Doctoral thesis from the Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden

Plasmodium falciparum-mediated modulation of innate immune cells: responses and regulation

Ioana Bujila

Stockholm 2016

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Cover illustration: Tyler Lieberthal, Department of Bioengineering, Imperial College,

London, United Kingdom.

The previously published paper is reproduced with permission of John Wiley and Sons.

© Ioana Bujila, Stockholm 2016

ISBN: 978-91-7649-296-3

Printed in Sweden by Holmbergs, Malmö 2016

Department of Molecular Biosciences, The Wenner-Gren Institute

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POPULÄRVETENSKAPLIG SAMMANFATTNING  

Ur ett globalt perspektiv är malaria en av våra vanligaste infektionssjukdomar. Infektionen

orsakas av parasiter ur släktet Plasmodium och sprids med hjälp av myggor. Fem olika

Plasmodium parasiter orsakar malaria hos människor och malaria orsakad av Plasmodium

falciparum är den dödligaste varianten. Det finns fortfarande inget välfungerande vaccin och

en av anledningarna kan vara att vi troligen inte har tillräckliga kunskaper om hur vårt

immunförsvar påverkas av malaria.

Denna avhandling handlar om vårt tidiga immunförsvar, främst två viktiga immunceller;

dendritiska celler och monocyter, och hur de påverkas av Plasmodium falciparum infektion.

En av de viktigaste funktionerna dendritiska celler har är att systematiskt avsöka individen för

invaderande bakterier, virus och parasiter och sen presentera ”sitt fynd” för andra

immunceller och på så sätt aktivera det förvärvade immunförsvaret. Denna process kallas för

antigenpresentation och för att genomföra den måste dendritiska celler genomgå en

mognadsprocess. I studie I visar vi att hemozoin, en biprodukt som bildas när Plasmodium

parasiterna infekterar och uppehåller sig i våra röda blodkroppar, i ett tidigt stadium kan

hämma mognadsprocessen hos dendritiska celler. Hemozoin har flera angreppspunkter, bland

annat så kommer inte viktiga receptorer upp på cellytan som behövs för att dendritiska celler

ska kunna migrera och presentera ”sitt fynd” för andra immunceller. Vidare så kommer inte

heller viktiga mognadsmolekyler upp på cellytan vars funktion är att delta i

antigenpresentationen. Studie II är en uppföljning på studie I. I denna studie undersöker vi

hur hemozoins hämmande effekt på mognadsprocessen av dendritiska celler kan regleras på

gennivå. När hemozoin ”äts upp” av dendritiska celler så sätts flera olika signalsystem igång

som till slut leder till produktion av proteiner nödvändiga för immunförsvaret. En viktig del i

denna signalprocess är transkriptionsfaktorer vars uppgift är att binda till specifika DNA-

sekvenser i cellkärnan. Transkriptionsfaktorer är nödvändiga för att gener ska kunna uttryckas

och slutligen ge upphov till proteiner. För att detta ska ske måste DNA:t vara tillgängligt för

inbindning och detta sköts av kromatinremodulerare som kan öppna och stänga kromatin. Vi

visar att hemozoin påverkar dendritiska celler så att de inte kan rekrytera nödvändiga

transkriptionsfaktorer och kromatinremodulerare till DNA-sekvenser av gener som är viktiga i

mognadsprocessen av dendritiska celler. På så sätt bildas troligtvis inte de proteiner som

behövs för att genomföra antigenpresentationen. I studie III undersöker vi svaret hos

monocyter med hjälp av genomsekvensering av Fulani och Mossi, två olika etniska grupper i

Burkina Faso. Fulani har i olika studier visat sig ha ett relativt bättre immunologiskt svar mot

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Plasmodium falciparum infektion i jämförelse med Mossi. Detta trots att de lever i samma

geografiska områden och har samma exponering till infekterade myggor. Vi visar att när

Fulani blir infekterade så uppreglerar de många viktiga gener. Det är gener som bland annat

deltar i det immunologiska svaret och dess olika signalvägar, men även gener som reglerar

processen som slutligen leder till produktion av proteiner. Fynden från denna studie ger oss

nya kunskaper om vilka molekyler, signalvägar och regulatoriska mekanismer som kan vara

viktiga för ett relativt bättre svar mot malaria hos människor.

Sammantaget visar denna avhandling att det tidiga immunförsvaret och dess olika

celltyper, dendritiska celler och monocyter, påverkas av infektioner med Plasmodium

falciparum. Dendritiska celler är hämmade i en av sina viktigaste funktioner till följd av

interaktioner med parasitprodukter och denna påverkan sker redan på gennivå. Vidare visar vi

i monocyter vilka signalvägar och regulatoriska mekanismer som kan vara viktiga för ett

relativt bättre svar mot det som är en av våra vanligaste och dödligaste parasitsjukdomar.

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LIST OF PAPERS

This thesis is based on the original papers listed below, which will be referred to by their

roman numerals in the text:

I. Bujila I, Schwarzer E, Skorokhod O, Weidner J.M., Troye-Blomberg M and Östlund

Farrants A.K.

Malaria-derived hemozoin exerts early modulatory effects on the phenotype and

maturation of human dendritic cells.

Cell Microbiol. 2015 Sep 8

II. Bujila I, Rolicka A, Schwarzer E, Skorokhod O, Troye-Blomberg M and Östlund

Farrants A.K.

Exposure to Plasmodium falciparum-derived hemozoin leads to impairment of

transcriptional activation upon dendritic cell maturation

Manuscript in preparation

III. Bujila I, Chérif M*, Sanou S.G*, Vafa M, O’Connell M.A, Ouédraogo N.I,

Lennartsson A, Troye-Blomberg M and Östlund Farrants A.K.

Transcriptome and DNA methylome analysis of two sympatric ethnic groups with

differential susceptibility to Plasmodium falciparum infection living in Burkina Faso. * Shared second authorship

Manuscript in preparation

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List of papers (not included in the thesis)

The following original articles are included in the authors’ works but do not concern the main

topic of this thesis.

IV. Topalis P, Mitraka E, Bujila I, Deligianni E, Dialynas E, Siden-Kiamos I, Troye-

Blomberg M and Louis C.

IDOMAL: an ontology for malaria.

Malar J. 2010 Aug;10;9:230

V. Kamugisha E, Bujila I, Lahdo M, Pello-Esso S, Minde M, Kongola G, Naiwumbwe

H, Kiwuwa S, Kaddumukasa M, Kironde F and Swedberg G.

Large differences in prevalence of Pfcrt and Pfmdr1 mutations between Mwanza,

Tanzania and Iganga, Uganda – a reflection of differences in policies regarding

withdrawal of chloroquine?

Acta Trop. 2012 Feb;121(2):148-51

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TABLE OF CONTENTS POPULÄRVETENSKAPLIG SAMMANFATTNING  ............................................................................  3  

LIST OF PAPERS  ...........................................................................................................................................................  5  

ABBREVIATIONS  .........................................................................................................................................................  9  

INTRODUCTION  ........................................................................................................................................................  10  

THE HUMAN IMMUNE SYSTEM – A BRIEF OVERVIEW  ....................................................................  10  

PATTERN RECOGNITION  .....................................................................................................................................  12  

ANTIGEN-PRESENTING CELLS AND THE MHC COMPLEX  .............................................................  13  

Monocytes ...................................................................................................................................... 14  

Dendritic cells ................................................................................................................................. 14  

Dendritic cells: from immature to mature  ...........................................................................................................  15  

MALARIA  .......................................................................................................................................................................  16  

The life cycle of Plasmodium falciparum ...................................................................................... 16  

The immune response against Plasmodium falciparum ................................................................. 18  

Hemozoin, a parasite-derived molecule ......................................................................................... 18  

Hemozoin-mediated effects on the response of monocytes and dendritic cells  ...........................................  19  

DIFFERENTIAL SUSCEPTIBILITY TO MALARIA IN ETNIC GROUPS IN WEST AFRICA  .  22  

EPIGENETICS AND TRANSCRIPTIONAL REGULATION – A BRIEF OVERVIEW  .................  23  

POST-TRANSLATIONAL MODIFICATIONS OF HISTONES  ...............................................................  24  

Enzymes and chromatin remodeling complexes affecting chromatin structure ............................. 25  

DNA METHYLATION  ..............................................................................................................................................  26  

EPIGENETIC MECHANISMS AND THE IMMUNE SYSTEM  ...............................................................  26  

PRESENT STUDY  .......................................................................................................................................................  29  

OBJECTIVES  .................................................................................................................................................................  29  

Specific aims .................................................................................................................................. 29  

METHODS  ......................................................................................................................................................................  30  

Experimental procedure for Paper I and II ..................................................................................... 30  

Experimental procedure for Paper III ............................................................................................. 30  

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RESULTS AND DISCUSSION  ...............................................................................................................................  31  

Paper I ............................................................................................................................................. 31  

Paper II ........................................................................................................................................... 33  

Paper III .......................................................................................................................................... 36  

GENERAL CONCLUSIONS  ................................................................................................................................  41  

FUTURE PERSPECTIVES  ...................................................................................................................................  42  

ACKNOWLEDGMENTS  .......................................................................................................................................  44  

REFERENCES  ...............................................................................................................................................................  46  

     

