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
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
2 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
3 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.
4 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
5 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
6 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
7 RESULTS AND DISCUSSION ...... 31
Paper I ...... 31
Paper II ...... 33
Paper III ...... 36
GENERAL CONCLUSIONS ...... 41
FUTURE PERSPECTIVES ...... 42
ACKNOWLEDGMENTS ...... 44
REFERENCES ...... 46
8 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
9 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
10 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).
11 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, Regulatory/anti-inflammatory properties. NK-, T-, and B-cells 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 Triggers chemotaxis, transendothelial cells 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 Hematopoietic growth factor (16). fibroblasts
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
12 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.
13 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).
14 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
15 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).
Immature DC Mature DC
High intracellular levels of MHC class II High surface levels of MHC class II High endocytosis Low endocytosis Low CD86 and CD83 expression High CD86 and CD83 expression CCR5 expression CCR7 expression
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
16 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.
Pre-erythrocytic stage
Erythrocytic stage
Figure 2. The P. falciparum life cycle. Modified from Jones et al (55). Reprinted with the permission of Nature Publishing Group.
17 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
18 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-
19 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).
20
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.
21 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.
22 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.
23
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
24 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).
25 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
26 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
27 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).
28 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).
29 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).
30 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.
31 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.
32 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).
33 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
34 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
35 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
36 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
37 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
38 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).
*
0.04 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 0.03 gene acidic ribosomal phosphoprotein P0. The horizontal line represents the median and statistical 0.02 analysis was done with Mann-Whitney non- parametric U test. Asterisks indicate significant 0.01 differences (*P< 0.05). CCR7 Fold Change CCR7 Fold
0.00 Dogon Inf. Dogon Fulani Inf. Fulani
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
39 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.
40 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.
41 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
42 areas or are born in countries with no malaria would further aid in understanding the stability of the immunological responses against P. falciparum infection.
43 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),
44 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
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