different family of small GTPases [103]. Phagocytosis mediated by
CLRs is not as well studied as phagocytosis mediated by FcR or CR. Dectin-1 mediated
phagocytosis seems to use different signalling pathways as it does not require Syk kinases
although it is dependent on tyrosine phosphorylation [104].
It is known that DCs lose their ability to take up antigen from the environment while
maturing, however this seems to be preceded by a brief period of migratory arrest and
enhanced uptake [105]. In line with this model, some evidence suggests that the ability to take
up antigen is regulated by activation of proteins downstream TLR signalling [106]. A clear
link between antigen processing and migration is the fact that gene knock outs of the invariant
chain, a crucial component for antigen processing, migrate faster than wild type DCs [107]. It
has also been shown that APCs become transiently paralyzed by microbial stimuli in order to
increase antigen sampling locally [108,109]. After the brief uptake period, maturation and
migration to lymph nodes will ensue. This enables the APC to carefully sample the
environment, process and select the antigen to be presented before setting of in search of
naïve T cells.
27
Antigen processing and presentation
Presentation of endogenous peptide fragments of cytosolic proteins on HLA-I molecules is
performed by all nucleated cells of the body. The HLA-I molecules are aided by a chaperone
molecule called β2-microglobulin to increase stability of the complex. Because the HLA-I
processing pathway leads to the transport of cytosolic proteins to the cell surface, this process
is called the cytosolic pathway. The cytosolic proteins are taken up by the proteasome and
degraded to peptide fragments. These fragments are then transported to the endoplasmatic
reticulum through the transporter protein TAP. In the endoplasmatic reticulum the peptide
fragments from the proteasome are then loaded on the HLA-I molecule and follow the normal
secretory pathway through the Golgi to reach the cell surface.
In contrast to the cytosolic pathway, professional APCs use the endocytic pathway to process
particles that are taken up from the extracellular environment and present peptide fragments
on HLA-II molecules. In APCs, extracellular components are transported into the cell through
endosomes therefore; this pathway is called the endocytic pathway. When an antigen is taken
up through an endosome the endosome is fused with lysosomes containing digestive enzymes
in an acidic environment. This endolysosome is where the pathogen is degraded by proteases.
Meanwhile, an HLA-II molecule that is contained in the endoplasmic reticulum bound to a
chaperone molecule called the invariant chain travels to the lysosome. In the lysosome the
chaperone is digested by proteases leaving only a fragment called class II-associated invariant
chain peptides (CLIP) in the peptide groove. The CLIP fragment is then replaced by a
selected peptide fragment from the antigen on the HLA-II molecule. The HLA-II-antigen
complex can then be transported to the APC surface where it is ready for encountering T cells
[110,111].
Proteolysis is an important step in antigen processing and it is believed that excessive protease
activity might destroy the antigen while moderate proteolysis may lead to enhanced antigen
28
presentation. It has been shown that an antigen that is more resistant to proteolysis induces
more potent T-cell responses than a protease-sensitive antigen [112]. Importantly, the levels
of MHC-II complexes on mature DCs are 10-100 fold higher than on monocytes or B cells
emphasising the important role of DCs in antigen presentation [113].
In line with the notion that DCs are the most potent APCs, it has been demonstrated that DCs
exhibit lower levels of protease activity than MΦs [114]. In addition, it has been shown that
DC phagosomes become transiently alkaline thus preserving the antigen from protease
activity and enhancing antigen presentation, particularly in cross presentation (see below)
[115].
Cross-presentation
Some APCs are capable of processing antigen through an alternative pathway that enables
them to present exogenous antigens on HLA-I molecules. This phenomenon is restricted to
particular subsets of professional APCs that induce antigen-specific CD8+ T cells and is
known as cross-presentation or cross-priming [116]. Cross-priming is induced by receptors
that recognize viruses, in particular TLR9 [117,118]. It has been shown that viral infection
can lead to cross-presentation by DCs and activation of CD8+ T cells without T-cell contact
but dependent on type I IFN production [119].
There is no consensus on the precise mechanism of cross-presentation and different pathways
have been proposed. The phagosomes and endosomes of DCs are constituted by the
endoplasmatic reticulum membrane. This may enable exogenous protein transportation into
the cytosol where proteins could go through the proteasome and TAP, back into the
endosomes and then be loaded on HLA-I molecules as any endogenous proteins are
[120,121]. This has been proposed to be TAP-dependent through the same mechanism that
dislocates misfolded proteins from the endoplasmic reticulum to be degraded [122]. However,
TAP-independent pathways have also been suggested to explain cross-priming [123].
29
Finally, it has been proposed that DCs migrating to lymph nodes to activate CD4+ T cells can
transfer exogenous antigens to resident DC populations in lymph nodes and that these cells
activate CD8+ T cells [124,125]. This hypothesis is strengthened by the fact that splenic DCs
can cross-present antigens more efficiently than other DC subsets [126].
Signal 2
The expression of co-stimulatory molecules on APCs is fundamental to their function since
they are the providers of signal two needed for the activation of T cells. APCs express a
variety of co-stimulatory molecules that constitute what is called the immunological synapse.
Two important co-stimulatory molecules are the members of the B7-family, CD80/B7.1 and
CD86/B7.2 that bind CD152/CTLA4 and CD28 on T cells. Such binding can mediate
activating or inhibitory signals [127]. For example, ligation of CD28 by CD80 and CD86
leads to T-cell activation while ligation of CD152 suppresses activating signalling in T cells
[128]. Furthermore, evidence indicates that priming of T cells without proper co-stimulation
[129,130] or in the absence of activating cytokines will lead to their differentiation into Tregs
or anergic T cells [131].
Two members of the TNFR family are also known to affect T-cell responses, CD134/OX40
and CD137/4-IBB. CD4+ T cells are more prone to secrete Th2 cytokines if the APC that
activates them express high amounts of OX40L that binds OX40 on activated T cells [132]. If
an APC expresses 4-1BBL, they preferentially induce activation of CD8+ cells and secretion
of IFN-γ [133]. In addition, it has been shown that OX40 can antagonise the induction of
peripherally induced Tregs while both OX40 and 4-1BBL can support proliferation of
naturally occurring Tregs [134].
Another important molecule for co-stimulation is the ICOS. Ligand binding to ICOS has been
shown to be important for triggering T cells as potent activators of B cells [135]. In addition,
30
ICOS seems to be able to mediate suppression since ligand-binding to ICOS is important in
generating Tregs [136].
Signal 3
After it had been established that signals 1 and 2 are necessary for the activation of naïve T
cells, it soon became clear that a third signal was also needed for the triggering of naïve T
cells into potent effector cells [92]. This signal is constituted by the cytokines secreted by
APCs and varies depending on the manner of activation of the APC. It is now known that
each defined Th lineage has specific requirements for their generation. For example, Th1 cells
can only be induced if IL-12 is secreted while IL-4 is essential to establish a Th2 lineage.
Cytokines
Cytokines are soluble mediators of inflammation secreted by leucocytes and epithelial cells.
As they serve to communicate between leucocytes they are called interleukins (ILs).
Traditionally they have been given names according to the function they were discovered for
but as they are sequenced they are renamed to interleukins. Some key cytokines will be
introduced below and their function will be further discussed when discussing inflammation.
IL-1β
IL-1β is a potent pro-inflammatory cytokine and a powerful pyrogenic factor that does not
seem to play a role in homeostasis. IL-1β induces increased expression of adhesion molecules
on the endothelium at a site of inflammation and acts as a bone marrow stimulant increasing
the number of circulating neutrophils [137]. Monocytes, MΦs and DCs produce IL-1β in
response to inflammation. It is produced as pro-IL-1β and needs to be cleaved in order to
become the active from of IL-1β [138]. In this context, it has recently been shown that
31
inflammasomes play a vital role in the induction of caspase-1 leading to the subsequent
realease of IL-1β [81].
TNF-α
TNF-α is a member of the TNF super family of proteins, which was initially discovered for
killing of tumour cells, and acts as a potent pro-inflammatory cytokine. TNF-α can trigger
apoptosis via the TNF receptor that activates pro-caspases thus inducing cell death. This
cytokine is crucial in infectious diseases as it efficiently promotes inflammation and helps to
combat the pathogen.
TNF-α is a central player in inflamed tissues in the early phase of inflammation, where it is
known to induce the production of many other pro-inflammatory cytokines [139]. However,
high levels of this cytokine are responsible for tissue damage and pathogenicity [140]. In the
late phase of inflammation, TNF-α can induce an IRF1-dependent autocrine loop of type I
IFN secretion, thus sustaining an inflammatory response and lowering the threshold of TLR
responses [141].