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ABBREVIATIONS APC Antigen-presenting cell BRG1 Brahma-related gene-1 CCR C-C chemokine receptor ChIP Chromatin immunoprecipitation DC Dendritic cells G6PD Glucose-6-phosphate dehydrogenase GM-CSF Granulocyte-macrophage colony-stimulating factor HAT Histone acetyltransferase Hb Hemoglobin HDAC Histone deacetylase HZ Hemozoin Igs Immunoglobulins IFN Interferon IL Interleukin IRF Interferon regulatory factor LPS Lipopolysaccharide MAPK Mitogen-activated protein kinase MCP-1 Monocyte-chemotactic protein-1 mDC Myeloid dendritic cells MHC Major histocompatibility complex MyD88 Myeloid differentiation primary response gene 88 NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells nHZ Natural hemozoin NK Natural killer NO Nitric oxide PAMP Pathogen-associated molecular pattern PBMC Peripheral blood mononuclear cell pDC Plasmacytoid dendritic cells PRR Pattern recognition receptor RNA-seq RNA-sequencing SNP Single nucleotide polymorphism Tc T cytotoxic TGF Transforming growth factor Th T helper TLR Toll-like receptor TNF Tumor necrosis factor Treg T regulatory TRIF TIR domain-containing adaptor protein inducing interferon-β TSS Transcription start site

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INTRODUCTION  

THE HUMAN IMMUNE SYSTEM – A BRIEF OVERVIEW

The mammalian immune system is very intricate and consists of multiple biological structures

and processes. The immune system must be able to detect a variety of agents and to

distinguish self from non-self as well as pathogenic organisms from non-pathogenic

organisms. It also has a homeostatic function, which includes elimination of damaged and/or

dead cells.

There are several barriers that together with immune cells and organs protect an organism

from invading pathogens. They consist of the skin and mucosa, antibacterial molecules, such

as defensins, and the commensal bacterial flora. Various immune cells, known as leukocytes,

can be found circulating throughout the body in the blood stream or lymphatic system and in

specialized immunological compartments such as the spleen, thymus and lymph nodes.

Leukocytes are generated through the process of hematopoiesis that takes place in the bone

marrow. Cells are in need of communication in order to coordinate immunological responses

and this is achieved with the help of soluble proteins referred to as cytokines and chemokines

(Table 1).

The immune system is traditionally divided into two compartments, the innate and the

adaptive immune system, which differ in their initiation speed, their specificity and their

ability to “remember” infectious events referred to as immunological memory.

The innate immune system is found in nearly all life forms. It responds through preexisting

molecules including complement factors and acute phase proteins and phagocytic cells, such

as neutrophils and macrophages. Recognition is mainly mediated through germ-line encoded

pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). Other PPRs include

the C-type lectin receptors, RIG-I-like receptors and NOD-like receptors (1). The innate

leukocytes include phagocytes, such as macrophages, monocytes, neutrophils and dendritic

cells (DC). Additional leukocytes are natural killer (NK) cells, mast cells, eosinophils and

basophils. The innate immune system may be sufficient to clear invading pathogens. If not, it

can limit the infection until the adaptive immune response has expanded enough to deal with

the threat.

The adaptive immune system is only found in jawed vertebrates and the effector cells are

B- and T-cells. These cells have antigen-specific receptors that are not germ-line encoded but

instead de novo generated leading to the high specificity of the adaptive immune system. The

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activation of both B- and T-cells involves recognition of antigens, which in regards to B-cells

can be direct recognition of the antigen, while in regards to T-cells, the antigen must be

processed and presented by antigen-presenting cells (APCs) in association with major

histocompatibility complexes (MHCs).

One major role for B-cells is the production of immunoglobulins (Igs). Igs can be

expressed on the B-cell surface as part of the B-cell receptor as well as secreted in a soluble

form, which is referred to as antibodies. Antibodies recognize antigens and participate in

neutralization of pathogens, activation of the complement cascade and enhancement of

phagocytosis. There are five different classes or isotypes of antibodies, IgA, IgD, IgG, IgM

and IgE. Activation of B-cells leads to the generation of antigen-specific antibody-secreting

plasma cells or long-lived memory cells. B-cells also produce a wide variety of cytokines in

order to provide “help” to other cells in the immune system.

Activated T-cells can directly kill other cells through the release of cytotoxic granula or by

induction of apoptosis (programed cell death) and these are known as CD8+ T-cells or T

cytotoxic (Tc) cells. CD4+ T-cells or T helper (Th) cells provide “help” to immune cells

through the production of various cytokines and chemokines. The differentiation of CD4+ T-

cells into various subsets is of pivotal importance for the host defense and for the regulation

of the immune response. Th1 cells are important in cell-mediated immunity; they produce

cytokines, such as interleukin (IL)-2, tumor necrosis factor (TNF)-α and interferon (IFN)-γ

and support the elimination of intracellular pathogens. Th2 cells are important in the humoral

immune response; they produce IL-4, IL-5 and IL-13 and support the elimination of parasites,

such as helminthes (2). T regulatory (Treg) cells are important in dampening immune

responses by suppressing “excessive” and thus potentially harmful immunological responses

and in maintaining unresponsiveness to self-antigens. They can exert their dampening

function through the production of IL-10 and transforming growth factor (TGF)-β (3). Yet

another type of T-cells are the unconventional γδ T-cells that bridge the innate and the

adaptive immune system. They display a limited antigen receptor repertoire, but provide

adaptive T-cell effector functions preceding the activation of the conventional T-cell subsets

(4). The adaptive immune response leads to the production of long-lived memory cells, which

have the ability to immediately “attack” the pathogen upon re-encounter. The pool of memory

cells differs among individuals depending on which infections a person has encountered

during his/her lifetime.

The innate and the adaptive immune system are intertwined, where the quality of the innate

immune response will regulate the following adaptive immune response (5).

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This thesis will focus on the innate immune response and different functions of DC and

monocytes in general and more specifically in response to Plasmodium falciparum (P.

falciparum) infection and parasite-derived molecules.

Cytokines/chemokines Primarily producing cell(s) Main function(s) IL-1β DC, activated monocytes and macrophages Pro-inflammatory, part of the acute phase

response (6).

IL-2 Mainly T-cells T-cell memory and supporting dev. of Treg cells (7).

IL-4 Granulocytes, T-cell subsets Th2 responses (8).

IL-10 DC, monocytes, macrophages, NK-, T-, and B-cells

Regulatory/anti-inflammatory properties. Negative feedback molecule (9).

IL-12 DC, monocytes, macrophages and B-cells Induce IFN-γ production in NK- and T-cells, induce diff. of naïve T-cells into Th1 cells (10).

MCP-1/CCL2 Monocytes, T-cells, fibroblasts, endothelial cells

Triggers chemotaxis, transendothelial migration to inflammatory sites (11).

TNF-α See IL-1β, NK-, T-, and B-cells in addition Pro-inflammatory, part of the acute phase response (12).

IFN-γ NK- and T-cells Control of viral infection, intracellular bacteria and tumor malignancies. Th1 type of immune response, APC activator (13).

IFN-α pDC Anti-viral defense (14).

TGF-β Various cell types Multifunctional controlling proliferation, differentiation in many cell types, regulatory functions (15).

GM-CSF Macrophages, T-cells, endothelial cells and fibroblasts

Hematopoietic growth factor (16).

Table 1. Cytokines/chemokines covered in this thesis: name, primarily producing cell(s) and main function(s).

PATTERN RECOGNITION

Recognition is mainly mediated through PRRs, which are able to distinguish between

different classes of pathogens through recognition of various structures and molecules, such

as components of bacterial cell walls or viral nucleic acids (17,18). PRRs are found in various

cellular compartments, such as plasma membranes, endosomes and lysosomes, thus enabling

effective recognition of both extracellular and intracellular threats.

The TLRs are the best characterized and to date, ten different TLRs have been described in

humans. Some are found on the cell surface (TLR1, TLR2, TLR4, TLR5 and TLR6) and

some in intracellular compartments (TLR3, TLR7, TLR8, TLR9). TLRs respond to pathogen

associated molecular patterns (PAMPs) or danger associated molecular patterns (DAMPs) as

first described by P. Matzinger (19). PAMPs recognized by cell surface TLRs include

lipopolysaccharide (LPS) from gram-negative bacteria, flagellin and fungal zymosan, while

intracellular TLRs recognize dsRNA, ssRNA and CpG-rich DNA. All TLRs except the

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intracellular TLR3, signal via the adaptor protein myeloid differentiation primary response

gene 88 (MyD88), whereas TLR3 signals via TIR domain-containing adaptor protein

inducing interferon-β (TRIF). TLR4 is capable of signaling through both MyD88 and TRIF.

Downstream events include phosphorylation by mitogen-activated protein kinases (MAPKs)

(20)   and activation of transcription factors, such as activator protein-1 (AP-1) and nuclear

factor kappa-light-chain-enhancer of activated B cells (NF-κB) leading to cytokine- and

chemokine production (21,22). Other transcription factors engaged downstream of TLR-

signaling includes the interferon regulatory factors (IRFs) whose activation leads to the

production of type I IFNs (23).

Another PRR is the NLRP3-inflammasome, which is part of the NOD-like receptor family.

Inflammasomes are multi-protein complexes, where the effector protein caspase-1 is

responsible for the cleavage of pro-IL-1β into its active form (24).

