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Doctoral thesis from the Department of Immunology, The Wenner-Gren Institute, Stockholm University, Sweden Human genetic factors involved in immunity to Plasmodium falciparum infection Manijeh Vafa Homann Stockholm 2008 Human genetic factors involved in immunity to Plasmodium falciparum infection All previously published papers were reproduced with permission from the publishers. © Manijeh Vafa Homann, Stockholm 2008 ISBN 978-91-7155-614-1 Cover illustration: Palle Ryde Printed in Sweden by Universitetsservice AB, Stockholm 2008 Distributer: Stockholm University Library ii To my parents, Mohammad Esmael Vafa and Sara Madadi And to my husband, Peter Homann Manijeh Vafa Homann iii Human genetic factors involved in immunity to Plasmodium falciparum infection To be conscious that you are ignorant is a great step of knowledge. Aristotle (384-322 BC) iv SUMMARY Development of clinical immunity can be influenced by host genetic factors. Some genetic disorders in erythrocytes have been shown to confer protection against malaria. Inter-ethnic differences in malaria susceptibility offer an approach to identify genes involved in immunity to malaria. The Fulani ethnic group, living in several African countries, has been shown to be more immunologically responsive to, and less frequently infected by Plasmodium falciparum, than their sympatric ethnic groups. Fewer malaria attacks and higher spleen rates have also been seen in the Fulani compared to the non-Fulani. IL-4 and IL-10 are two cytokines with crucial roles in the regulation of both inflammatory- and antibody responses. IL-4 -590 C/T and IL-10 -1087 A/G single nucleotide polymorphisms (SNP) have been shown to result in variable production of the respective cytokine. This study investigates; the associations between the above mentioned polymorphisms and malariometric indexes in the Fulani and the Dogon in Mali; the correlations between malaria-specific IgG, IgG1-4, IgE, as well as total IgE and parasite prevalence, multiplicity of malaria infection as well as splenomegaly; and the impact of IL-4 590 variants on the levels of the studied antibodies. The allele and genotype frequencies of both studied SNPs differed significantly between the two groups. The Fulani IL-4 T allele carriers had a significantly higher infection prevalence compared with those carrying the CC genotype. No correlation between antimalarial antibody levels and parasite prevalence was seen in any of the communities. In the Fulani, the increase in total IgE levels was related to the presence of infection. Malariaspecific IgG4 levels were negatively correlated to the number of clones within the Fulani. The Fulani IL-4 T allele carriers had higher total and malaria-specific IgE levels, compared to the CC genotype carriers. Taken together, these results suggest that the amount of antibodies may not be the key element in the protection against malaria. Rather, specificity, function or affinity might be of more importance in this context. IgG4 might be involved in protection against malaria. The impact of IL-4 -590 variants on the antibody levels may be affected by other genetic/epigenetic/epistatic or environmental factors. The role of IgE in malaria remains to be clarified. Multiplicity of infection (MOI) is the number of various P. falciparum strains co-infecting a single individual. MOI has been shown to be influenced by age, seasonal variation and transmission intensity. Both negative and positive effects of MOI on the risk of clinical malaria have been reported. We investigated the impact of erythrocyte variants on the MOI, the correlation between MOI and malaria morbidity, and association between MOI and age, parasite density and aslo seasonal variation. MOI increased after the transmission season in all subjects, except in α-thalassaemic and in G6PD-mutated children. MOI was positively correlated to parasitemia, and did not vary over age (in the range of 2 to 10 years). No relation between MOI and clinical attack was noted. These data suggest that α-thalassaemia may protect against infection by certain parasite strains. G6PD-mutated individuals may resist against increase in MOI after the transmission season due to rapid clearance of infection at an early stage. Furthermore HbAs and the ABO system may have an impact on protection against severe disease, but do not seem to affect MOI in asymptomatic individuals. Further studies are required to confirm the age-independency of MOI and to better clarify the relations between MOI and clinical attacks. Manijeh Vafa Homann v Human genetic factors involved in immunity to Plasmodium falciparum infection ORIGINAL ARTICLES This doctoral thesis is based on the following original papers, which in the text, are referred to by their roman numerals. vi I. Manijeh Vafa, Bakary Maiga, Klavs Berzins, Masashi Hayano, Sandor Bereczky, Amagana Dolo, Modibo Daou, Charles Arama, Bourema Kouriba, Anna Färnert, Ogobara K. Doumbo and Marita Troye-Blomberg, Associations between the IL-4 590 T allele and Plasmodium falciparum infection prevalence in asymptomatic Fulani of Mali. Microbes Infect. 