Type I IFNs
These cytokines were named interferons over 50 years ago because they were discovered to
interfere with viral replication [142]. They are found in all vertebrates where they have been
investigated and many subgroups have been discovered [143]. Although there are about 20
different type I IFNs, IFN-α and IFN-β are the classical antiviral IFNs. Type I IFNs can be
produced by a wide variety of cells. Viral recognition triggers transcription of type I IFNs
which when bound to their receptors trigger the JAK/STAT pathway leading to transcription
of IFN stimulated genes (ISG). The most potent producers of IFN-α are pDCs and NK cells as
a response to viral infection. IFN-α acts on immune cells to elicit anti-viral responses and also
functions as an analgesic.
32
The triggering of RIG-I, MDA5, TLR3, TLR7, and TLR9 activates the transcription factors
AP1, NF-kB, IRF3 and IRF7 leading to secretion of type I IFNs [144]. It has been suggested
that DC secretion of type I IFNs can act in an autocrine manner, thus enhancing activation of
DCs. This was supported by the fact that DCs from type I IFN knockout mice exhibited an
impaired phenotype and were less potent activators of T cells [145]. Although it seems clear
that type I IFNs can play an important role in DC maturation they do not seem to be able to
induce potent maturation on their own [146,147].
IL-6
Upon its discovery this cytokine was called B-cell stimulatory factor-2, but it was renamed as
an interleukin as the cDNA was sequenced [148]. Pro-inflammatory cytokines such as TNF-α
and IL-1β induce IL-6, while its secretion is inhibited by the Th2 cytokines IL-4 and IL-13.
Nevertheless, this cytokine can skew CD4+ T cells to become IL-4 secreting cells and it has
therefore been regarded as a Th2 cytokine [149]. This effect was shown to be induced by
disruption of the autocrine IFN-γ loop that leads to Th1 activation through induction of
SOCS1 [150]. It has now become clear that IL-6 is a key factor in the induction of the
transcription factor RORγt and in the differentiation of naïve T cells to the Th17 lineage
effector cells [151]. Recently, it has been suggested that IL6 is an important regulator of the
balance between Th17/Tregs [152,153].
IL-12 family
IL-12 is the classical Th1-inducing cytokine secreted by neutrophils, monocytes, MΦs and
DCs and induces IFN-γ release in NK cells and T cells. IL-12 was the first member of the IL-
12 family of interleukins discovered and initially called NK-cell stimulatory factor [154].
More recently, IL-23 [155] and IL-27 [156] were added to the IL-12 family. All three are
heterodimers and IL-12 and IL-23 share the p40 subunit. IL-12 is composed of the p40 and
33
the p35 subunits while IL-23 is composed by the p40 and the p19 subunit. Similarly, the
corresponding receptors are composed by two chains and both IL-12 and IL-23 receptors
share IL-12Rβ1 chain, while the second chain is different for each receptor. In this way the
function these cytokines exert by stimulating the cognate receptors is different.
For instance, IL-23 creates autoimmune inflammation in a mouse model for multiple sclerosis
while IL-12 does not [157]. Secretion of the IL-12 family cytokines can be differentially
regulated depending on the nature of the microbial stimuli. This is evidenced by the fact that
LPS can induce both IL-12 and IL-23 release by DCs whereas peptidoglycan induces IL-23
but not IL-12 secretion [158].
IFN-γ
IFN-γ is an important and well-studied cytokine that mediates strong pro-inflammatory
responses. It was originally termed macrophage-activating factor and is the only member of
the type two IFN class. IFN-γ secretion is induced in activated T cells and NK cells by IL-12
and IL-23 and release of this mediator leads to a classical Th1 response while supressing Th2
responses [159]. NK and T cells produce IFN-γ and release generally follows in response to
virus, intracellular bacteria or tumor cells [160].
IL-4 & IL-13
IL-4 is secreted by granulocytes and various T-cell subsets [161] and represents the hallmark
cytokine for Th2 responses. This mediator was originally known as the B-cell differentiation
factor [162]. IL-13 is another Th2 cytokine with very similar effects to IL-4 but seems to be
more important for immunity against certain infections such as different species of
Leishmania [163]. The IL-4 receptor α-chain can bind both IL-4 and IL-13. However different
receptor complexes can be composed by different subunits enabling both cytokines to mediate
different responses [164].
34
IL-10
Perhaps the most important cytokine that regulates immune responses is IL-10. It was
originally known as “cytokine synthesis inhibitory factor” because it was shown to inhibit
Th1 cells and was therefore regarded as a Th2 cytokine [165]. It is now known that IL-10 can
be secreted by T cells, B cells, DCs and MΦs during an infection. The inhibition of IL-1 and
TNF-α is a very important anti-inflammatory effect of IL-10, while it also inhibits the
production of many other cytokines, even its own production [86].
The importance of IL-10 as a regulator of immune responses was evaluated in IL-10 deficient
mice. These mice remained healthy until their gut was colonized by bacteria. After
colonization, the animals developed chronic enterocolitis [166] indicating that the immune
responses were not properly regulated. The potency of IL-10 is emphasized by the fact that
some viruses, such as Epstein Barr and human cytomegalovirus, encode an IL-10 homologue
that is released in order to escape immune responses [167].
TGF-β
Another soluble mediator that is a key factor for regulating immune responses is the
transforming growth factor (TGF)-β. There are various isoforms of TGF-β and they are all
members of the TGF-β superfamily. They are secreted in an inactive form often called the
latent TGF-β and it has been suggested that the regulation of activation of the latent form is
the most potent form of regulation of TGF-β [168]. TGF-β is a pleiotropic cytokine that is of
outmost importance for preserving homeostasis. This is emphasized by the fact that mice
lacking the TGF-β1 protein die prematurely of excessive inflammatory tissue damage [169].
APC-derived cytokines in T-cell differentiation
IL-12 secretion by DCs and MΦs is tightly linked to the induction of IFN-γ in naïve T cells
and commitment of these cells to the Th1 lineage [170,171]. IL-12 priming of T cells also
35
impairs their capability of secreting IL-4 [172], thus further favouring Th1-type responses
rather than Th2. Microbial stimuli such as LPS and Toxoplasma gondii are required and
sufficient for the production of IL-12 by DCs and to initiate their migration to T-cell areas of
the spleen [173,174].
In addition, IL-12 and IFN-γ production is known to be important for the protection against
intracellular infections like mycobacteria and genetic defects in the Th1 axis leads to
susceptibility to disease [175]. Conversely, IL-23 does not induce Th1 responses while it
plays an important role in the protection against extracellular bacteria, such as Klebsiella
pneumoniae [176]. It has now been shown that this is an effect of the induction of Th17
responses [177-180].
INFLAMMATION
Upon sterile or pathogen-mediated tissue damage, necrotic and surrounding cells secrete
inflammatory mediators that in turn recruit and activate immune cells. The subsequent release
of inflammatory mediators leads to an increased permeability of the surrounding vessels, thus
increasing the blood flow at the site of inflammation. Simultaneously, circulating immune
cells start to adhere to the vessel walls as a consequence of increased expression of adhesion
molecules such as selectins and integrins on endothelial cells. The selectins induce rolling of
blood cells on the vessel wall and the integrins mediate transendothelial migration.
Granulocytes, that are normally the first immune cells that are recruited at the site of
inflammation, release their granules containing toxic compounds and phagocytosis of
microbes is induced, leading to their own death. The granulocytes also release proteases that
will start to liquefy the foci of inflammation thus inducing the formation of pus. Monocytes
will also phagocytose pathogens and simultaneously process antigens for subsequent antigen
presentation. Similarly, tissue specific APCs such as DCs that are activated by pathogens and
36
inflammatory mediators, will carry antigens to the lymph nodes in order to activate
appropriate T cells.
In the early phases of inflammation the proinflammatory cytokines secreted such as IL-1, IL-6
and TNF-α serve to increase inflammation and temperature. Later on, the secretion of soluble
factors will be skewed to regulatory cytokines, such as IL-10, in order to decrease
inflammation. In the normal situation the pathogen is removed, the inflammation resolved,
and homeostasis restored. However, chronic inflammation may persist that can lead to severe
tissue damage.
Leukocyte movement
Inflammation implies recruiting leukocytes to the site of injury or infection. In order for cells
to find their final destination, signalling molecules are secreted that guide the movement of
leukocytes. These molecules are small proteins called chemokines. The name derives from
their ability to induce chemotaxis i.e cell movement towards a gradient of a certain substance.
The chemokines are classified according to structural properties and the C symbolises the
location of highly conserved cystein residues in the peptide chain of chemokines. Chemokines
are divided into two major subgroups; the CC and the CXC [181]. More recently, two smaller
subgroups have been added; i.e. the C and the CX3C subgroups [182]. The chemokine
receptors (CCRs) are coupled to a G protein for intracellular signalling [183] that contain a
characteristic seven transmembrane domain.
Inflammatory CCRs
Inflammatory CCRs often have various ligands and many of them are shared (Table 3). This
is the case for CCR1, CCR2 and CCR5 that typically bind inflammatory mediators such as
macrophage inflammatory protein (MIP)-1α and MIP-1β, Monocytes-Chemoattractant
Protein (MCP)1-4 and Regulated on Activation Normal T Expressed and Secreted Protein
37
(RANTES) [184]. Cells expressing any of these CCRs will sense inflammatory chemokines
and start to move towards the site of inflammation.