ANTIGEN-PRESENTING CELLS AND THE MHC COMPLEX

 

Antigen-presentation can be carried out by professional APCs expressing MHC class II, such

as monocytes, macrophages, DC and B-cells. Monocytes can be precursors of certain subsets

of macrophages and DC. Macrophages are found in all tissues throughout the body and show

extensive functional diversity. Beside an important role as professional APCs, macrophages

participates in homeostasis and tissue repair (25). Antigen-presentation can also be carried out

by non-professional APCs expressing MHC class I, which includes all nucleated cells. Non-

professional APCs present antigens/peptides of cytosolic and nuclear origin on the cell

surface in association with MHC class I molecules to naïve CD8+ T-cells. Professional APCs

present antigen/peptides that are derived from proteins degraded in the endocytic pathway in

association with MHC class II molecules on the cell surface to CD4+ T-cells. In addition,

some APCs have the ability of cross-presentation in which exogenous antigens/peptides can

be presented in association with MHC class I molecules. Cross-presentation is thought to be

important in the immune response against viruses (26).

Monocytes and DC constitute the topic of this thesis and will therefore be further discussed

in the following sections.

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Monocytes

 

Monocytes originate from myeloid progenitors and after differentiation in the bone marrow

they enter the blood circulation, where they represent approximately 10% of the leukocyte

population. They are recruited to sites of inflammation and/or infection where differentiation

into DC and macrophages contribute to immune responses and tissue repair (27).

The circulating monocyte population is heterogeneous and three major subsets have been

described so far. The characterization is based on their expression of CD14 (co-receptor for

LPS) and CD16 (also known as Fc-γ receptor III). They are referred to as classical monocytes

(CD14++CD16-), intermediate monocytes (CD14hiCD16+) and non-classical monocytes

(CD14dimCD16++) (28). There are distinct functions attributed to the different subsets, where

classical and intermediate monocytes are involved in inflammation and leukocyte recruitment

while non-classical monocytes are involved in sensing tissue damage (29). Gene expression

profiling has revealed that classical monocytes are highly versatile and able to respond to a

large variety of self and microbial ligands. Upon LPS stimulation, monocytes produce high

levels of for example the chemokine monocyte-chemotactic protein-1 (MCP-1) (30).

Dendritic cells

 

DC are sentinels of the immune system in which they migrate to sites of

inflammation/infection, capture and process antigens followed by migration to secondary

lymphoid tissues where they present “their finding(s)” to naïve T-cells. They can respond to a

variety of stimuli leading to differentiation and maturation as well as having a high capacity to

produce various cytokines. DC are also capable of maintaining tissue homeostasis (31). Ralph

Steinman and Zanvil Cohn first described DC in the 1970s, a finding that in 2011 was

awarded with the Nobel Prize. Using mixed leukocyte reaction (MLR), Steinman et al could

show that DC were highly effective in stimulating allogenic T-cells (32).

Myeloid DC (mDC) and plasmacytoid DC (pDC) have been described in human peripheral

blood (33). mDC express myeloid markers and are capable of producing high levels of IL-12,

a cytokine necessary for potent T-cell priming. pDC do not express myeloid markers and are

able to secrete high levels of type I IFNs, such as IFN-α, in response to viral infections (34).

Different DC subsets have different expression of various PRRs, such as TLRs. In humans,

mDC express TLR1-8 and TLR10 while pDC express TLR1, TLR6-7 and TLR9-10 (33,35).

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The observation that monocytes could acquire DC-like features in vitro if cultured with IL-4

and granulocyte-macrophage colony-stimulating factor (GM-CSF) was an important step in

DC research (36). Under inflammatory conditions in vivo, monocytes have the ability to

differentiate into DC (37,38). DC are, together with macrophages and B-cells, professional

APCs and one cannot state enough the importance of DC in antigen-presentation considering

the fact that they exhibit 10-100 fold higher levels of MHC class II compared to B-cells (39).

Furthermore, DC are capable of cross-presentation which enables them to present exogenous

antigens on MHC class I molecules thus stimulating CD8+ T-cells (40).

Dendritic cells: from immature to mature

 

In the periphery, DC are immature and very efficient in capturing antigens, which is achieved

through phagocytosis or receptor-mediated endocytosis. Antigen-uptake induces phenotypic

and functional changes in which DC transform from immature to mature (Figure 1). The

process of maturation consists of a series of events. DC go through morphological changes,

such as the loss of adhesive structures, cytoskeleton reorganization and higher cellular

motility as reviewed in Trombetta et al (41). Further, DC decrease their endocytic capacity

and redistribute MHC molecules from intracellular compartments to the cell surface during

the maturation process. Immature DC can quickly internalize MHC class II molecules while

maturation and/or inflammatory stimuli can keep MHC class II molecules stably expressed on

the cell surface thus enabling T-cell contact (42). The maturation process also includes a

switch in the expression of certain C-C chemokine receptors (CCRs) and the secretion of

various chemokines. A classical example is the switch from CCR5 expression in immature

DC to CCR7 expression in mature DC, thus enabling migration to secondary lymphoid tissues

(42,43). Furthermore, co-stimulatory molecules, such as CD80 and CD86, are up-regulated

during the maturation process (44). An additional feature of mature DC is the expression of

CD83, a transmembrane molecule part of the immunoglobulin superfamily (45). The function

of CD83 remains enigmatic, but using RNA interference to down-regulate CD83 expression

in human monocyte-derived DC rendered the cells less able to induce allogenic T-cell

proliferation (46).

Cytokine production by DC contributes to the commitment of naïve CD4+ T-cells into

more specialized T-cell subsets. One example is the release of IL-12, which influences naïve

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T-cells to take the Th1-route (47,48). Another example is the production of IL-10 and TGF-β,

which induces generation of Treg cells (49).

Figure 1. Characteristics of immature and mature DC. Illustration by Tyler Lieberthal.

MALARIA

Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium. Five

species are known to infect humans; P. falciparum, P. vivax, P. ovale, P. malariae and P.

knowlesi, where P. knowlesi was previously only known to infect macaques (50,51). P.

falciparum predominates in Africa and is responsible for the majority of morbidity and

mortality associated with malaria (52) while P. vivax is more widespread, but less deadly. P.

ovale and P. malariae are less frequently encountered.

The life cycle of Plasmodium falciparum

The parasite has several developmental stages in its two-host life cycle that during

development requires both Anopheles mosquito vectors and human hosts (Figure 2). P.

falciparum goes through ten morphological transitions in five different host tissues (53). In

humans, the life cycle includes the pre-erythrocytic stage, the erythrocytic stage and the

gametocyte stage. The pre-erythrocytic stage, which is asymptomatic, begins with the bite of

an infected female Anopheles mosquito. When the infected mosquito takes a human blood

meal, it inoculates sporozoites into the skin and the blood stream. The inoculum is generally

low and only a small percentage of sporozoites reach the liver in order to infect hepatocytes

and commence the asexual replication cycle. Some sporozoites enter the lymphatic circulation

instead of the blood stream and travel to draining lymph nodes (54). After reaching the liver

parenchyma, sporozoites pass through several hepatocytes before infecting one hepatocyte

Immature DC

High intracellular levels of MHC class II High endocytosis

Low CD86 and CD83 expression CCR5 expression

Mature DC

High surface levels of MHC class II Low endocytosis

High CD86 and CD83 expression CCR7 expression

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and form merosomes. The merosomes rupture after 5-16 days depending on parasite species

and release thousands of merozoites into the blood stream where they invade erythrocytes.

This initiates the symptomatic erythrocytic stage, which is responsible for all clinical

manifestations associated with malaria. Inside erythrocytes, merozoites undergo a series of

maturation steps beginning with ring stage followed by maturation into trophozoites and

schizonts. The schizont ruptures and releases 16-32 new merozoites that can infect new

erythrocytes. This cycle of rupture and reinfection takes 48-78 h depending on parasite

species and yields the typical fever episodes that are characteristic for malaria. Importantly,

not all parasites mature into schizonts. Some parasites differentiate into male and female

gametocytes that can be taken up by a new feeding mosquito and undergo fertilization in the

mosquito midgut. Newly formed sporozoites are then transferred to the salivary glands and

can be injected again when the mosquito takes a new blood meal.

Figure 2. The P. falciparum life cycle. Modified from Jones et al (55). Reprinted with the permission of Nature Publishing Group.

Pre-erythrocytic stage

Erythrocytic stage

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The immune response against Plasmodium falciparum

 

The complex nature of malaria immunity has been known since historic passive transfer

studies were conducted in the 1960s, where antibodies were shown to play a role in

controlling P. falciparum blood stage infection (56). Malaria immunity develops only after

repeated exposure, is not sterile and is both species and parasite-stage specific (57). Briefly,

upon first encounter with the parasite, the individual becomes ill. In malaria endemic areas,

children under the age of five and pregnant women are especially vulnerable. After repeated

exposure, older children and adults develop protection from the severe form of illness and

death, although they can still be asymptomatic parasite carriers. Important immune

mechanisms during the pre-erythrocytic stage include sporozoite-specific antibodies and

CD8+ T-cells and their production of IFN-γ, which can eliminate intrahepatic parasites.

During the erythrocytic stage, humoral responses are thought to be important by blocking

erythrocyte invasion, initiating antibody-dependent cellular killing and binding of antibodies

to the surface of infected erythrocytes thus enhancing clearance (58-61). In addition, γδ T-

cells have been shown to be important in controlling parasitemia during the erythrocytic stage

of P. falciparum infection (62,63). Adaptive immune responses aside, a potent and long

lasting immune response to P. falciparum infection will inevitably also depend on a proper

functioning innate immune response. The innate immune response to P. falciparum infection

is able to clear parasites, thus controlling infection through a pro-inflammatory cytokine-

mediated response, however such a response must be properly balanced as not to be

detrimental to the host (64).