2007; 9: 1043-8 II. Manijeh Vafa, Elisabeth Israelsson, Bakary Maiga, Amagana Dolo, Ogobara K. Doumbo and Marita Troye-Blomberg. Relationship between immunoglobulin isotype response to Plasmodium falciparum blood stage antigens and parasitological indexes as well as splenomegaly in sympatric ethnic groups living in Mali. Acta Tropica, under revision III. Manijeh Vafa, Elisabeth Israelsson, Bakary Maiga, Amagana Dolo, Ogobara K. Doumbo and Marita Troye-Blomberg. Impact of the IL-4 -590 C/T transition on the levels of Plasmodium falciparum specific IgE, IgG, IgG subclasses and total IgE in two sympatric ethnic groups living in Mali. Manuscript IV. Manijeh Vafa, Marita Troye-Blomberg, Judith Anchang, Andre Garcia and Florence Migot-Nobias, Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal: relation to transmission, age and erythrocyte variants. Malaria Journal 2008; 7:17 TABLE OF CONTENTS SUMMARY ................................................................................................................................................................................... V ORIGINAL ARTICLES ............................................................................................................................................................ VI ABBREVIATIONS .......................................................................................................................................................................1 INTRODUCTION .........................................................................................................................................................................3 THE IMMUNE SYSTEM ............................................................................................................................................. 3 Innate immunity ................................................................................................................................................ 3 Cells of the innate immune system ................................................................................................................................ 4 Acquired immunity ............................................................................................................................................ 5 T lymphocytes ................................................................................................................................................................ 5 B lymphocytes ................................................................................................................................................................ 8 Antibodies and Ig class switching .................................................................................................................................. 9 FcRs .............................................................................................................................................................................. 11 Cytokines ......................................................................................................................................................... 11 IFN-γ ............................................................................................................................................................................. 12 IL-4 ............................................................................................................................................................................... 12 IL-10 ............................................................................................................................................................................. 13 MALARIA .............................................................................................................................................................. 13 Life cycle of malaria parasite ......................................................................................................................... 14 Clinical features of malaria infection ............................................................................................................ 15 IMMUNE RESPONSE TO MALARIA .......................................................................................................................... 16 Clinical immunity ............................................................................................................................................ 16 Innate immunity to blood stages ..................................................................................................................... 16 Acquired immunity to blood stages ................................................................................................................ 18 Role of antibodies in P. falciparum immunity ............................................................................................................ 18 CD4+ T lymphocytes .................................................................................................................................................... 19 Cytokines ......................................................................................................................................................... 