Lymphoid CCRs
In contrast to the inflammatory CCRs, the lymphoid CCRs such as CXCR4 and CCR7, do not
have many known ligands. CCR7 is expressed on naive T cells and it has been shown that
lack of this receptor severely impairs primary immune responses [185]. Both T and B
lymphocytes express CXCR4 and this receptor has a dramatic effect on the development of B
cells [186]. A ligand for CXCR4 is stromal derived factor (SDF)1/CXCL12, while MIP-
3β/CCL19 and secondary lymphoid tissue chemokine (SLC)/CCL21 bind to CCR7 and are
mainly expressed in lymphoid tissues. Both CCR7 and CXCR4 are highly expressed on
mature DCs, allowing their migration to secondary lymphoid organs [36].
Receptor Ligand Location iDC mDC
CCR1 CCL3/MIP-1α, CCL5/RANTES Inflammation + -
CCR2 MCP-1-4 Inflammation + -
CCR5 MIP-1α, CCL4/MIP-1β, CCL5/RANTES Inflammation + -
CCR7 CCL19/ELC/MIP-3β, CCL21/SLC Lymphatic tissue - +
CXCR4 CXCL12/SDF-1α 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-immature DC, mDC-mature DC, MCP-monocyte chemoattractant
protein, MIP-Macrophage inflammatory protein, SDF-stromal derived factor . References: [182,184]
38
RELATED BACKGROUND
APCs IN INFLAMMATION AND THE LINK TO ADAPTIVE IMMUNITY
Inflammation is the trigger that activates APCs through a variety of different receptors. The
ligation of PAMPs or DAMPs to APC receptors triggers intracellular activation signals. The
sum of these signals decides what action the APC will take.
Antigen presentation is the act that links pathogen recognition by innate immune cells to the
initiation of the adaptive immune responses. Professional APCs are all capable of activating
cells of adaptive immunity. However, different APC subsets can be triggered by distinct
signals and have become specialized in activation of specific branches of the adaptive system.
So far, the mechanisms that orchestrate the activation of adaptive responses are only partially
clarified.
Monocytes
As discussed earlier, human monocytes in circulation are commonly divided into the classical
CD14++
CD16- and the non-classical CD14
+CD16
+ monocytes. The CD14
+CD16
++ subset
expresses higher levels of HLA-DR and was shown to produce higher levels of TNF-α [187]
but lower levels of IL-10 [188] as compared to classical monocytes. Their murine
counterparts have been defined with many similarities. Perhaps the major difference is that in
humans the non-classical represent only a small part of the circulating monocytes while in
mice the subsets are equally distributed [189]. The CD16+ subset is more potent in inducing
T-cell responses in a mixed leukocyte reaction (MLR) than the CD16- monocytes [190].
Monocytes are precursors of MΦs and can give rise to inflammatory [191] and anti-
inflammatory MΦs [192] and to inflammatory DCs [46]. The differentiation of monocytes
seems to be differently regulated during homeostasis and under inflammatory conditions.
Monocytes do not seem to replenish MΦ in the lung [193], or in the spleen, DCs [194] under
39
steady state conditions. Monocytes can, however, be an important source of DC during
inflammation [10,49,195]. In this context, it has also been shown that the non-classical
monocytes are more prone to trans-endothelial cell migration and differentiation into
inflammatory DCs [196].
Macrophages
Classical activation of MΦs, induced by IFN-γ, leads to differentiation of M1-MΦs that
secrete high levels of IL-12, the most potent Th1 inducing cytokine, and low levels of the
anti-inflammatory protein IL-10. In contrast, MΦ activation by IL-4 and IL-13 leads to M2
polarization and secretion of high levels of IL-10 and low IL-12. In addition, upon stimulation
with IFN-γ and CD40 ligand (CD40L), M1-MΦs secrete high levels of pro-inflammatory
mediators including IL-1β and TNF- while M2-MΦs secrete low levels of these cytokines
but high levels of IL-10. M2-MΦs do, however, secrete higher levels of pro-inflammatory
chemokines perhaps indicating a role in recruitment of other cells and in the regulation of
homeostasis [197].
The roles of M1-MΦs in promoting inflammation and Th1 differentiation are emphasized by
the fact that IFN-γ also enhances the expression of TLR4 and downstream signalling proteins
such as MyD88 [198]. In contrast, the Th2 cytokine IL-13 inhibits LPS-induced caspase-1
activity and modulates gene expression of various genes in the IL-1β system [199] indicating
a dampening function on inflammatory responses.
Selective depletion of MΦs at different stages of inflammation in a model of liver injury has
shown that these cells are important both during the injury and the recovery phase [200].
Specifically, it has been suggested that classical MΦs are involved in tissue degradation that
lead to injury [201] while alternative activation of MΦs promote tissue healing [202].
Generally, alternatively activated MΦs are thought to play an important role in the
maintenance of homeostasis. The fact that the receptors that are upregulated during alternative
40
activation have various endogenous ligands has been used as an argument to strengthen this
view [203].
Dendritic cells
DCs are well known to become potently activated by microbial stimuli which lead to
maturation, antigen processing and activation of naïve T cells, thus initiating adaptive
immune responses [91]. This could lead to activation of distinct T-helper subsets such as the
previously described Th1, Th2 and Th17 [2,3][204]. Finally, it is becoming clear that DCs
also play an important role in the induction and maintenance of tolerance through the
induction and maintenance of regulatory leukocytes [205,206]. These different pathways of
initiation of adaptive immunity are highly dependent on cytokines.
Myeloid DCs
Myeloid DCs express a broad repertoire of PRRs and respond to bacterial, parasitic, fungal
and viral infections. They also have the capacity to tailor inflammatory and T-cell responses
depending on the nature of the antigen [207]. For example, upon LPS stimulation DCs up
regulate maturation markers and secrete high levels of IL-12, which is known to induce Th1
responses [208]. The secretion of IL-12 by DCs not only initiates Th1 responses but is also
critical for generating IgM-secreting plasma cells [209]. In addition, DC-derived IL-12 can
induce class switching to IgG and IgA [210].
Although the specific characteristics of circulating mDC subsets are being investigated their
different roles are not yet completely clarified. BDCA-3+ DCs have been shown to express
lower levels of HLA-DR and co-stimulatory molecules than the BDCA-1+ subset, indicating
lower levels of activation [39]. In fact, a potential immunomodulatory role for the BDCA-3+
subset has been suggested during malaria infection [211].
41
In addition, BDCA-3+ DCs of sheep, mice and humans express XCR1 and selectively respond
to the ligand XCL1 expressed preferentially in CD8+ memory T cells [212]. These findings
support the view that these cells may be the human counterpart of the murine DCs devoted to
cross-presentation of viral antigens to CD8+ T cells. In line with this hypothesis, it was shown
that XCR1 knock outs have impaired early CD8+ T-cell responses [213].
Plasmacytoid DCs
The pDCs are specialized to handle viral infections and have several features that enable them
to respond quickly and potently to viruses. They were originally called natural IFN-producing
cells since they release vast amounts of type I IFNs upon recognition of nucleic acids [214].
The pDCs seem to play a crucial role in humoral immunity; high levels of type I IFN
secretion by pDCs can induce plasma cell differentiation and Ig production [215,216] and can
also induce class switching of B cells [217]. Furthermore, pDCs possess intracellular
endosomes containing HLA-I molecules that are quickly mobilized, loaded with antigen and
sent to the surface to activate CD8+ T cells upon viral infection, thus contributing to the
development of an efficient effector response [218].
The pDCs are also able to induce both Th1 and Th2 responses in vitro. It was shown that
virus-stimulated pDCs secreted high levels of IFN-α and lead to Th1 responses while IL-3-
stimulated pDCs upregulated OX40L, secreted low levels of IFN-α and led to Th2 phenotype
[219].
Tolerogenic DCs
As the most important initiators of adaptive immunity, DCs must not only be able to activate
potent inflammatory responses, but should also be able to induce tolerance. DCs that travel
the periphery sample the environment and constantly present self-derived and harmless
environmental peptides on their HLA-II molecules. These cells can, as long as they remain in
42
an immature state, maintain peripheral tolerance [205] indeed antigen presentation in the
absence of co-stimulation can lead to T-cell anergy [220].
Apoptotic cells display phosphatidylserine on their surface, which is recognized by various
receptors on APCs and can lead to uptake of apoptotic bodies by these cells [221]. The uptake
of material from apoptotic cells does lead to antigen presentation but not maturation of DCs
[222,223]. DCs migrate to local lymph nodes during steady state conditions to present self-
antigen derived from apoptotic cells to T cells without inducing their activation [224,225].
The phenotypic characterization of tolerogenic DCs is still not well defined, however ILT3,
ILT4 and IDO may be surface indicators of tolerogenic DCs [226-228].