Hemozoin, a parasite-derived molecule

Hemozoin (HZ), also known as malaria pigment, is a parasite-derived crystalline by-product

of hemoglobin (Hb) digestion (Figure 3). Inside host erythrocytes, the parasite digests up to

80% of Hb, which constitutes an important source of energy and nutrients (65,66). This

digestive process generates free heme, which is harmful for the parasite. The parasite lacks

heme oxygenase, which is needed for heme degradation, thus there is a need for a different

method of heme-detoxification. Heme-detoxification is achieved through the conversion of

free heme into crystalline molecules referred to as HZ. The lysis of mature parasite-infected

erythrocytes leads to the release of HZ into the circulation. HZ contains, beside the crystalline

  19

heme core; lipids, bioactive lipoperoxidation products and proteins of both parasite and host

origin and this HZ is referred to as natural HZ (nHZ). HZ can also be highly purified, which is

defined as free of co-purified molecules or modified, which contains or lacks specific

molecules of host and parasite origin.

Purification of nHZ is a laborious task and the usage of the synthetic analog β-hematin is a

way to overcome the difficulties in isolating nHZ. It is also a method to study the importance

of the crystalline structure itself in immune-modulation. β-hematin however structurally

similar to nHz, is not identical in size and shape of the crystals thus leading to differences in

the immune-modulatory effects of the two molecules (67).

Figure 3. Scanning electron micrograph of P. falciparum-derived nHZ depicting its crystalline structure (left) and Phalloidin stain of DC depicting nHZ phagocytosis and localization (right). Adapted from Olivier et al and Bujila et al (65,68). Phalloidin stain of DC reprinted with permission from John Wiley and Sons.

Hemozoin-mediated effects on the response of monocytes and dendritic cells

Exposure to parasite-derived molecules, such as nHZ, has been shown to have both

suppressing and activating effects on the response of monocytes and DC.

In monocytes, HZ elicits various effects as reviewed in Schwarzer et al (69). Examples of

inhibitory effects seen in in vitro studies include the impairment of the ability to generate

oxidative burst upon appropriate stimulation after nHZ-exposure (70). Phagocytosis of nHZ

by monocytes has been shown to impair the cell surface expression of MHC class II and the

mRNA expression of inducible nitric oxide (NO) as well as impairment of NO production

after appropriate stimulation (71,72). In addition, augmented production of IL-10 by

monocytes following nHZ-exposure has been shown to negatively affect the production of IL-

  20

12 (73). Examples of stimulatory effects include the production of TNF-α and IL-1β after HZ-

exposure and induced mRNA expression of IL-1β and MCP-1 (74,75).

Several studies have shown that DC exposed to nHZ or β-hematin have impaired ability to

mature and subsequently to prime naïve T-cells. Monocytes differentiated into monocyte-

derived DC in the presence of nHZ were shown to have impaired cell surface expression of

MHC class II, CD80 and CD83 (76). In a previous study from our group, DC exposed to β-

hematin were shown to be hampered in their cell surface expression of MHC class II and

CD83, but not CD80. In addition, β-hematin-exposed DC did not produce IL-12 and were

thus unable to induce proliferation of allogenic T-cells (77). On the contrary, purified HZ has

in various studies been shown to induce DC activation. Coban et al showed that exposing

monocyte-derived DC to purified HZ induced cell surface expression of CD86 and CD83.

The effect of β-hematin did not differ from that of unstimulated controls. The DC also

produced IL-12 in response to purified HZ (78). Studies from the same group showed that

murine bone marrow-derived DC produced large amounts of IL-12p40 and various

chemokines in a dose-dependent manner in response to purified HZ. They also showed that

the molecular mechanisms underlying the response to purified HZ included TLR9 and the

adaptor protein MyD88, since HZ-induced responses were impaired in TLR9-/- and MyD88-/-

mice (79). Later Parroche et al showed that it was not HZ itself that activated TLR9, rather

the associated malarial DNA. It was suggested that HZ acts as a carrier of malarial DNA into

intracellular compartments where it can activate TLR9 (80). Others have been able to show

that HZ is neither a TLR9-ligand nor a carrier of malarial DNA (81). The DNA-protein

complex responsible for the effects observed in DC were instead attributed to parasite

nucleosomes (histone-DNA) (82). In addition NLRP3-inflammasomes have been shown to

play a role in DC activation, where β-hematin-exposure of bone marrow-derived macrophages

and DC led to production of IL-1β in an NLRP3-dependent manner (Figure 4) (83).

  21

Figure 4. Innate sensing of P. falciparum and parasite-derived molecules. HZ traffics parasite-derived nucleic acids into the phagolysosome. HZ is thought to be recognized by the NLRP3 inflammasome and TLR9, although HZ-dependent activation of TLR9 is controversial. TLRs signals through the adaptor proteins MyD88 and TRIF, eventually leading to the activation of NF-κB and IRFs and further the induction of for example pro-inflammatory cytokines and type I IFNs. Modified from Gazzinelli et al (84). Reprinted with the permission of Nature Publishing Group.

  22

DIFFERENTIAL SUSCEPTIBILITY TO MALARIA IN ETNIC GROUPS IN WEST

AFRICA

In order to study P. falciparum effects in vivo, the Fulani ethnic group is of particular interest.

They have lower susceptibility to P. falciparum infection compared to other ethnic groups

such as the Dogon in Mali (85) and the Mossi and Rimaibé in Burkina Faso (86,87). The

different groups live in sympatry and their interethnic differences in susceptibility and

immune responses to P. falciparum infection are well established. The Fulani have repeatedly

been shown to have lower parasite rates, less clinical symptoms, higher spleen rates, lower

number of parasite clones, higher titers of malaria-specific antibodies and higher cytokine

production compared to other sympatric ethnic groups (85,86,88-92). The Fulani in Mali have

higher percentage of activated memory B-cells and higher percentages of plasma cells upon

P. falciparum infection compared to the Dogon, which might help to explain previous

findings of higher antibody titers in this group (93). Furthermore, the Fulani have higher

numbers of IL-4- and IFN-γ-producing cells (91) and functional deficit of their Treg cells (94)

compared to neighboring sympatric ethnic groups. In addition, children of Fulani or Dogon

ethnicity have been shown to respond differently in the activation of various APC subsets and

TLR responses upon P. falciparum infection (95). Hence, the Fulani are considered to have a

more protective response against P. falciparum infection.

The observed interethnic differences cannot be explained by differential exposure. It has

been shown that the entomological inoculation rate, measured in both Burkina Faso and Mali,

was similar in the different villages. There are no differences in the usage of impregnated bed

nets or intake of antimalarial drugs between the different groups (85,87). Efforts have been

made to investigate underlying genetic variations in order to explain the differential

susceptibility to malaria between Fulani and other ethnic groups. Higher frequencies of

classical malaria-resistance genes, such as hemoglobin S and C, α-thalassemia and glucose-6-

phosphate dehydrogenase (G6PD) deficiency, cannot explain the observed differences (96).

Furthermore, single nucleotide polymorphisms (SNPs) in immune competent genes associated

with cytokine- and antibody responses could not comprehensively explain the observed

interethnic differences (97-103). The underlying protective mechanisms probably involve

both genetic and epigenetic components, where the involvement of epigenetic mechanisms

warrants further investigations.

  23

EPIGENETICS AND TRANSCRIPTIONAL REGULATION – A BRIEF OVERVIEW

 

In 1942, Waddington stated that phenotype arises from genotype through programmed

changes. The definition of epigenetics nowadays is; heritable information during cell division

other than the DNA sequence itself (104). In other words, epigenetics refers to heritable

changes that alter gene expression levels without altering the DNA sequence.

The immunological response has the potential to do great harm, such as excessive

inflammation and tissue damage, and therefore it needs to be tightly regulated. It is evident

that the chromatin landscape together with transcription factors and transcriptional co-

regulators play an important role in the regulation of inducible genes, such as genes involved

in immunological and inflammatory responses (105,106). Furthermore, there are multiple

layers of regulatory checkpoints occurring post-transcriptionally including mRNA splicing,

mRNA polyadenylation and mRNA stability as reviewed in Carpenter et al.  Such mechanisms

could be important in modifying the potency and duration of the immunological response

(107). There are several “new players” in the immune regulatory field, such as long

noncoding RNA, which is essential for the regulation of various classes of immune genes

(108). Chromatin structure and post-translational modifications will be further discussed in

the following sections.

Chromatin comprises of DNA wrapped approximately 146bp around a core of eight

histone proteins, which constitutes the nucleosome. The histone core is comprised of

duplicates of the histone proteins H2A, H2B, H3 and H4 (109). Histone proteins and DNA

together form the 10 nm chromatin fiber, which is further packed into the 30 nm fiber. This

packaging is achieved by the linker histone H1, which pulls nucleosomes closer together

(110). Nucleosome structure is highly conserved among eukaryotes (111). Chromatin

structure can either be loosely packed into euchromatin (open chromatin) or more densely

packed into heterochromatin (closed chromatin) (Figure 4) (112). Euchromatin enables

transcription by making the DNA more accessible (113). DNA accessibility is a very tightly

regulated process and post-translational modifications of histones, chromatin remodeling

complexes and DNA methylation are ways of regulating DNA accessibility.

  24

Figure 4. Organization of chromatin with different chromatin states of active transcription (euchromatin) and restricted transcription (heterochromatin). Adapted from Wong et al (114). Reprinted with the permission of BMJ Publishing Group Ltd.  