20 RELATED BACKGROUND .....................................................................................................................................................22 HUMAN GENETIC FACTORS AND MALARIA IMMUNITY ......................................................................................... 22 RBC variants ................................................................................................................................................... 22 The ABO blood group system ..................................................................................................................................... 22 The haemoglobinopathies ............................................................................................................................................ 23 G6PD deficiency .......................................................................................................................................................... 25 Others............................................................................................................................................................................ 25 Human leukocyte antigen (HLA) .................................................................................................................... 25 Ethnicity .......................................................................................................................................................... 26 FcγR gene polymorphisms .............................................................................................................................. 27 Cytokine gene polymorphisms ........................................................................................................................ 28 GENETIC DIVERSITY OF MALARIA PARASITE ........................................................................................................ 28 Multiplicity of malaria infection..................................................................................................................... 29 MSP2 ............................................................................................................................................................... 30 PRESENT STUDY ......................................................................................................................................................................31 AIM OF STUDY ...................................................................................................................................................... 31 METHODOLOGY .................................................................................................................................................... 32 RESULTS AND DISCUSSIONS.................................................................................................................................. 33 Paper I............................................................................................................................................................. 33 Paper II ........................................................................................................................................................... 34 Paper III .......................................................................................................................................................... 36 Paper IV .......................................................................................................................................................... 38 CONCLUDING REMARKS AND FUTURE PERSPECTIVES ..........................................................................................40 ACKNOWLEDGMENT .............................................................................................................................................................42 REFERENCES ............................................................................................................................................................................44 Manijeh Vafa Homann vii Human genetic factors involved in immunity to Plasmodium falciparum infection viii ABBREVIATIONS ADCC ADCI APC BCR CM CR1 CSR CTLA4 DC DP ELISA FcR Foxp3 G6PD GPI Hb HC HLA IFN Ig IL iRBC IRF-1 LC MHC MOI MSA MSP NK NKR NO PAMPs PCR PfEMP1 PfRBCs PRRs RBCs SNP STAT Tc TCR TGF Th TLR TNF Treg Antibody-dependent cellular cytotoxicity Antibody-dependent cellular inhibition Antigen-presenting cell B-cell receptor Cerebral malaria Complement receptor 1 Class switching recombination Cytotoxic T lymphocyte-associated antigen-4 Dendritic cell Double positive Enzyme-linked immunosorbent assay Fc receptor Forkhead box P3 Glucose-6-phosphate dehydrogenase Glycosylphosphatidylinositol Haemoglobin Heavy chain Human leukocyte antigen Interferon Immunoglobulin Interleukin Infected-Red-Blood Cell Interferon-regulatory factor-1 Light chain Major-histocompatibility complex Multiplicity of infection Merozoite-surface antigen Merozoite-surface protein Natural killer NK receptors Nitric oxide Pathogen-associated molecular pattern Polymerase-chain reaction P. falciparum-erythrocyte membrane protein 1 P. falciparum-infected red-blood cells Pattern-recognition receptors Red-blood cells Single-nucleotide polymorphism Signal transduction and activator of transcription