Endogenous ligands
As we have seen, APCs and other hematopoietic cells interact intimately to decide whether or
not to respond to a specific microbe/antigen and what type of responses to induce. However,
non-hematopoietoc cells also play a role in this context. One example is the mucosal surfaces,
which constitute the interface between the outside world. For instance, epithelial cells of the
intestine express various TLRs and can secrete soluble factors that participate in inflammatory
responses [229]. Furthermore, under homeostatic conditions, intestinal cells [230-232] as well
as liver parenchyma [233] maintains a DC population that is tolerogenic [234].
Two important factors for induction of tissue tolerance are thymic stromal lymphopoietin
(TSLP) and transforming growth factor (TGF)-β [235]. TSLP induces a mature phenotype on
DCs, with up regulation of HLA-II and co-stimulatory molecules but no secretion of
inflammatory cytokines [236].
There is no doubt that non-hematpoietic cells participate in inflammatory reactions but their
role is not entirely understood. On the one hand, epithelial cells have been shown in vitro to
be necessary for potent antiviral Th1 responses [237]. On the other hand, it has been shown
43
that tissue responses to LPS are capable of inducing inflammation but not DC activation and
therefore these responses are not sufficient to trigger adaptive immunity [238].
In addition, heat-shock proteins (HSPs) are endogenous ligands that can act as trigger for
inflammation and the most studied is a family called molecular chaperones. Chaperones are
involved in folding of proteins but are also released during stress conditions and have
important stimulatory functions on innate immunity [239].
High mobility group box 1 (HMGB1) is also an important mediator of inflammation. This
nuclear DNA-binding protein is released upon necrotic cell death and is known to be involved
in both sepsis and autoimmune diseases. HMGB1 can bind to various receptors, such as
TLR4, involved in inflammatory responses [240].
The manipulation of APCs by cytokines
Different pathways of initiation of adaptive immunity are highly dependent on APC
differentiation and functionality which is in turn affected by various cytokines.
It is well established that DCs are capable of secreting high amounts of pro-inflammatory Th1
cytokines like IL-12 and type I IFNs and that they are essential in orchestrating adaptive T
cell responses. However, the effect of these cytokines on APCs can have profound effects on
the development on immunity.
For instance, if immature DCs are exposed to TNF-α they become phenotypically mature
expressing high levels of HLA and co-stimulatory molecules but they do not secrete IL-12
[241]. In fact, they can even induce IL-10 secreting antigen-specific T cells that protect from
autoimmune disease [242,243]. In the presence of fibroblasts TNF-α favors the differentiation
of monocytes into DCs rather than MΦs as well as induce maturation of pDCs and mDCs
[244,245].
There are other important inflammatory mediators that can affect the differentiation and
functionality of APCs. Fibroblast secretion of IL-6 is capable of skewing monocyte
44
differentiation from DCs to MΦs. This cytokine also impairs migration of mature MoDCs and
inhibits DC maturation and subsequent T-cell activation [246,247]. IL-6 may therefore play a
role in the specific differentiation and function of APCs [248].
A crucial cytokine for immune regulation is IL-10 and a well-recognized anti-inflammatory
effect of IL-10 is to dampen APC functions. For example, a major role is to contain
inflammation and this is achieved by preventing MΦs from killing and by inhibiting cytokine
secretion by MΦ and DCs [86]. In fact, DCs that are matured in the presence of IL-10 exhibit
reduced expression of HLA-II, co-stimulatory molecules and impaired secretion of
inflammatory cytokines [220,249-251]. In addition, DCs matured in the presence of IL-10
induce suppressive T cells that do not require IL-10 or TGF-β to exert their suppressive
function [252], and are therefore different from the Th3 and Tr1 subsets.
MALARIA
Malaria burden
Malaria is one of the most devastating infectious diseases in the world and it is tightly
connected to poverty [253,254]. According to the WHO malaria report form 2010, there were
225 million cases of malaria reported in 2009 as compared to 233 million cases in the year
2000. Though the number of cases has varied during this time period the number of deaths has
been steadily decreasing to781 thousand from 985 thousand during the same time period. In
Africa, 85% of the deaths occur in children below the age of 5 and about 98% of the cases are
caused by Plasmodium falciparum [255,256]. Pregnancy associated malaria is another aspect
of the infection that defines pregnant women as an important risk group for malaria infection
[257].
45
Parasite life cycle
The parasite is transmitted from the definitive host, mosquitoes of the genus Anopheles, to
intermediate hosts such as reptiles, birds and mammals. The four “classical” species infecting
humans are Plasmodium falciparum, vivax, malariae, ovale. A fifth species, Plasmodium
knowlesi, was known to infect macaques but has also been reported to infect humans [258-
264]. The sporozoites are transmitted to humans from the salivary glands of the infected
mosquito (Figure 4).
Figure 4. The Malaria Life cycle
Reprinted from. Ménard, Robert, 2005: Knockout malaria vaccine? (Nature 433, 113-114). with permission from
Nature Publishing Group
The sporozoites immediately infect the liver upon entering the blood stream and develop into
merozoites. The merozoites leave the liver and infect-red blood cells (RBCs), where they
undergo replication. This is the blood stage of the infection and in the RBCs the parasites
develop from ring stage, to trophozoites and finally schizonts. Thousands of new merozoites
are released into the blood stream at the rupture of each schizont. Among the merozoites there
46
is a small portion of gametocytes released that are necessary for sexual stages in the mosquito
gut.
Hemozoin
The free heme groups that are left upon degradation of haemoglobin are toxic to the parasites.
Therefore, they are transformed into malarial pigment or hemozoin (Hz) that are innocuous
crystals [265]. APCs take up the Hz released by the parasites. In order to create in vitro
models of malaria stimulation, hemozoin crystals can be synthetically formed from hemin
[265] and the product is usually referred to as β-hematin or synthetic (s)Hz. It has been shown
that sHz induces recruitment of leukocytes, mainly monocytes and neutrophil populations,
and trigger potent pro inflammatory responses [266-268]. The immunogenicity of Hz in mice
and in humans will be discussed in the next section.
Innate Immune responses to malaria
MyD88, a central adaptor protein in TLR signalling, has been suggested to play an important
role in the pathogenesis of the malaria. Different parasite-derived or modified molecules have
been shown to bind TLRs and elicit innate immune responses [269]. For example
glycosylphosphatidylinositol anchors (GPIs) of protozoan parasites has been shown to bind
TLR2 [270] and TLR4 [271].
It has also been suggested that Hz activates cells through TLR9 [272]. It was then
demonstrated that Hz functions as a carrier of malarial DNA to the endolysosomes and that
malarial DNA attached to Hz is responsible for TLR9 activation [273]. More recently, it was
postulated that it is not Hz, but rather a protein-DNA complex, later discovered to be the
nucleosome, that triggers TLR9 activation [274,275].
Polymorphism in the TLR4 gene predisposes to the severe form of malaria in children [276]
and polymorphisms in TLR4 and TLR9 genes are associated with increased risk of low birth
47
weight in the offspring of pregnant women infected with malaria [277]. These findings
suggest that TLRs contribute in determining inflammatory responses upon malaria infection.
In this context it has been shown that secretion of pro- and anti- inflammatory cytokines is
increased upon specific TLR stimulation of whole blood that was primed by malaria antigens
[278].
Taken together, these studies indicate that P. falciparum modulates TLR activity and that
innate immunity, in particular TLRs, plays an important role in the responses against malaria.
Antigen-presenting cells in malaria
APCs are crucial initiators of adaptive immunity, express high amounts of TLRs and may be
fundamental in host responses to malaria. In fact, DC-mediated suppression of T-cell
functionality has been suggested to be the cause of the poor T-cell mediated immune
responses seen in malaria [279,280]. Observations in vivo have shown that children with acute
malaria have a lower expression of HLA-DR on peripheral blood DCs as compared to healthy
children [281]. Moreover, a possible immunomodulatory role has been suggested for the
BDCA-3+ subset of mDCs upon malaria infection in Kenyan children [211].
Observations in vitro have shown that APCs exposed to Hz increase both pro- and anti-
inflammatory mediators as reviewed in [282]. In addition, Hz can mediate activation of pDCs
via TLR9, although it is unclear whether the stimulus that activates these cells is Hz itself,
parasite DNA or both in a complex [272,273,283]. As mentioned above, more recent studies
suggest that TLR9 is bound by a histone-malarial DNA complex [274,275].
Whether or not Hz is responsible for activation of TLR9 there are documented immunological
effects of Hz. Hz-loaded monocytes have been shown to secrete higher levels of TNF-α and
IL-1β [284] than unstimulated cells, but also lower levels of IL-12p70 by an IL-10-dependent
mechanism [285]. Monocytes also exhibit inhibited differentiation to DCs [286] as well as
impaired up-regulation of HLA-II molecules when exposed to Hz [287]. LPS-induced
48
maturation of human MoDCs was shown to be impaired upon prior exposure to high amounts
of P.falciparum-infected red blood cells (iRBCs) [288]. However, when immature DCs were
exposed to parasite-derived Hz, enhanced maturation was observed [289].