 

POST-TRANSLATIONAL MODIFICATIONS OF HISTONES

Histone modifications play an important role in regulating chromatin structure and as such in

exposing DNA for transcription, replication or DNA repair. Histone proteins have lysine-rich

tails that can be chemically modified in various ways including acetylation, methylation,

ubiquitinylation, phosphorylation and ribosylation (115).

Histone acetylation is associated with transcriptional activation. Acetylation can neutralize

positively charged lysines, thus weakening the affinity between histone proteins and DNA,

leading to a more open chromatin structure, and thus more accessible DNA. H3K9ac and

H3K27ac are examples of histone modifications that are associated with transcriptional

activation, where the latter distinguishes active enhancers from inactive enhancers (116).

Histone methylation can be associated with both transcriptional activation and silencing.

The function depends on the location and degree of methylation, which can be mono-, di- and

tri-methylation (113,117). H3K4me3 is enriched at enhancers and correlates with

transcriptional activation. H3K9me3 and H3K27me3 are often found at promoters in

heterochromatin (118,119). In addition, H3K27me3 levels have been shown to be higher at

silent promoters compared to active promoters in human T-cells. Similarly, H3K9me2 and

H3K9me3 levels were higher in silent genes compared to active genes, while high H3K9me

levels were detected at active promoters, thus highlighting some of the complexity in regards

  25

to histone modifications (120). Promoters can show a combination of histone acetylations and

methylations, such as H3K4me, H3K4me2, H3K4me3 and H3K9ac at active promoters in

human T-cells (121). Histone modifications are highly dynamic and added or removed by

various enzymes.

 

 

Enzymes and chromatin remodeling complexes affecting chromatin structure

 

Acetylation of histones is carried out by histone acetyltransferases (HATs), whereas histone

deacetylases (HDACs) remove acetyl groups, which usually leads to transcriptional silencing

(122). HATs include the Gcn5-related N-acetyl transferase family which further includes

Gcn5 and PCAF, the CBP/p300 family and the MYST family which consists of TIP60 and

Mof (123). There are four classes of HDACs, class I-IV (124). Various sets of enzymes are

involved in methylation and demethylation processes and they are usually specific for the

sites they modify (125,126).

Chromatin remodeling complexes are DNA translocases and they are essential in

regulating nucleosome positioning and thus chromatin structure. Chromatin remodeling

complexes are able to “displace” histone octamers from DNA or translocate octamers onto

adjacent DNA segments, thus exposing underlying DNA sequences. There are currently four

different families of ATP-dependent chromatin remodeling complexes; SWI/SNF, CHD,

ISWI and INO80 (127). Catalytic ATP-subunits, such as the brahma-related gene-1 (BRG1)

in the SWI/SNF complex, enables the reposition of nucleosomes (128). Furthermore, the

complexes contain non-catalytic subunits that are needed for targeting and regulation of

specific nucleosome positioning processes. Chromatin remodeling complexes recognize

various histone modifications, DNA sequences and RNA signals that targets them to specific

genomic sites (129).

  26

DNA METHYLATION

 

DNA methylation is involved in various processes including silencing of transposable

elements, regulation of gene expression, genomic imprinting and X-chromosome inactivation.

DNA methylation refers to the addition of a methyl group to the 5’ position of the pyrimidine

ring of certain cytosines that are adjacent to guanines in the DNA, known as CpG

dinucleotides. Most of our DNA across the genome is methylated (70%-80% of all CpG sites)

with the exception of densely clustered CpG sites referred to as CpG islands. CpG islands

most often mark the site of promoters and 5’ ends of genes (130). CpG islands are not the

only regions for DNA methylation-dependent regulatory processes. The greatest variation in

methylation levels occurs in CpG island shores, which are found 2kb upstream or downstream

of CpG islands. High-throughput DNA methylation analysis has also reveled the presence of

methylation sites known as shelves which are located 2-4kb upstream or downstream of CpG

island shores (131). Methylated promoters are associated with transcriptional repression

through various mechanisms such as interference with transcription factor-binding and

recruitment of proteins and repressor complexes that bind methylated CpGs (130). DNA

methylation patterns can provide information about ongoing gene activity in normal and

diseased cells and as such, they could prove to be useful as biomarkers (132).

EPIGENETIC MECHANISMS AND THE IMMUNE SYSTEM

It is now established that the cooperation between certain transcription factors, complexes

regulating chromatin structure and epigenetic mechanisms, such as histone modifications and

DNA methylation, is of major importance in the regulation of genes involved in the

immunological response (105,133). Epigenetic mechanisms are essential in the process of

hematopoiesis, as well as in the differentiation of the various myeloid and lymphoid cell

subsets (131). They have also been shown to be important in the differentiation and cytokine

expression of Th-cells (134).

When monocytes differentiate into monocyte-derived DC, the expression of the classical

monocyte marker CD14 is lost and instead expression of the DC-specific marker CD209/DC-

SIGN is up-regulated, which is important for establishing contact between DC and T-cells.

CD14 expression on monocytes is associated with active histone marks, such as H3K9ac.

Upon differentiation, the loss of CD14 is accompanied by the loss of H3K9ac with a

  27

reciprocal loss of repressive histone marks and increase of H3K9ac at the CD209/DC-SIGN

gene locus (135). Another study of monocyte differentiation into monocyte-derived DC

showed that levels of H3K4me3 decreased during differentiation in regards to monocyte-

specific genes and increased in regards to DC-specific genes (136). Histone modifications

have also been shown to be important in suppressing IL-12 production in a model of severe

peritonitis-induced sepsis of murine DC (137). In addition, a comparison of classical

monocytes between sepsis patients and healthy controls showed different patterns of

H3K4me3, H3K27me3 and H3K9ac in immune relevant genes between the two groups (138).

HATs have been shown to play a role in NF-κB-mediated inflammation as reviewed in

Ghizzoni et al (139). Treatment of murine DC with HDAC inhibitors strongly inhibited the

expression of IL-12 and it was further shown that although acetylation was increased at the

IL-12p40 locus, the necessary transcription factors were not recruited and thus transcriptional

activation was hampered (140). Similarly, HDAC inhibitors down-regulated the expression of

numerous genes involved in the immunological response in human and murine macrophages

and DC and in vivo, HDAC inhibitors increased the susceptibility to bacterial and fungal

infection, but led to protection against septic shock (141). Taken together, HATs and HDAC

inhibitors have been shown to be important in regulating the immunological response.

Chromatin remodeling complexes also have an important role in immune regulation.

Macrophages stimulated with LPS show kinetically distinct waves of transcriptional

activation of immunologically important genes. BRG1, the catalytic subunit of the chromatin

remodeling complex SWI/SNF, is needed for the activation of secondary response genes, but

not for the activation of primary response genes (142,143).

Differentiation and maturation of DC is coupled with loss of DNA methylation across

many regions, enhancers and transcription factor-binding sites that are associated with DC

linage commitment and response to immune/inflammatory stimuli (144). Following bacterial

infection, rapid changes in DNA methylation have been shown to occur as part of the

regulatory response to infection in DC (145).

Immune memory has been considered to be a hallmark trait of the adaptive immune

system. However, as our understanding of the immune system deepens, the distinction

between the innate and the adaptive immune system becomes less static. Cells of the innate

immune system are thought to be able to acquire memory-like features, a phenomenon known

as trained immunity. Trained immunity refers to enhanced responsiveness to a secondary

challenge due to priming by an initial challenge. Epigenetic modifications are thought to be

essential for the process and sustainment of trained immunity (131,146). One example comes

  28

from Candida albicans-derived β-glucan “training” of monocytes, which has been shown to

be associated with changes in the activating mark H3K4me3 at the promoter region of genes

important in the pro-inflammatory response (147).

  29

PRESENT STUDY

OBJECTIVES

The overall aim of this thesis was to examine the influence of P. falciparum infection and

parasite-derived molecules on the response of innate immune cells and how infection or

parasite-derived molecules affected the regulation of the anti-parasitic response.

Specific aims

 

• To study the early effects of nHZ-exposure on DC phenotype and functionality and to

compare the response to that of the synthetic analog β-hematin (Paper I).

• To investigate how the nHZ-induced effects on DC phenotype and functionality could

be transcriptionally regulated (Paper II).

• To evaluate the influence of P. falciparum infection on the transcriptome and

methylome of sympatric ethnic groups with known differential susceptibility to P.

falciparum infection living in Burkina Faso (Paper III).

  30

METHODS

 

A general description of the methods and the cohort material is provided below and in more

detail in each paper.

Experimental procedure for Paper I and II

 

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy anonymous blood

donors by Ficoll-Paque gradient centrifugation. CD14+ monocytes were magnetically isolated

followed by differentiation into monocyte-derived DC. Cell phenotype was analyzed by flow

cytometry. The presence of podosome structures was assessed by Phalloidin staining and

functional capacity was investigated in co-cultures with autologous CD3+ T-cells. mRNA

levels were analyzed by qPCR and cytokine/chemokine levels were assessed in cell culture

supernatants by ELISA. For paper II, binding of transcription factors, BRG1 and histone

modifications were analyzed by chromatin immunoprecipitation (ChIP).

Experimental procedure for Paper III

 

Young adolescent men belonging to the Fulani or Mossi ethnic group in Burkina Faso were

recruited to the study. P. falciparum infection was determined by rapid diagnostic test and

later confirmed by qPCR. CD14+ monocytes were magnetically isolated from whole blood.