T cytotoxic T-cell receptor Transforming-growth factor T helper Toll-like receptor Tumor-necrosis factor Regulatory T-cell Manijeh Vafa Homann 1 Human genetic factors involved in immunity to Plasmodium falciparum infection 2 INTRODUCTION The immune system The immune system consists of a substantial number of components and mechanisms by which humans are protected from invaders. It detects a wide variety of foreign agents and discriminates them from self components. The immune system is branched into two arms, the innate and the acquired immune system. Innate immunity The innate immune system provides an immediate defence against infections and has been found in almost all multicellular plants and animals. The innate immune system consists of physical, chemical and cellular barriers. The invaders that manage to pass the nonspecific host anatomical barriers, encounter a number of cells, with variable capacities in defence mechanisms, such as neutrophils, macrophages, dendritic cells (DCs) and natural killer cells (NK). The innate immune system detects infections through an array of soluble and membrane bound sensors called pattern-recognition receptors (PRRs). PRRs recognize conserved microbial components, which are usually crucial for microbial survival, known as pathogen-associated molecular patterns (PAMPs). The scavenger receptors (SRs) are members of the PRRs family, present on macrophages, DCs and certain epithelial cells. SRs not only recognize different microbial ligands, but also detect endogenous and modified hostderived molecules [1]. Scavenger receptors are structurally diverse and accordingly categorized into eight classes (A to H) [2]. Pathogens evading from extra-cellular recognition, face another line of detection in the host cytosol. Nucleotide binding and oligomerization domains (NOD)-like receptors (NLRs) and retionid acid-inducable gene I (RIG-I)-like receptors are two recently identified cytosolic PRR that recognize PAMPs in the intracellular compartments [3]. Toll-like receptors (TLRs) are the most studied PRRs. They recognize a wide range of microbial motifs at the cell surface or within the endosomes. TLRs are highly expressed by macrophages, DCs and B cells. Hence they also play an important role in the induction of the adaptive immune response. Eleven TLRs have been identified in humans Manijeh Vafa Homann 3 Human genetic factors involved in immunity to Plasmodium falciparum infection until now. Based on the cellular localization, TLRs are classified into two groups. TLR 3, 7, 8 and 9 are expressed in endosomes, while the second group, including TLR1, 2, 4, 5 and 6, are present on the surface of many different cells [4, 5]. Complement proteins, mannose-binding lectin (MBL) and C-reactive protein (CRP) are other members of the PRR family. Once PRRs detect PAMPs, multiple pro-inflammatory signalling pathways are activated and an effective response against the intruder is mounted [3]. Cells of the innate immune system Macrophages and DCs take up, degrade and present antigens, associated with major histocompatibility (MHC) molecules, on their surface to activate the cells of the acquired immune system. Thus these cells are also called antigen presenting cells (APCs). MHC molecules are glycoproteins found on the surface of cells and are categorized into class I and II. Class I MHC is expressed by almost all nucleated cells, while MHC class II is expressed by professional antigen-presenting cells (i.e. macrophages, DCs and B cells). DCs are leukocytes specialized in presenting antigens. They can activate naïve T and B cells, and thus link the innate and the adaptive immune responses. Immature DCs are widely scattered in peripheral tissues, at the entrance sites of pathogens, and are very efficient in engulfing antigens. Following the ingestion of antigen, DCs travel to lymph nodes, where they present antigens to T cells. Meanwhile, they mature, and their ability to take up antigens is reduced. Instead they get more efficient in presentation of antigens through the upregulation of MHC class II and co-stimulatory CD80 and CD86 molecule expression [6]. Macrophages also express more MHC class II molecules, after activation, but in contrast to DCs, exhibit a greater phagocytic ability. NK cells are large, granular lymphocytes, widely distributed in the body, and can be recruited to the inflammation sites in different tissues. They are the first line of defence against various viral infections. NK cells are capable to induce the death of self cells, which have been modified upon intracellular infections and malignant transformation. NK cells discriminate between target and non-target cells using a set of activating (e.g. NKG2D, NKp30, NKp 44, NKp 46 and CD16) or inhibitory receptors, such as KIR-inhibitory receptors (KIR stands for killer-cell immunoglobulin-like receptor) [7]. Inhibitory receptors recognize 4 MHC class I molecules, as a marker of self cells, and inhibit the activation of NK cells. The expression of MHC class I molecule is usually down-regulated in infected or transformed cells. Thereby, interaction of activating receptors with the ligands on infected or transformed cells, in the absence of MHC class I, leads to NK cells activation. Activated NK cells release