Thus, both pro- and anti- inflammatory effects have been attributed to malarial stimuli when
in contact with APCs. Many factors such as the heterogeneity of APCs used, different parasite
strains, different types of Hz employed and the time of exposure of malarial stimuli to APCs
could explain these discrepancies [290,291]. Taken together, existing data suggest that the
function of various APC subsets is influenced by P.falciparum and that immune responses to
malaria may be hampered by the modulation of APC functions.
Cytokines and clinical implications in malaria
For a successful elimination of parasites it is important to have an early innate response
including secretion of pro-inflammatory cytokines like IL-12 and TNF-α. For optimal
clearance of the parasite, that initial response should be followed by an effective adaptive
immune response including high titres of antimalarial antibodies to clear the parasitemia. On
the other hand, pro-inflammatory cytokines may also play an important role in determining
pathology, in particular in the development of severe malaria. In fact, low levels of IL-10 and
high levels of TNF-α have been associated with disease severity and in severe cases in
Gabonese children IL-12 was found to be lower as compared to the mild cases [292]. In
addition, the development of cerebral malaria has been associated with high plasma levels of
IL-1β [293] and higher levels of IL-6 and IL-10 were associated with severe malaria as
compared to uncomplicated malaria [294].
Fulani and Dogon in northern Mali
The study area in Sahel, northern Mali, lies in Dogon country but in this particular area the
Dogon farmers settled down only 50 years ago. The Fulani, a traditionally nomadic pastoral
49
people, settled in this area at least 200 years ago with their cattle. The Fulani have been shown
to be less susceptible to malaria than their sympatric counterparts i.e. other ethnicities living
under similar geographical, economic and social conditions [295]. In addition to the relative
protection of symptomatic disease, the Fulani have higher proportions of malaria specific IL-4
and IFN-γ producing cells as compared to their sympatric neighbours. They also have
increased spleen rates and exhibit higher malaria-specific antibody titres [295-298]. Recently,
a lower Treg activity has been shown in a population of Fulani in Burkina Faso as compared
to a neighbouring sympatric group [299]. So far, no satisfactory explanation for the relative
protection to malaria infection of the Fulani has yet been found.
REUMATHOID ARTHRITIS
Reumathoid arthritis (RA) is a chronic inflammatory autoimmune disease of the joints.
Approximately 1% of the world’s population is affected and RA is more common in the
female population [300].
Clinically, RA is characterized by joint pain, stiffness and swelling due to synovial
inflammation and effusion. The joint is composed by the two cartilage covered bone ends
separated by synovial fluid and lined by the synovial membrane. In the healthy state there are
synoviocytes sitting on the membrane lining the joints. In RA, for yet unknown reasons, the
joint is infiltrated by leukocytes and the synoviocytes start to proliferate [301] (Figure 5). In
contrast to other forms of arthritis, RA synovitis tends to disobey tissue boundaries,
infiltrating articular bone and cartilage, thus inducing a pannus formation. Eventually the
tissue, cartilage and the underlying bone are destroyed and the disease causes disability,
cardiovascular complications and premature death if insufficiently treated [302].
50
Figure 5. Healthy (left) joint and joint affected by rheumatoid arthritis (right).
Reprinted from Strand et al. Nature Reviews Drug Discovery 6, 75–92 (January 2007) | doi:10.1038/nrd2196
with permission from Nature Publishing Group
The aetiology of RA is still unknown and therefore there is no curative treatment. In order to
alleviate the symptoms of the disease, anti-inflammatory drugs can be used but they do not
affect disease progression. In the past decade, several new anti-rheumatic drugs have become
available for the treatment of RA. In particular, biological agents that target specific pro-
inflammatory cytokines such as TNF- have proven to be efficient in combination with
methotrexate [303]. Nevertheless, anti-TNF- treatment exhibits notable side effects and is
inconvenient for patients to use because it is administered by injection. In addition, TNF
inhibition is not always effective, and the extent of therapeutic response varies between
different individuals [302].
51
Role of MΦs and DCs in RA
Although the cause of RA has not yet been found, research over the past decades has lead to a
better insight into the pathogenesis of the disease. Among others, APCs appear as crucial cells
in determining this inflammatory condition. Large amounts of cells that are found in the
inflamed joint are MΦs and their numbers correlate to tissue destruction [304,305]. The main
mechanism for MΦ involvement in tissue destruction is likely by driving inflammation
through secretion of pro-inflammatory cytokines, such as TNF-α and IL1-β, that are known to
be important for disease progression [306].
Joint destruction starts by cleavage of the proteoglycans by aggrecanases, followed by
degradation of the collagen backbone by matrix metalloproteinases (MMPs) [307]. It has been
shown that synovial MΦs, that are present in large numbers in the cartilage-pannus-junction
[305], are important in driving the secretion of aggrecanases and MMPs [308].
Once the cartilage is degraded the bone erosion starts to take place. One central mechanism of
bone destruction is the generation of osteoclasts that origin from cells of myeloid lineage
[309]. M-CSF is produced by synovial endothelial cells and fibroblasts and its production is
increased by inflammatory mediators such as TNF-α and IL1-β [310,311]. The receptor
activator of NFκB (RANK) is essential for the formation of osteoclasts from myeloid
precursors [312]. It has been shown that monocyte/ MΦs of synovial tissues develop into
osteoclasts in the presence of M-CSF and RANK [313]. In support of the role of MΦs in bone
erosion, it has been shown that the radiological progression of joint destruction correlates with
the degree of synovial MΦ infiltration [304]. Thus, MΦs can contribute to RA pathogenesis
by inducing inflammation at early time points and amplifying cartilage destruction and bone
erosion at later time points.
It is known that resting, immature DCs are present in healthy tissues, including the synovial
tissue [314] and it has been shown that DCs can prime autoimmune responses in local lymph
52
nodes [315] which could lead to development of autoantibodies and chronic inflammation.
Autoantibodies against citrullinated protein antigens are present in about 70% of RA cases
[316] and are associated with enhanced tissue injury [317] and worse outcome [318,319].
In support for a role of DCs in maintaining inflammation during RA, pDCs and mDCs are
reduced in the circulation of RA patients as compared to healthy controls while present in
synovial tissue in higher amount [320,321]. In addition, mDCs from RA patients were highly
responsive to endogenous and exogenous ligands and produced greater quantities of pro-
inflammatory cytokines than those from healthy controls [322,323]. Although MΦs and
mDCs are believed to be the most important cells for triggering inflammation and
cartilage/bone damages in this chronic disorder, recent findings also describe a role for pDCs.
In fact, when investigating the correlation between pDCs, rheumatoid factor and
autoantibodies, the number of pDCs in the synovium was positively correlated to serum levels
of rheumatoid factor and anti-citrullinated autoantibodies. This subset is known to play a
crucial role in the development of humoral immunity [215] and these observations suggest a
role for pDCs in triggering humoral autoimmunity in RA [321].
FMS-like tyrosine kinase 3 ligand (Flt-3L) has been shown to be strongly expressed in
synovial fluid of RA patients and induce erosive arthritis in mice [324]. Flt3-L is known to
induce differentiation of human CD11c+ DCs and increase their numbers in circulation [325]
thus providing a possible link between DCs and disease progression.
Finally, targeting of co-stimulatory molecules with blockers of the B7 family has proven
effective in reducing the clinical signs and symptoms of inflammation in RA patients [326].
This further emphasizes the role of co-stimulation by APC in initiating and perpetuating
inflammation during this autoimmune disorder.
In summary, various studies indicate APCs as crucial mediators of critical events in RA
pathogenesis.
53
THE STUDIES
GENERAL AIM
As discussed in previous chapters professional APCs are of crucial importance in the
generation of adaptive immune responses. Much recent research is focused on clarifying the
precise mechanisms of these processes and their role in infection and autoimmunity. We have
tried to create in vitro or ex vivo approaches that allowed us to investigate the behaviour of
these cell types in response to specific stimuli or under the influence of anti-inflammatory
drugs.
SPECIFIC AIMS
Paper I
The aim of this study was to investigate whether the effect of an anti-inflammatory
pharmaceutical compound might act trough modulating the properties of APCs of myeloid
lineage. We hypothesized that the anti-inflammatory effects of the drug, previously observed
in animal models, may be through suppression of APCs.
Paper II
The goal of this study was to characterize the behaviour of MoDCs in malaria by challenging
these cells with iRBCs or sHz in vitro and to analyse their phenotype, migratory behaviour
and cytokine secretion. We hypothesised that an impaired activation of DCs by the parasite or
products derived from infection may contribute to the poor development of immunity to
malaria.