Simultaneous isolation of total RNA and DNA was performed on monocytes and the

remaining CD14- population. RNA-sequencing (RNA-seq) was performed on CD14+

monocytes and the CD14- population from the same individual, where total RNA was

ribodepleted and converted into cDNA using random primers. Sample selection was done as

following: Samples with mixed infections, G6PD-deficiency and carriers of hemoglobin C

were excluded from the analysis. We included samples from infected and uninfected Mossi

individuals and infected and uninfected Fulani individuals. RNA-seq was performed on a total

of 12 samples; 12 CD14+ samples (of note; one monocyte sample from an infected Fulani

individual was not successfully sequenced) and 12 CD14- samples. After sequencing, the

reads were aligned to a reference genome (human, GRCh37). The DNA methylome analysis

was performed on a total of 12 CD14+ samples of infected and uninfected Fulani and Mossi

individuals with the Infinium Human Methylation450 BeadChip (Illumina).

  31

RESULTS AND DISCUSSION

 

 

Paper I

 

One of many key questions left to be resolved in regards to human immune responses against

P. falciparum infection concerns APCs, such as DC, and the potential of parasite-derived

molecules to modulate DC function, maturation and the ability to activate naïve T-cells. It is

clear that the parasite-derived molecule HZ plays an essential part in the biology of

Plasmodium infections and two important antimalarial drugs, chloroquine and artemisinin,

targets the formation of HZ as part of their mode of action (148).

In this paper we investigated the response of DC exposed to nHZ and the effects that this

interaction had on the ability of DC to properly mature. We found that nHZ exerted rapid

immune-modulatory effects on DC phenotype and maturation. To establish a more complete

picture, we examined both transcriptional events and protein expression. We could show that

DC produced high levels of the chemokine MCP-1 in response to nHZ and as such they

posses the potential to migrate and further recruit leukocytes, including other immature DC, to

the site of infection or inflammation (149). DC exposed to nHZ did not down-regulate the cell

surface expression of CCR5. Together with the observation that DC after exposure to nHZ did

not up-regulate the cell surface expression of the lymphoid homing CCR7 indicates a failure

to properly mature, as the transition from CCR5 to CCR7 expression is a hallmark marker of

DC maturation. Sallusto and colleagues have shown that the loss of CCR5 on the cell surface

is rapid and in line with this we also observed a substantial loss 4 h following LPS-

stimulation. This down-regulation is not a result of transcriptional changes as the authors

observe retained CCR5 mRNA levels even after 40 h of stimulation. This result was

confirmed in our study were there were no detectable differences in mRNA levels of CCR5

between the different stimuli for up to 24 h. The rapid loss of CCR5 on the cell surface is

rather attributed to auto-desensitization of the receptors due to chemokine production by

maturing DC (150).

We could also show that following nHZ exposure, there was no cytoskeletal redistribution

of actin, such as loss of adhesive podosome structures, which is associated with DC

maturation. However, nHZ-exposed DC showed features of partial maturation as indicated by

higher cell surface expression of molecules essential for antigen-presentation, such as MHC

class II and the co-stimulatory molecule CD86 compared to unstimulated cells.

  32

The function of the maturation marker CD83 is not fully known, but its importance for the

interaction between DC and T-cells has been shown in various studies (46,151). We could

clearly demonstrate that DC exposed to nHZ did not up-regulate CD83 expression, neither on

a transcriptional level nor on the cell surface. In fact, nHZ selectively inhibited LPS-induced

expression of CD83. To investigate the functional implications of the observed partial

impairment of DC maturation, we co-cultured DC exposed to nHZ with autologous CD3+ T-

cells. We could show that nHZ-exposed DC were unable to induce proper T-cell activation.

We could also show that unlike nHZ, the synthetic analog β-hematin tended to have an

effect on the down-regulation of CCR5 cell surface expression and led to significant increases

in the percentage of cells expressing the maturation marker CD83.

Reported immune-modulatory effects of HZ on innate immune cells are rather

contradictory (65,152,153). One explanation could be the HZ itself, which can be highly

purified, modified or natural. Adding to the complexity, nHZ and the synthetic analog β-

hematin differ substantially in their chemical composition, where β-hematin exclusively

comprises of a poly-heme crystal (154). The nHZ used in this study is considered to have in

vivo relevance as this is the HZ-composition that is taken up by phagocytic cells (155).

Furthermore, as many as 42 human plasma proteins, obtained from plasma of malaria

patients, have been identified to bind with high affinity to nHZ, of which several have known

immune-modulatory functions (156).

In conclusion, we showed that exposure to nHZ renders DC partially immature and that the

effect of nHZ differs to some extent from that of β-hematin in regards to the maturation

process. Therefore, any results obtained from studies using β-hematin should be carefully

interpreted and extrapolated. Maybe β-hematin should be considered as a control to nHZ,

rather than a malarial molecule in its own right.

The data provided herein further illustrates the modulation of DC phenotype, maturation

and function induced by nHZ and describes a possible implication of nHZ-induced DC

impairment on the overall poor induction of effective immune responses against P. falciparum

infection.

  33

Paper II

 

In paper II we followed up on our findings from paper I, where we showed that nHZ affected

the maturation process of DC. Stimulation with LPS led to increasing mRNA levels of genes

important in DC maturation including the chemokine receptor CCR7, the co-stimulatory

molecule CD86 and the maturation marker CD83, whereas nHZ-exposure did not. Therefore,

we investigated possible underlying transcriptional events that could shed light on the

regulation of nHZ-induced impairment of DC maturation. The fact that HZ in all its different

preparations is capable of modulating the effects of several different cell types and various

receptors add to the complexity of studies addressing underlying molecular events following

HZ-exposure. HZ has been shown to activate numerous innate sensors and thus various

signaling pathways as reviewed by Gazzinelli and colleagues (84). Briefly, HZ has been

suggested to trigger TLR9, TLR4 and the NOD-like receptor NLRP3 (79,83,155,157).

Affected transcription factors downstream of TLR-signaling include NF-κB and IRFs, where

NF-κB has multiple important functions in the immune response (158). Higher activation of

the NF-κB pathway has been shown in PBMCs from patients with uncomplicated P.

falciparum infection compared to healthy controls and moreover several studies have

demonstrated that HZ can mediate activation of the NF-κB pathway in human monocytes and

murine macrophages (159-161). These findings promoted the investigation of the binding of

NF-κB subunits to the promoter region of genes important in DC maturation, such as CCR7,

CD86 and CD83, which are all NF-κB target genes (162-164). We could not detect any

recruitment of NF-κB subunits (p105/p50 or p65) to the promoter region of genes important

for DC maturation, neither at the transcriptional start site (TSS) nor at NF-κB binding sites

following 2 h of nHZ-exposure. IRF3, part of the IRF family of transcription factors, has been

implicated in various aspects of immune regulation including the regulation of type I IFNs

and various chemokines (23) and IRF3 activation has been observed upon P. falciparum

infection (165). Recruitment of IRF3 to the promoter region of CCR7, CD86 and CD83 was

also lacking following nHZ-exposure, unlike what was seen following stimulation with LPS.

These findings point towards an inability of nHZ-exposed DC to recruit certain transcription

factors to the promoter region of genes important in the maturation process. The general lack

of transcriptional activation of the genes in question following nHZ-exposure that we

observed in paper I, could be associated with this inability. Overall, optimal responses against

invading pathogens most likely involve the activation of both the NF-κB and the IRF pathway

(166,167).

  34

Transcriptional regulation also involves chromatin remodeling since the condensed structure

of chromatin can hinder access of proteins to the underlying DNA and chromatin remodeling

complexes, such as the SWI/SNF complex with its catalytic subunit BRG1, are important in

this process. These complexes use ATP hydrolysis to slide nucleosomes and play a major role

in the regulation of gene expression, DNA replication and DNA repair (168). BRG1 has been

implicated in the regulation of gene expression in the immune system. Firstly, SWI/SNF

complexes and NF-κB have been shown to be linked through the action of the nuclear protein

Akirin2 and this interaction was shown to be important for TLR-mediated expression of pro-

inflammatory genes in murine macrophages (169). Secondly, Holley et al could show that

BRG1 had a role during activation, but not differentiation of murine B-cells and that

especially TLR-pathways and JAK/STAT cytokine signaling pathways were BRG1-

dependent (170). Macrophages stimulated with LPS show kinetically distinct waves of

transcriptional activation of genes important in the immune response. Chromatin remodeling

events involving BRG1 were shown to be required for the activation of secondary response

genes (142,143). CCR7, CD86 and CD83 were transcriptionally activated by LPS 2-4 h after

stimulation as shown in paper I and thus these genes most likely are secondary response

genes. In light of the importance of BRG1-dependent chromatin remodeling events in the

immunological response, we investigated the recruitment of BRG1 to the promoter region of

genes important in DC maturation. We observed that nHZ-exposed DC did not recruit BRG1

to the promoter region of CCR7, CD86 and CD83, which was in contrast to DC stimulated

with LPS. As a result of the lack of BRG1-recruitment, we suggest that necessary remodeling

events are hampered following nHZ-exposure, which might have an effect on DNA-

accessibility and further affect transcriptional activation. It is tempting to speculate that nHZ

has the capacity to actively inhibit recruitment of certain transcription factors and BRG1 to

the promoter region of genes important in DC maturation through so far unknown

mechanisms. We show in paper I that nHZ can inhibit LPS-induced DC maturation in regards

to the percentage of cells expressing CD83 on their cell surface. Whether this effect occurs

already at a transcriptional level and involves inhibition of certain transcription factors and

BRG1 is currently under investigation.

mRNA levels of CCR5 were unaffected following nHZ-exposure and LPS-stimulation as

shown in paper I. However, in the present study we observed recruitment of both IRF3 and

BRG1 to the promoter region of CCR5 following stimulation with LPS. The CCR5 promoter

has two promoters with different transcription factor-binding sites. Kuipers and colleagues

have shown that neither NF-κB nor IRFs (IRF1) were involved in the activation of CCR5

  35

expression. Instead, they could show that CCR5 expression was activated by the

cAMP/CREB pathway, which has its binding sites in the upstream promoter (171). Although

we could detect recruitment of IRF3 and BRG1 to the CCR5 promoter following LPS-

stimulation, it might no be relevant for the transcriptional regulation of CCR5, thus

highlighting the complexity of the gene regulatory network and the importance of studying

gene expression in specific and kinetically distinct contexts.