perforin and granzymes containing cytotoxic granules, which induce programmed cell death in the target cells [8]. NK cells are identified as CD3-CD56+ lymphocytes. Human NK cells consist of two subsets, CD56bright and CD56dim NK cells. The latter subset is mainly found in the blood and spleen and display stronger cytotoxic effector functions than CD56bright cells, which are more abundant in the lymph nodes [7]. NK cells can rapidly get activated and produce pro-inflammatory cytokines, in particular interferon (IFN)-γ. Therefore, they can influence the adaptive immune response by inducing T helper (Th) 1 type of responses [9]. Acquired immunity While the innate immune system recognizes patterns shared by groups of nonself molecules, T- and B lymphocytes more specifically identify foreign molecules through the specificity of their antigen receptors, T- and B-cell receptors (TCR and BCR). The TCR is a heterodimer of either αβ or γδ chains, which recognizes antigen and is responsible for the specificity of the T cell. This heterodimer is associated with CD3, a multicomponent signaltransducing complex. CD3 triggers intracellular signalling, induced by binding of the antigen to the TCR. In contrast to the BCR, which can identify various cell-surface or soluble antigens without prior modifications, the TCR recognizes antigen-derived peptides in association with MHC molecules, presented on the surface of other cells [10]. T lymphocytes T lymphocytes are derived from bone-marrow-resident stem cells, and develop into self-tolerant, functional T cells through a multistage differentiation process in the thymus. These stages are distinguished by the expression of the key surface molecules, CD4 and CD8. The very first immature cells express none of these molecules and therefore are called double negative (DN). At this stage, αβ and γδ lineages are generated based on the expression of the αβ or γδ subunits of the TCR complex. The αβ lineage differentiates into a CD4CD8 double positive (DP) stage. Further positive and negative selections occur that lead to deletion of non Manijeh Vafa Homann 5 Human genetic factors involved in immunity to Plasmodium falciparum infection functional and autoreactive TCRs. Eventually, MHC-restricted and self-tolerant CD4 and CD8 single positive (SP) T cells are generated and leave the thymus. Thus, mature αβ T lymphocytes comprise of CD8+ T cytotoxic (Tc), CD4+ helper- and regulatory T cell lineages [11]. Despite the expression of antigen-receptors, γδ T cells display innate characteristics and form an independent population of lymphocytes [12]. γδ T cells, in most instances, do not detect peptide antigens presented on the surface of APCs, as αβ T cells do. Instead they recognize alkalamines, nonpeptideprenyl pyrophosphates and phosphorylated uridine and thymidine compounds [13]. CD8+ T lymphocytes participate in responses to infections, through their cytotoxicity and cytokine production. They can produce IFN-γ, tumor necrosis factor (TNF)α, interleukin (IL)-2, IL-4, IL-5, IL-10 and transforming growth factor (TGF)-β. Based on their migration capacity and cytokine production, CD8+ T cells are named Tc1 and Tc2. CD8+ T lymphocytes exert their cytotoxicity through two mechanisms. They can induce apoptosis in target cells by the expression of FasL/CD95L, that makes the target cell to terminate the expression of the receptor Fas/CD95, and thereby initiating apoptosis. They also can kill the target cell via exocytosis of lytic granules that forms pores in the membrane of the target cell, which then allows the lytic granules to enter the cell and to induce apoptosis. CD8+ T cells recognize antigens presented by MHC class I, and need help from DCs and CD4+ T cells to be fully activated. However, the exact mechanism behind this has not yet been fully understood. It has been proposed that DCs present antigen to CD4+ T cells in associations with MHC class II. CD4+ T cells differentiate, proliferate and up-regulate the expression of the CD40L which interacts with CD40 molecules on DCs. This enables DC to activate and transform CD8+ T cells to effector cells that identify antigens in association with MHC class I molecules. A suppressive CD8+ T cell subset, with regulatory function has also been reported [14]. Upon activation, after interaction of TCR with antigen-MHC, naïve CD4+ T cells can differentiate into Th1, Th2, Th17 or regulatory T cell (Treg), according to cytokine milieu (Fig 1). Once differentiated, each lineage is characterized by its own cytokine profile. In the presence of IL-12 and through the activation of signal transduction and activator of transcription (STAT)-4 and T-bet, CD4+ T cells differentiate into a Th1 lineage that produces 6 IFN-γ, a key cytokine in response to intracellular infections. Naïve CD4+ T cells become Th2, when IL-4 is present and this process is regulated by STAT-6 and GATA-3. Th2 cells produce IL-4, a cytokine with an important role in the host defence against helminth infections. IL-23 synergizes with IL-1 and IL-6, inducing Th17 differentiation, a process regulated by STAT-3 and retinoic acid-related orphan receptor (RORγt) transcription factors. Th17 cells secrete the pro-inflammatory cytokine IL-17, and have been proposed to participate in