Paper III
The aim of this study was to investigate if the relative protection of the Fulani to malaria
infection could be related to the properties of APC. We hypothesised that early innate
54
responses, for example via TLRs, and the prevalence or activation status of different APC
subsets of the Fulani children could be related to the enhanced protection against malaria seen
among the Fulani.
55
METHODS A brief description of the methods used in the different projects is outlined below. For more
elaborated descriptions please see the materials & methods of each paper.
Paper I
Monocytes were purified from peripheral blood mononuclear cells (PBMCs) obtained from
healthy blood donors using Ficoll separation. The monocytes were then cultured in the
presence of different growth factors in order to generate different kind of APCs. The APCs
where then allowed to mature after which their phenotype, cytokine secretion and
immunostimulatory ability were assessed. The anti-inflammatory drug rabeximod was added
at different stages of differentiation and both cells and supernatants obtained at different time
points were analysed. The immunophenotype of the cells was analysed by FACS upon
staining for the relevant surface markers. The levels of secreted cytokines were measured in
the supernatant using cytometric bead array (CBA). The phagocytic capacity of the APCs was
assessed by allowing cells to internalize FITC-labelled Dextran or yeast particles. The
immunostimulatory abilities of the cells were assessed in an allogeneic MLR.
Paper II
Monocytes were purified from PBMCs obtained 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 migratory properties were
assessed using a Boyden chamber, their culture supernatant was analysed for cytokine
secretion using CBA and their immunostimulatory abilities was assessed through an
allogeneic MLR.
56
Paper III
A total of 77 Malian children belonging to Dogon and Fulani ethnic groups were recruited
upon the informed consent of their parents. Giemsa stained slides where made and the
children were divided into groups of malaria infected or non-infected. Of the Dogon children
a total of 40 were enrolled of whom 20 were infected and 20 were non-infected. Of the Fulani
14 infected children were recruited and 23 non-infected making a total of 37 enrolled. Venous
blood samples were collected from all enrolled children and plasma was taken apart and
frozen for future analysis of cytokine and antibody levels in circulation. PBMCs were isolated
and stained for APC subtypes, activation markers and their phenotype was analysed by FACS.
In addition PBMCS were stimulated with TLR-specific ligands and the supernatants of the
stimulated cells were analysed for cytokine secretion using CBA and ELISA.
57
RESULTS AND DISCUSSION
Paper I
The levels of cytokines and growth factors present during the generation of APCs are what
decide what type of APC they will become. APCs can develop into functionally distinct
subsets promoting different kinds of responses during inflammation. In addition, the manner
of activation of the APC decides what type of response the APC promotes. Thus, the manner
of their activation will affect what type of effector cells the T cells become. These T cells are
crucial for generating potent plasma cells capable of secreting large amounts of antibodies.
Therefore, APCs also play an important role in triggering humoral responses.
RA is a chronic inflammatory disease characterized, among other things, by autoreactive
antibodies. There is increasing evidence that TLRs may be involved in Th lineage decisions
and the maintenance of inflammation in RA [327,328]. This, and the fact that blocking of co
stimulation has yielded promising results in patients non responsive to TNF treatment
emphasize the role of antigen presentation in the development and maintenance of RA [329].
We investigated the effects of a newly developed anti-inflammatory drug, Rabeximod, on
APCs in vitro to evaluate its mode of action. In order to investigate the effect of Rabeximod
on DCs we generated MoDCs. An inflammatory subset of MΦs resembling the M1-type
described in the literature was generated by employing allogeneic stimulation of monocytes.
This stimulation lead to the differentiation of monocytes into MΦs that secrete IFN-, TNF-α,
IL-6, and IL-13 and efficiently support antigen presentation to allogeneic T cells. These cells
will be referred to as allostimulated (Allo)-MΦs. Another subset resembling the M2-MΦ was
also generated using the growth factor M-CSF that will be referred to as anti-inflammatory
(AI)-MΦs.
As expected from the literature [29] the phenotypic analysis showed that the MΦs retained
their CD14 expression while the MoDCs expressed low CD14 but high CD1a. When
58
Rabeximod was added to monocytes at day 0 before addition of the differentiation stimuli the
MoDCs and Allo-MΦ were impaired in their differentiation whereas differentiation of AI-MΦ
was unaffected. This suggests that Rabeximod might impair the differentiation of APC with
pro- but not anti- inflammatory properties.
A key feature of APCs is the ability to take up antigen from the environment. The capacity of
each APC subset to take up antigen was assessed by allowing them to internalize FITC-
labelled Dextran particles. This assay revealed that MoDCs cultured in the presence of
Rabeximod where less efficient at taking up antigen whereas both MΦ subsets remained
unaffected.
The most potent initiators of T-cell proliferation were shown to be DCs already 30 years ago
[32]. The ability of each APC subset to induce proliferation of allogeneic T cells was assessed
in allogeneic MLRs. As expected, the MoDCs exhibited superior capacity to activate T cells
as compared to the MΦ subsets. Between the two MΦ subsets the Allo-MΦ were more potent
than the AI-MΦ at inducing T-cell proliferation. Both MoDCs and Allo-MΦs exhibited
impaired allostimulatory capacity upon exposure to Rabeximod, while AI-MΦs remained
unaffected by the drug. These findings again indicated that pro-inflammatory responses may
be selectively affected by the drug.
Rabeximod on its own however, does not seem to affect cytokine secretion by MΦ other than
decrease IL-6 production of MoDCs. It is possible that Rabeximod also affects IL-10
secretion but the variations in the results suggest that this effect may be donor dependent.
These results are in agreement with a previous study showing that Rabeximod did not affect
cytokine production in the absence of LPS in murine PBMCs [330]. Because of the crucial
importance of APCs early on in inflammation, our findings are in agreement with previous
work in a mouse model where the most potent effect of the drug was observed during the
early phases of inflammation [331].
59
Taken together, the results presented here show that Rabeximod impairs the generation and
immunostimulatory ability of inflammatory APCs but do not affect immunomodulatory APC
subsets. Therefore, this may have an alleviating effect on the disease course of RA and
deserves further evaluation.
Paper II
Immunity to malaria is only acquired after many years of repeated exposure and this
immunity is rapidly lost. The reasons underlying the slow acquisition of immunity to malaria
is not well understood. Potent and long lasting immune responses are dependent on adaptive
immunity and the generation of memory lymphocytes. For these cells to be generated DCs are
required to potently induce naïve T cells to become effector cells. Depending on the
conditions under which an APC activates a naive T cell it may differentiate into different
kinds of effector cells or even become anergic. Consequently, to brief a TCR-MHC
interaction or lack of co-stimulatory molecules and/or cytokine secretion may lead to anergic
T cells. Therefore the phenotype and functional properties of APCs during an infection are
crucial in defining the ensuing immune response.
The effects of Hz or iRBCs on monocytes and MoDCs have been investigated in various
studies as reviewed in [332,333]. It has been shown that MoDCs that have been exposed to
high doses of iRBCs do not mature properly in response to LPS [288] and a similar effect on
MoDCs has been seen by Hz [286].
This study was designed to analyze the effect of sHz and iRBCs on phenotypic and functional
properties of MoDCs with regards to maturation, migration and antigen presentation. We
choose a combination of TNF-α and prostaglandin E2 (hereafter referred to as TNF-α/PGE2)
as positive control because of the enhanced migratory capacity seen in MoDCs exposed to
these stimuli [334].
60
A clear dose response pattern was observed upon incubation with sHz with regards to
maturation markers CD80, CD83 and HLA-DR on MoDCs. However, only the surface
markers CD80 and CXCR4 were significantly increased. Although a slight up regulation of
CD83, HLA-DR and CCR7 was also observed. This up regulation of maturation markers
upon sHz stimulation was markedly lower than the one observed after stimulation with TNF-
α/PGE2. MoDCs were also analyzed for kinetics of the expression of surface markers on
MoDCs was assessed after 12, 36 and 60 hours of stimulation with sHz.
The results indicated that up regulation of CD83 by sHz was transient as compared to the up
regulation of the same marker induced by TNF-α/PGE2. The surface marker CD83 is
important for DC maturation and can influence DC-T cell interactions [35]. Therefore, these
findings may indicate that MoDCs exposed to sHz are only partially and transiently activated
and consequently become poor inducers of adaptive immune responses.
A similar phenotype with only marginal up regulation of maturation markers was found when
MoDCs were incubated with iRBCs including significantly increased CXCR4 expression.
These results are in line with other findings reporting of a partial maturation of DCs upon
incubation with malaria-derived products [289,335].
Because of the increased expression of the lymphoid CCRs observed upon stimulation with
sHz we assessed the migratory ability of these cells towards the ligands for CCR7 and
CXCR4, i.e 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 fact that partially mature MoDCs increased their migration towards chemotactic ligands,
important for homing to lymph nodes, in vitro may indicate that partially mature circulating
DCs are induced to travel to the lymph nodes during a natural P. falciparum infection.