Histone modifications are highly dynamic unlike our more static inherited genetic

variation. The plasticity of histone modifications makes them interesting targets to manipulate

during disease conditions. Histone modifications contribute significantly to the regulation of

gene expression in P. falciparum parasites as reviewed by Doerig et al (172). Histone

modifications are also shown to contribute to the regulation of antigenic variation in P.

falciparum parasites, an essential process for immune evasion and pathogenesis (173). The

effects of P. falciparum-derived molecules in regards to histone modifications in human

innate immune cells are largely unexplored. We investigated the enrichment of various

histone modifications, both activating and silencing, at the promoter region of genes

important in DC maturation. Overall, DC exposed to nHZ did not enrich for histone

modifications, neither activating nor silencing, at the promoter region of CCR7, CD86 or

CD83. On the contrary, we observed possible enrichment of the silencing mark H3K27me3 at

the TSS of CD83 following nHZ-exposure and this observation together with the lack of

enrichment of the activating mark H3K4me3 could be associated with the hampered induction

of CD83 mRNA levels observed in paper I following nHZ-exposure. Stimulation with LPS

yielded different enrichment patterns of histone modifications. One example is an overall

suggested enrichment of H3K4me3 in the promoter region of CCR7, CD86 and CD83, genes

that we in paper I showed are stably induced by LPS for up to 24 h.

Paper II is an ongoing study and so far sample size and variation hampers our data

interpretation. Furthermore, despite the fact that LPS is a potent inducer of DC maturation,

which makes it a good positive control in paper I, its use for comparison in studies

investigating transcriptional events following nHZ-exposure is complicated. One reason is

that nHZ is recognized by multiple receptors unlike LPS that is recognized by TLR4. In

addition, their cellular uptake is different, where nHZ is taken up by phagocytosis

(78,174,175).

Taken together, our findings in paper II suggest an inability of nHZ-exposed DC to recruit

certain transcription factors and BRG1 that are needed for transcriptional activation to the

promoter region of CCR7, CD86 and CD83. In addition, exposure to nHZ did not lead to the

  36

enrichment of various histone modifications to the promoter regions in question. These

observations might help to shed light on the molecular mechanisms underlying the impaired

DC maturation upon nHZ-exposure that we observed in paper I.

 

Paper III

In paper II we found indications that the impairment of DC maturation following exposure to

parasite-derived molecules could be due to hampered transcriptional events in vitro.

In paper III, we asked ourselves what molecular mechanisms could be underlying the

response to naturally acquired P. falciparum infection in innate immune cells in vivo. More

specifically, we speculated about the molecular mechanisms that could help to explain the

relatively better protection against P. falciparum infection observed in the Fulani population

in parts of West Africa in comparison to other sympatric ethnic populations. The relatively

better protection against P. falciparum infection in the Fulani group compared to other groups

living under similar social and geographical conditions is well established (86,88-95). Less

established are the underlying mechanisms, genetic or perhaps epigenetic, for this relatively

better protection. We believe that characterization of transcriptional events that could play a

role in governing a relatively more protective response against P. falciparum infection could

aid in the development of better tools and therapies to fight malaria.

The impact of P. falciparum infection on the human genome is substantial and has

contributed to the acquisition of malaria resistance genes in exposed populations (176,177).

The observed interethnic differences to malaria can neither be explained by vector exposure

or usage of protective measures (bed nets, antimalarial drugs) nor by malaria resistance genes

(85,87,96). Fulani have been shown to be genetically distinct from other sympatric groups,

such as the Mossi in Burkina Faso (178). Studies have been conducted addressing the

association between SNPs and immunological responses in sympatric groups as reviewed in

Arama et al (179). To date, these studies have not been able to provide a comprehensive

explanation of the underlying mechanisms governing the relatively better protection against

malaria in the Fulani group, thus suggesting the involvement of so far unknown genetic or

epigenetic factors. To address this knowledge gap, we investigated the transcriptome by

whole genome sequencing and the methylome by DNA Methylation Array of Fulani and

Mossi individuals naturally infected with P. falciparum in comparison to uninfected

individuals living in Burkina Faso. We focused on monocytes as they have been shown to be

  37

important during P. falciparum infection, where they contribute to both protection and

pathogenesis (180-183). We isolated CD14+ monocytes from whole blood, from which we

simultaneously obtained total RNA and DNA. The same procedure was used for the

remaining CD14- population that mainly consisted of T-cells, B-cells and NK-cells. We

divided the study participants in four groups: uninfected Mossi, infected Mossi, uninfected

Fulani and infected Fulani. We firstly wanted to investigate if there were any transcriptional

differences between monocytes and the CD14- population and we found that gene expression

profiles differed substantially between monocytes and CD14- cells. This finding highlights the

importance of using specific cell populations and not whole blood or PBMCs in gene

expression studies where the latter approaches do not allow for discrimination of cell-specific

contributions in the immunological response and regulatory pathways.

Since there were no significant changes in the CD14- population between the different

groups, we further focused on different gene expression patterns in monocytes. We showed

that gene expression patterns were significantly altered between uninfected Fulani and

infected Fulani, where the majority of altered transcripts were up-regulated upon infection.

Changes in gene expression in the Mossi group regardless of infection status or in comparison

to Fulani individuals did not reach statistic significance. Gene Ontology (GO) analysis

revealed that up-regulated genes in infected Fulani were involved in biological processes such

as translational elongation, vesicle-mediated transport, intracellular signaling cascade and

actin cytoskeleton organization. Molecular functions included enzyme binding, small GTPase

regulator activity and transcription factor binding. When we classified genes based on their

general function (UniProtKB/Swiss-Prot database) we found multiple classes of genes up-

regulated upon infection in Fulani individuals such as genes associated with RNA/DNA,

transcription factors, genes involved in signal transduction, chromatin structure,

immunological responses, protein trafficking and apoptosis to mention a few. There is a

consensus in the field that the balance between pro-inflammatory and anti-inflammatory

responses to malaria is important in order to successfully resolve the infection (57,184). We

found that Fulani individuals upon infection up-regulated gene transcripts that are known to

be involved in the pro-inflammatory response including several transcripts connected to TLR-

signaling such as components of the NF-κB, IRF and MAPK pathway. This finding correlated

well with the fact that the Fulani have been shown to have a more pro-inflammatory response

in regards to cytokine- and chemokine production downstream of TLR-signaling in response

to P. falciparum infection (92). In addition, a study of TLR-responses using PBMCs from

Fulani and Dogon in Mali showed that the TLR-responses were hampered in Dogon

  38

individuals upon infection, while no inhibition was observed in Fulani individuals (95). A

result that further strengthens the involvement of the NF-κB pathway in the relatively more

protective response against P. falciparum infection is our observation of enrichment of

binding sites for NF-κB subunits in up-regulated genes in Fulani individuals upon infection.

Several transcripts involved in the anti-inflammatory response were up-regulated including

genes involved in the regulation of the NF-κB pathway and genes coding for receptors for

anti-inflammatory cytokines, such as the IL-10- and IL-4 receptor. We speculate that Fulani

individuals are able to balance pro- and anti-inflammatory responses to P. falciparum

infection and this ability could contribute to better disease outcomes. Of note, however

beneficial this response is against P. falciparum infection, it could be associated with

autoimmunity in the Fulani group (185-187).

The cellular response to invading microbes is complex and involves the cooperation of

signaling pathways with chromatin structure, regulatory elements involved in transcription

and epigenetic modifications (106,188). We observed that a large portion of the up-regulated

genes in Fulani individuals upon infection was related to chromatin structure and chromatin

remodeling. Several components of chromatin remodeling complexes, including SWI/SNF,

were up-regulated. As previously discussed in paper II, the SWI/SNF complex with its

catalytic subunit BRG1 has been shown to be involved in the regulation of NF-κB-dependent

genes. There are several transcripts up-regulated in infected Fulani that are correlated with

transcriptional activation, such as subunits of the methyltransferase COMPASS complex that

is responsible for H3K4 methylation and the acetyltransferase CBP/p300, which also targets

NF-κB-dependent genes and IRF-dependent genes (189-192). Taken together, there could be

an association between the up-regulation of various chromatin remodeling complexes and

enzyme complexes, and the overall higher transcriptional activation in Fulani individuals

upon infection.