responses against extra-cellular bacteria and fungi, and also to mediate autoimmune diseases. TGF-β1 upregulates forkhead box P3 (Foxp3) expression and induces the differentiation of naïve CD4+ T cells to become Treg, which produce TGF-β1 [15]. This process is regulated by STAT-5. CD4+ T cell subsets can regulate each other via changes in the cytokine milieu. Figure 1: Modified from Chen and O’Shea [15] Treg cells are named after their functional ability to regulate/suppress immune responses, and can develop in the thymus (natural Treg or nTreg) or be induced in the periphery (inducible or iTreg). Foxp3, IL-2 production and CD25 (IL-2 receptor α chain) Manijeh Vafa Homann 7 Human genetic factors involved in immunity to Plasmodium falciparum infection expression are essential for Treg development, and their function and survival. Naturally occurring CD4+CD25+ Treg cells are key elements of self-tolerance maintenance. In the presence of TGF-β, uncommitted CD4+ T cells in periphery can convert into iTreg and release the regulatory cytokine IL-10 (Tr1 subset) or TGF-β (Th3 subset). In vitro studies have shown that suppression of CD4+CD25- effector T cells, by Treg cells, depends on cell-cell contact rather than soluble mediators. Membrane bound TGF-β, the cytotoxic T lymphocyteassociated antigen-4 (CTLA-4) and the glucocorticoid-induced tumour necrosis factor (GITR), are suggested to be important in this contact. Tregs have been shown to directly kill activated CD4+ and CD8+ T cells in a perforin- or granzyme-dependent manner. It has been proposed that upon cell-cell contact, Tregs modulate APCs function and make them unable to activate effector T cells. Moreover, Tregs mediate immune-response suppression via the induction of naïve T-cell differentiation into regulatory cells rather than pathogen-specific effector cells. This phenomenon is called “infectious tolerance” and was suggested to be mediated by IL-10 or TGF-β [16]. Another subset of T lymphocytes, which does not fit well in acquired immunity is the NKT cell subset. These cells are derived from DP thymocytes, and are mainly present in the bone marrow and liver, but can also be found in other lymphoid compartments. NKT cells share characteristics of both NK and T cells [17]. NKT cells get activated upon interaction of their TCR with glycolipids associated with the MHC class I-like molecule CD1d, which is expressed by APCs. NKT cells are known to be major producers of IFN-γ, a crucial cytokine for early activation of macrophages [18]. B lymphocytes B-cell development from hematopoietic cells occurs before birth in the foetal liver and subsequently in the bone marrow. In the bone marrow, B cells develop from pro-B to pre-B cells and eventually to membrane-bound IgM-expressing immature B cells. Naïve mature B cells leave the bone marrow and travel to secondary lymphoid tissues, where they become activated. Following activation, B cells become either memory or plasma cells. Memory B cells are more abundant than naïve resting B cells and can be rapidly re-stimulated by antigens. They often harbour membrane-bound IgG and IgA. Plasma cells are antibody- 8 producing cells. They are large cells, enriched in endoplasmic reticulum, ribosome and golgi. Plasma cells do not express membrane Ig and do not divide [19]. Antibodies and Ig class switching The BCR is present on the surface of B cells, and is a complex of immunoglobulin (Ig) associated with two accessory proteins, called Igα and Igβ. The BCR identifies and binds the antigen, and Igα and Igβ trigger intra-cellular signalling induced by binding of the antigen [10]. Antibodies are expressed and secreted by activated B lymphocytes. Antibodies are glycoproteins with a common structure, but differing in size, charge and amino acid sequence. The basic structure consists of two identical light chains (LC) which are bound to two identical heavy chains (HC) via disulfide bonds and non-covalent interactions. The amino-terminal portion of the chains, called variable (V) region, vary amongst different Igs and determine the antibody specificities. The carboxyl-terminal portions of the chains constitute the constant (C) region and are rather conserved. According to the sequence patterns of the constant region, the heavy chain is grouped into five classes: μ, δ, γ, ε and α, each named an isotype, and the light chain is found in two isoforms, κ and λ. In the process of Ig synthesis, rearrangements in gene segments (VHDJH in HC and VLJL in LC) and somatic mutations produce variations in amino acid sequences in the amino-terminal portions of the chains. This generates 106 to 107 6 different variable regions, which reflects the generation of 7 a repertoire of Igs with 10 to 10 various specificities. Based on the heavy chain, antibodies are also classified into five classes, IgM, IgD, IgG, IgE and IgA. In humans, minor variations in amino acid sequences of the α and the γ heavy chains result in further classification of IgA and IgG into two (IgA1 and IgA2) and four (IgG1-4) subclasses, respectively. Rearrangement in the Ig heavy chain occurs during B cell development from pro-B to pre-B cell. In the pre-B cell, the membrane bound μ chain and the surrogate light chain (a complex of two proteins which are