However, MoDCs failed to properly up regulate CCR7 when they were exposed to sHz before
61
TNF-α/PGE2 was added to the culture. Expression of CCR7 not only induces chemotaxis
towards lymphoid organs but has also been reported to enhance maturation, survival and
migratory speed of DCs [336]. Therefore we hypothesize that malaria, or products derived
from infection, could negatively affect DC responses in the presence of other maturation
stimuli.
The cytokines released by MoDC in response to sHz and iRBCs were also analyzed. An
increased release of IL-6, IL-10, TNF-α but not IL-12 was seen when MoDCs were incubated
with 20 µg sHz. Similarly, incubation of DCs with iRBCs increased the secretion levels of the
same cytokines. In addition stimulation of MoDCs with iRBCs lead to secretion of IL-1β that
was not observed upon stimulation with sHz.
It has been shown that IL-10 secreted by monocytes/MΦs upon contact with Hz decreases the
capacities of PBMCs in culture to secrete IL-2, IL-12 and IFN-γ [337], thus leading to
impaired induction of potent Th1 type responses. Consequently, lack of potent Th1 induction
would significantly impair the induction of a potent adaptive immune response. Secretion of
pro-inflammatory cytokines is generally important in the initial phases of malaria infection.
However, when high levels of pro-inflammatory cytokines are maintained they have been
associated with inflammatory induced tissue injury [338]
Finally, we assessed the ability of MoDCs to activate allogeneic T cells in an MLR after
contact with sHz or iRBCs. We found that cells that had been in contact with sHz induced
proliferation to a degree similar to the cells incubated with medium alone or uninfected RBCs.
In contrast, proliferation induced by MoDCs stimulated with iRBCs was similar to that of
cells stimulated with TNF-α/PGE2. The fact that iRBC-stimulated MoDCs induced
considerably higher T-cell activation despite their immature phenotype indicates that some
other mode of action must be involved. It is possible that a hitherto unknown mechanism or
soluble factor related to malaria infection may influence these processes.
62
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. However, MoDCs exposed to
sHz did not induce proliferation of allogeneic T cells whereas those exposed to intact infected
erythrocytes did. This suggests different modes of action of these activation patterns.
The fact that iRBCs induce secretion of IL-1β also indicates that differential inflammatory
responses are triggered by iRBCs as compared to sHz. It is known, that the Nalp3
inflammasome is activated by sHZ suggesting that caspase1 may be activated by both
mechanisms. However the production of pro-IL-1β is also required which may or may not be
triggered by sHz.
These in vitro observations suggest that DC responses in malaria are potent enough for DCs to
make contact with naïve 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.
This experimental model does not appear to yield such potent MoDCs but seem to be more in
line with studies of murine malaria.
In fact, it has been shown that DCs migrate to CD4 T-cell areas of the spleen upon blood
stage infection [339]. However, it was also shown that the P. chaubadi chaubadi parasite
induced only marginal maturation of DC [335,340]. Moreover, although these DCs displayed
an activated phenotype the formation of T-cell clusters and induction of differentiation was
impaired [340].
It is therefore possible that the actions of malarial products on MoDCs, with induction of
partial maturation and increased IL-10 secretion which in turn lead to impaired differentiation
of host T cells. The poor activation of T cells may then lead to impaired adaptive immune
responses and therefore insufficient clearance of the parasites.
63
We therefore hypothesise that the lack of stable CD83 expression and no secretion of IL-12
are signs of hampered activation of APCs by the parasite or by products derived from
infection and that this may contribute to the poor development of immunity to malaria.
Paper III
It is well known that the Fulani show less clinical symptoms of infection than sympatric
ethnic groups although they are exposed to the same parasite pressure [296]. Different aspects
of the immune responses to malaria of the Fulani have been investigated in different African
countries [295,341]. The Fulani exhibit higher antibody titres, enlarged spleen rates and
higher levels of cytokines in circulation compared to sympatric ethnic groups.
This study was designed to investigate some aspects of the innate immune responses in
children belonging to the Fulani ethnic group in Mali. Children from a neighbouring village
belonging to a different ethnic group, the Dogon, were recruited to the study as a control
population. The aim of the study was to investigate the prevalence and activation status of
APCs as well as responses to specific TLR stimuli of Fulani and Dogon children and if this
could be involved in their different response to malaria infection.
In addition to the data presented in the paper included in this thesis we confirmed that Fulani
children have higher levels of malaria specific antibodies (Figure 6 and Figure 7) and higher
levels of cytokines (Figure 8) in circulation as compared to Dogon. The Fulani had higher
levels of total antimalarial IgG and IgM and of the IgG subclasses IgG1, IgG2 and IgG3 but
not IgG4.
The levels of cytokines involved in inflammation were measured in serum of children
belonging to the two ethnic groups. It was found that Fulani children had higher levels of
IFN-α, IFN-γ, IL-6, IL-8 and IL-12(p70) than the Dogon regardless of infectious status
(Figure 8). These findings are in line with previous findings of our group in the adult
populations of these and other villages [342-344].
64
Figure 6. Specific antibody responses to P. falciparum 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 7. Malaria specific IgG subclass responses to P. falciparum in Fulani and Dogon children.
Blood plasma samples from 77 children were analysed for antimalarial IgG1 (A), 2 (B), 3 (C) and 4 (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.
65
Figure 8. 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- α (D) and IFN-γ (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.
We then analyzed four subtypes of blood DCs as well as their activation status in the children
recruited to the study. A decreased expression of the activation marker HLA-DR was found
on all DC subsets of infected Dogon children as compared to their uninfected peers.
Conversely, the expression of HLA-DR was increased in Fulani children undergoing infection
on BDCA-2+ and BDCA-3
+ subsets when compared to their uninfected peers. The expression
of CD86 followed the same pattern i.e. decreased in the infected Dogon children while it is
increased or unaltered in infected Fulani children.
66
We then analysed the frequency of the different subsets in circulation. An increase of
circulating BDCA-2+ and BDCA-3
+ DCs was observed in the infected Dogon as compared to
the uninfected Dogon. In contrast, a decrease of circulating BDCA-2+ and BDCA-3
+ DCs was
observed in the infected Fulani as compared to their uninfected peers.
The activation status of the monocytic subsets in circulation was reduced in terms of
decreased expression of HLA-DR and CD86 in infected children regardless of ethnicity.
However, this decrease was more pronounced in the Dogon, consequently Fulani children
undergoing infection maintained higher levels of activation markers on classical monocytes
than the infected Dogon.
The expression of TLRs differs between the different APC subsets. In order to further
characterize the responses of these cells we stimulated their PBMCs with specific ligands for
TLR4, TLR7 and TLR9 that is LPS, Imiquimod and CpG respectively. We then analysed the
supernatants of the PBMCs stimulated with these ligands for cytokine secretion.
We found that secretion of practically all investigated cytokines was supressed in Dogon
children undergoing infection while Fulani children seemed unaffected. Strikingly, the
secretion of IFNγ was completely abolished in Dogon children undergoing infection. Not
surprisingly the ratio of IFNγ/IL-10 was severely decreased in Dogon undergoing infection
but unaffected in the Fulani. This pattern of supressed pro-inflammatory responses of the
Dogon were confirmed when the ratio of TNF-α/IL-10 was calculated. A decreased ratio of
TNF-α/IL-10 upon stimulation of PBMCs with ligands to TLR7, TLR9 but not TLR4 was
observed. This might indicate that the TLR4 signalling pathway is differently affected than
the TLR7 and TLR9 pathways.
In summary, the activation status was increased in DCs of Fulani undergoing malaria
infection but decreased in Dogon undergoing infection while the prevalence of these cells in
circulation was decreased in infected Fulani and increased in infected Dogon. We therefore
67
hypothesize that 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. The increased
activation status of circulating DC subsets in infected Fulani children may indicate that these
cells have migrated to lymphoid organs to initiate adaptive immunity and are therefore no
longer found in circulation. Conversely, in the Dogon ethnic group, malaria infection may
impair activation of circulating DCs thus possibly affecting downstream cellular and humoral
immune responses.
We observed that PBMCs of infected Dogon children are suppressed in their responses to
specific TLR ligands but the cells from the Fulani remain unaffected when undergoing
infection. This is in line with our findings that DCs of Fulani in circulation seem to become
more activated upon infection while DCs of the Dogon are suppressed.
Taken together, these findings suggest that there is a difference in the prevalence and
activation status of blood DCs and cytokine secretion between the Fulani and the Dogon in
response to malaria infection. Because of the crucial importance of APCs in initiation of
adaptive immune responses these differences may very well be important for the relative
protection against malaria seen in the Fulani as compared to the Dogon.
68
GENERAL CONCLUSIONS It has become evident that APCs play a crucial part in the early stages of an immune response.
It is also increasingly evident that different manners of APC activation can have very different
consequences, and that what we commonly regard as phenotypic activation may not lead to
initiation of adaptive immunity. Microbial stimuli have been shown to induce potent
responses in DCs including secretion of IL-12 and initiation of adaptive immunity [173,174].