We further investigated whether DNA methylation could be involved in the regulation of

the relatively more protective response to P. falciparum infection. Although we detected

different methylation patterns in the different groups compared to each other, only a few loci

were involved and thus we conclude that DNA methylation alone is probably not contributing

to the regulation of the different response to P. falciparum infection.

The impact pathogens have on host transcriptional programs and the epigenome is evident.

Pathogen-induced transcriptional dysregulation has been observed during the pre-erythrocytic

stage of Plasmodium infection where different sets of genes were altered throughout the

infection process. Similar transcriptional dysregulation has also been observed upon infection

  39

with Toxoplasma gondii, Mycobacterium leprae and cytomegaloviruses as reviewed by

Silmon de Monerri et al (193).

The results presented in this paper provide important insights into monocyte biology in

well-characterized individuals naturally exposed to P. falciparum infection. In addition, this

study fills a knowledge gap, since little is known about the transcriptional events that govern a

relatively better protection against P. falciparum infection. Ockenhouse and colleagues have

shown that both common and divergent immune signaling pathways in PBMCs are apparent

between pre-symptomatic experimentally induced and symptomatic naturally infected

individuals. Common induced pathways included transcripts of the TLR-pathway and NF-κB

signaling. Divergent immune signaling pathways only found to be up-regulated in naturally

infected individuals included genes involved in immune regulation such as IL-10 and the

MAPK-signaling cascade (194), of which we were able to attribute some of these features to

monocytes.

In a different cohort of naturally P. falciparum infected Fulani and Dogon individuals in

Mali, we found significantly higher CCR7 mRNA levels in PBMCs from infected Fulani

individuals compared to their uninfected peers (unpublished data).

Figure 5. CCR7 mRNA level in PBMCs from Fulani or Dogon children in Mali (n=5-12/group). Fold change is in comparison to the housekeeping gene acidic ribosomal phosphoprotein P0. The horizontal line represents the median and statistical analysis was done with Mann-Whitney non-parametric U test. Asterisks indicate significant differences (*P< 0.05).

This finding could, in part, confirm that the higher transcriptional activity we have observed

in the Fulani group in Burkina Faso could also be apparent in the Fulani group in Mali.

Maybe some of the underlying protective mechanisms observed in the Fulani ethnic group

could partly be attributed to the presence of more mature DC capable of migrating to

secondary lymphoid tissues and present antigens to naïve T-cells. It has been previously

shown by our group that there were lower frequencies of circulating DC in infected Fulani

compared to their uninfected peers (95). The involvement of nHZ in this matter is currently

unknown. In addition, possible differences in nHZ-deposition in DC and other phagocytic

Dogon Inf. Dogon Fulani Inf. Fulani0.00

0.01

0.02

0.03

0.04

CCR7

Fol

d Ch

ange

*

  40

cells from sympatric ethnic groups with differential susceptibility to malaria have yet to be

investigated.

Samples from individuals in natural infection settings are likely to have been obtained later

in disease stage than those obtained from individuals participating in a clinical trial or in

controlled human malaria studies. Our samples were collected when the transmission season

was over, which may account for the lack of symptomatic individuals. Nevertheless, our data

aids in conceptualizing the processes and regulatory networks that are associated with a

relatively better protection against P. falciparum infection. A deeper understanding of the

underlying mechanisms governing such a relatively more protective response could in the

future aid in the development of therapies against malaria.

 

  41

GENERAL CONCLUSIONS  

The importance of the innate immune response to P. falciparum infection is evident. Parasite-

derived molecules are able to influence the phenotype and maturation process of innate

immune cells, such as DC. The modulation of innate immune cells by parasite-derived

molecules could be one of the underlying reasons for the poor induction of long-lasting

protective immunity that is characteristic of P. falciparum infections. The interaction between

cells of the innate immune system and parasite-derived molecules inevitable leads to changes

in transcriptional programs and this interaction is most likely hampering underlying

transcriptional regulatory mechanisms governing the process of maturation in vitro.

The results presented in this thesis have also set the foundation of the transcriptional

processes and regulatory networks that may contribute to a more protective response in vivo

in naturally infected individuals with differential susceptibility to P. falciparum infection.

Overall, the work presented in this thesis leads to a deeper understanding of P. falciparum-

induced modulation of the responses of innate immune cells and the underlying mechanisms

possibly regulating those responses.

  42

FUTURE PERSPECTIVES  

In light of the data presented in this thesis, several aspects of P. falciparum infection in

regards to innate immune cells in vitro and in vivo, would be interesting to study:

• The ability of nHZ to actively inhibit recruitment of certain transcription factors and

BRG1 upon LPS-induced DC maturation. It would be interesting to further also study

the possible inhibitory role of P. falciparum-infected erythrocytes.

• To further characterize the underlying regulatory mechanisms of the hampered DC

maturation upon nHZ-exposure in regards to enzymes affecting chromatin structure.

Questions to be addressed include the possible differences in enzyme activity between

cells exposed to nHZ and control cells and further to study the effects of enzyme

inhibitors in regards to genes and gene products important in DC maturation. As there

is no potent vaccine against malaria and drug resistance is emerging, HDAC

inhibitors, which are currently used in cancer treatments, are being evaluated as

potential new anti-parasitic drugs (195).

• We have identified different innate signaling pathways and regulatory networks that

govern relatively better responses against P. falciparum infection in naturally exposed

sympatric ethnic groups living in Burkina Faso. It would be interesting to strengthen

the relevance of our findings by comparing transcriptional events during the high

transmission season and then follow-up of the same individuals when there is no

transmission. In addition, healthy, non-immune Caucasians could be used as a control

to naturally exposed individuals in order to investigate how transcriptional responses

differs among the different groups. There are established differences in the humoral

response against P. falciparum infection between Fulani and control groups, so it

would be interesting to investigate B-cell specific transcriptional responses in

sympatric ethnic groups.

• On a larger scale, investigations of the possible loss of the relative better protection

against P. falciparum infection in Fulani individuals that have left malaria-endemic

  43

areas or are born in countries with no malaria would further aid in understanding the

stability of the immunological responses against P. falciparum infection.

  44

ACKNOWLEDGMENTS

What a journey this has been! My sincerest gratitude to all colleagues (past and present), co-

authors and collaborators who has crossed my path; without you this venture would not have

been possible.

To Ann-Kristin Östlund Farrants and Marita Troye-Blomberg for taking on “shared

custody” in my supervision. Thank you Anki for your “open door policy”, cookies and pep

talks whenever needed (which tended to be quite often at times). Thank you Marita for getting

me back on track when I felt lost and no matter where in the world I was (Burkina Faso,

South Africa…), I felt your support.

Thank you both for giving me this opportunity and the freedom to grow professionally and

personally in your labs, and simply for believing in me.

To Prof. Artur Scherf for agreeing to be my opponent and taking the time to go through and

discuss my thesis with me.

To all the professors at the former department of immunology: Eva Sverremark-Ekström

(thank you for making space for me in your lab and letting me disturb your post-docs and

students with my many questions and requests for fika), Carmen Fernandez, Eva

Severinson, and Klavs Berzins for your interest in my progress during this process.

To my co-authors and collaborators; especially Evelin Schwarzer, Oleksei Skorokhod and

Prof. Paulo Arese in Turin, for welcoming me to your lab and sharing your vast knowledge

on all things hemozoin. Andreas Lennartsson, for your enthusiasm over our joint project and

for our many ice-box meetings at Södra StationJ Jessica M. Weidner, the American whirl

wind ak.a microscope master, it was short but sweet. Prof. Mary O’Connell, Dr. Issa Nebie

Ouedraogo, Guillaume S. Sanou and Mariama Cherif for making my stay in Burkina Faso

a memory for life. Anna Rolicka, for our long hours bench side dealing with the hardship that

is ChIP.

To past and present students/post-docs at the department: Maria Johansson, Sophia

Björkander (thank you for reading my thesis and listening to me complain about it),

  45

Yeneneh Haileselassie, Claudia Carvalho-Queiroz, Sofia Nordlander, Manuel Mata

Forsberg, Charles Arama, Khaleda “Mukti” Qasi-Rahman, Stephanie Böhm, Anna

Vintermist, Antoni Ganez Zapater, Jaclyn Quin, Ulrika Holmlund, Sachie Kanatani,

Linda Westermark and many many more.

I especially want to mention Pablo Giusti for your tough love and many laughs, it just wasn’t

the same without you, Ebba Sohlberg my “newest” friend, but one of my dearest, you simply

rock! Stephanie Boström my former roomie, both you and your cakes gave me great support

over the years. Dagbjört “tanten” Petursdottir, with you I can always be my socially

awkward self. The Nespresso coffee machine “George”, there is no comfort like the one

found in a truly awesome cup of espresso.

To Margareta “Maggan” Hagstedt, for expanding my vocabulary with crosswords (eda =

bakström), the department just was not the same after you left!

To my friends of which I cannot mention all here, but that does not mean you do not hold a

most special place in my heart. There is no way of making it through a PhD without the best

of friends and I am truly lucky in that aspect. Anneli “dudeiiiiee” Lind, Cecilia “duracell-

kaninen” Jädert, Stina “Bullen” Klyft, Catharina “Cattis” Nyrén, Brita Ivarsdotter and

Karin Gustafsson “the travel mafia”.

To my family, Mamma, Pappa, Raz, Mami and Tati …words are so not enough! Vă iubesc

pe toți atât de mult.

On the road again

Just can’t wait to get on the road again

Goin’ places that I’ve never been

Seein’ things that I may never see again

And I can’t wait to get on the road again

Willie Nelson

  46

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