In mice it has been shown that CD8α- DCs express TLR7 but CD8α
+ DCs do not. Upon
injection of TLR7 ligand both subsets display a mature phenotype but only CD8α- DCs,
which respond directly to TLR7, secrete IL-12 [345]. In line with these studies it has been
shown that DCs secrete TNF-α and IL-6 in response to microbial antigens but only the TLR4-
mediated responses lead to IL-12 production [346]. These findings suggest that TLR ligation
is necessary and sufficient to mount an effective immune response.
Taking these data into consideration, Joffre et al. suggested that all DCs respond to
inflammatory stimuli by migrating to the lymph nodes but only those cells that have sensed
microbes directly induce effector T cells. The bystander DCs that have not directly sensed the
pathogen become activated but would instead present tissue antigens from the infectious foci
and “rescue” these antigens from the immune response thereby evading excessive and
detrimental self-responses [347]. These bystander cells may therefore display an activated
phenotype but be unable to induce efficient T-cell responses. Supporting this hypothesis is the
fact that un-activated DCs continuously present self-derived antigens from tissues or apoptotic
cells without leading to initiation of immunity [241].
We suggest that rendering APCs to act as bystander cells, i.e. not efficient in inducing proper
T-cell responses may be an effective way of subverting immune responses by pathogens such
as P. Falciparum. Moreover, inappropriate signalling while recognizing and presenting self-
69
antigens may activate APCs and lead to prolonged and detrimental immune responses, thus
leading to autoimmunity.
Our first study revealed that a drug that has shown anti-inflammatory effects in a murine
model of autoimmune arthritis acts efficiently by inhibiting inflammatory subsets of human
APCs in vitro. This could be an effective mechanism to break the maintenance of
inflammation during RA and contribute to decelerate disease progress. It also suggests a
central role for APCs in determining detrimental inflammation in autoimmunity.
Our second study revealed that products of malaria infection, such as Hz and iRBCs do
induce phenotypic activation and migration of MoDCs. We also observed that iRBCs induce
T-cell proliferation but none of these malaria-stimulated DCs produce IL-12. This might
indicate that malaria infection can somehow induce APCs to act as bystander APCs,
ineffective at activating T cells, even though they have had previous contact with the parasite.
In the third study, we compared the Dogon population of Mali to the Fulani, an ethnic group
that displays a relatively better protection against malaria. We observed that TLR responses of
Dogon children are severely suppressed and their APCs express decreased levels of activation
markers when undergoing malaria infection, whereas the Fulani exhibit unaltered TLR
responses and increased APC activation while undergoing infection. These differences in
TLR responses and APC behaviour may indicate an important role for APCs in malarial
immunity.
In summary, these data support the view that when APCs are improperly activated the ensuing
adaptive responses may become inefficient or excessive leading to pathology (Figure 9). This
suggests a central role for APCs in contributing to autoimmunity and dysregulated responses
to pathogens. Our observations may suggest that the malaria parasite has found a way to limit
APC responses to that of bystander cells and therefore insufficient long-term immunity is
initiated upon contact with malaria derived products. In addition, the Fulani ethnic group may
70
somehow have overcome this suppression and their APCs respond more potently to malaria
infection. This could lead to increased levels of IFN secretion and augmented adaptive
responses leading to higher titres of protective antibodies.
Figure 9. Consequences of immune activation under different circumstances. Different challenges to the immune system induce different responses in APCs. The responses elicited in APCs
lead to distinct T-helper responses according to the different situations. We suggest that malaria has a distinct
way of manipulating these responses so that efficient T-cell responses are dampened. Moreover, inappropriate
triggering of APC responses may play an important role in the induction of autoimmunity.
71
FUTURE WORK Our studies, together with previous findings, indicate that APCs are important players in the
orchestration of immune responses both in autoimmune disease and in malaria infection.
Much recent progress has been made in elucidating the mechanisms through which the
malaria parasite activates innate immune receptors, in particular TLR9. Because of the
functional differences observed in TLR responses between Dogon and Fulani, it would be
interesting to investigate known mutations in various TLR genes in order to unravel whether
these differences are genetically determined.
In addition, there are many details about the mechanisms surrounding APC responses to
malarial products that would be interesting topics for further research. For instance, detailed
information about the effects of different strains of parasites on different APC subsets and on
APC precursors. Since increased levels of BDCA-3+ DCs have been associated with severe
disease and an increase of that subset in circulation is seen in the Dogon that are more
susceptible to disease. More detailed investigations into the role of BDCA-3+ DCs in malaria
as compared to other circulating subsets of DCs is needed. More detailed knowledge is also
needed of the mechanisms behind the relatively better protection to malaria seen among the
Fulani, in particular the role of APCs. This knowledge would be of outmost importance for
understanding how a potent response to malaria is achieved and may also be helpful in
vaccine development.
Finally, and perhaps most important, is the underlying question of how different activation
patterns of APCs lead to different effector responses. In general, a better understanding of
APC activation and downstream responses would be invaluable for coming up with efficient
strategies to combat infections and immune disorders.
72
ACKNOWLEDGEMENTS This work would not have been possible to complete were it not for the invaluable support
and friendship of many wonderful individuals that I have crossed paths with during the years.
Therefore, I would like to thank the whole 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.
The most important person for the realization of my PhD studies has of course been my
supervisor Marita Troye-Blomberg. I thank you first for giving me the opportunity to work
with you but also for always taking your time to help me in every way and for sharing your
vast experience.
The next key person to the work behind this thesis from whom I learnt most practical aspects
and with whom I spent lots of time during the first years of my studies is my co supervisor
Stefania Varani. I thank you for your patience while working with my stubborn self through
all these years.
I would also like to thank all the other seniors at our or other departments for sharing their
knowledge and experience. In particular Carmen Fernandez, muchísimas gracias por todo tipo
de apoyo en la ciencia y en lo demas, I am also grateful to Klavs Berzins, Eva Sverremark-
Ekström and Eva Severinson and Ann-Kristin Östlund Farrants for great company and many
interesting discussions during the years.
My thanks also goes to the backbone of the department, Gelana Yadeta, Margareta ”Maggan”
Hagstedt, Ann Sjölund och Anna-Leena Jarva tack för all hjälp och för att ni stått ut med mina
galenskaper.
I next want to thank my friends, fellow malariologists and future doctors Charles Arama for
all the long talks, un monde meilleur est nécessaire et possible! Stéphanie Boström om du
hoppar av vetenskapen kan du ju alltid bli bagare ;) with both of whom I have spent good and
bad times all over the planet, I am really glad I have had the pleasure to work closely with
73
these two wonderful personalities during these years. And also to the latest addition in the
malaria team Ioana “thevampire” Bujila with whom I have also had the pleasure to work
closely together and shared many enjoyable experiences. A special thanks also to Lisa
Israelsson whose good spirits and pleasurable company I had the privilege to enjoy during the
first years.
Then there are those that I haven’t had the chance to work so close to but with whom I have
shared many happy and less happy moments Ebba “toomanynicknamestoremember”
Sohlberg, Maria Johansson, för kryssen, kaffet, kakorna och alla andra äventyr vi delat.
Then there is the long list of past and present students at the department special thanks also to
Petra Amoudruz, Halima Balogun, Jubayer Rahman, Salah Farouk and Amre Nasr for their
outstanding personalities and kind manners. I am also glad to have gotten to know Nancy
Awah, Olga Chuquimia Flores, Manijeh Vafa, Shanie Saghafian Hedengren, Natalija
Gerasimick, Yeneneh Haileselassie, Ylva Sjögren Bolin, Ulrika Homlund, Nora Bachmayer,
Yvonne Sjölund, Nnaemeka Iriemenam, and John Arko-Mensah that have contributed to
many good times in the department.
Then there are the people I have come in contact to for one reason or another that I have had
the privilege to work with or at least get to know like Anna Tjärnlund, Yohannes Assefaw-
Redda, Valentina Mangano, Sabina Dahlström, Samad Ibitokou and Maiga Boubacar.
My fellow malariologists from the Swedish Malaria Network including Pedro and Isabel of
the Portuguese resistance group, the “Chen girls” Sandra Nilsson and Kim Brolin, Anna
Färnert with students Ann Liljander and Klara Lundbom, finally the later additions of Maja
Malmberg and Mia Palmqvist.
I would also like to extend my gratitude to co-authors and all the people senior or students
who I have come in contact with during these years.
74
Last but not least are the people everywhere outside of the scientific world that I am fortunate
enough to share my life with. You are all too many to mention here but you have been every
bit as important as the people mentioned above.
Para toda mi extendida familia, pasada, presente y porvenir, de sangre o recuparada por los
caminos los llevare por siempre conmigo.
Till mina L(o)uisar, Mamma, Marre och Erre jag hade inte kunnat välja en bättre familj om
jag hade kunnat, har er att tacka för allt jag är och önskar er all lycka i världen!
75
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