non-covalently bound to form a light chain-like structure) associate with the Igα/Igβ heterodimer to build the pre-BCR. The light-chain gene is then rearranged, and intact IgM is displayed on the immature B cells. In an antigenindependent manner, naïve mature B cells begin to splice the variable region genes with the δ Manijeh Vafa Homann 9 Human genetic factors involved in immunity to Plasmodium falciparum infection chain constant region and express IgD (in addition to IgM) on their surface to form fully mature naïve B cells. All Igs consist of two regions, the Fab and the Fc. The Fab contains the variable region of the Ig, differs from one antibody to the other, and is the part of the antibody that identifies and binds antigenic epitopes. The Fc fragment is conserved in each class and subclass of the antibodies and mediates effector functions specific to that class. A set of antibodies of different classes can be produced in response to a particular antigen as a result of class switching recombination (CSR). In the IgH locus, the genes for the constant regions are positioned in the following order, Cμ, Cδ, Cγ, Cα and Cε. In CSR, one of these C regions recombines to the rearranged variable (VDJ) region to generate the respective isotype of the antibody. The first antibody produced after exposure to antigen is mainly of the IgM class. By substituting the CH regions of IgM with those of IgG, IgA and IgE, CSR modulates the effector functions of antibodies without changing their specificity. Antibody-class switching is mainly regulated by extra-cellular signals, in particular cytokines. [19-23]. Antibody-mediated effector functions Antibodies recognize and bind to antigens, but they can not remove or kill the pathogen by themselves. Antibodies interact with other proteins or cells to perform the effector function of a humoral response. Binding of antibody-antigen complexes to the Fc receptors (FcR) on the surface of phagocytic cells, promotes the phagocytosis of the antigen (opsonization). In humans, IgM, IgG1, IgG2 and IgG3 activate the classical pathway of complement. IgA1, IgA2 and IgE can activate the alternative pathway of complement. Complement refers to a set of serum glycoproteins which interact and cooperate with other immune molecules or cells to remove the antigen or kill the pathogen. IgG also mediates antibody-dependent cellular cytotoxicity (ADCC). ADCC is the initiation of cytotoxic activities of the effector cells against the pathogen, upon binding of antibody in complex with antigen, to the FcR on a number of immune cells, particularly NK cells. No effector function has been identified for IgD [19, 20, 24]. 10 FcRs Antibody-mediated responses are regulated by different FcRs. Unlike, CH regions of IgM and IgD, CH regions of IgG, IgE and IgA can bind to their respective receptors (Fcγ, Fcε and Fcα) on innate immune cells and promote their pro-inflammatory and killing functions. This links the specificity of adaptive responses to potent effector functions of the innate immune response. Co-expression of activating and inhibitory FcRs on innate and adaptive immune cells regulates the immune response and thus avoids tissue damage induced by exaggerated immune responses. In addition to immune cells, FcRs are expressed on endothelial cells, mesangial cells and osteoclasts [22, 25]. B cells only express inhibitory FcRs (FcγRIIB) [26]. Cytokines Cytokines are low molecular weight polypeptides produced by different cells, whereby cells communicate with each other. Cytokines are released in response to a variety of inflammatory stimuli such as infectious agents and their products, or in response to other cytokines. In general, a cascade of cytokines is produced by different cell types, eventually ending with the final immune-effector function. The majority of cytokines are transiently secreted and generally have a short half-life. Accordingly, cytokines are mostly produced and act locally, either on the same cell that produced them (autocrine effect) or on the adjacent cells (paracrine effect). A minority of cytokines enters the circulation and acts on distant tissues (endocrine or hormonal effect). According to their role in inflammation, cytokines are classified into pro- and anti-inflammatory cytokines. Cells at the site of inflammation such as DCs, macrophages and mast cells release a panel of pro-inflammatory cytokines, including IL-12, IL-18, IL-8, TNFα and IFN-γ, which causes the inflammation. Anti-inflammatory cytokines, like IL-4, IL-10 and IL-13, are able to down-regulate the production of pro-inflammatory cytokines. The balance between pro- and anti-inflammatory cytokines during the course of an inflammation determines the outcome of the inflammatory response. Importantly, the early inflammatory cytokine production also influences the subsequent development of the adaptive immune response [27, 28]. Cytokines are also involved in the regulation of the adaptive immunity. They play an important role in activating and regulating lymphocytes [27]. Cytokines are important Manijeh Vafa Homann 11 Human genetic factors involved in immunity to Plasmodium falciparum infection in the regulation of humoral response and antibody-class switching. For example in humans, IgG4 and IgE production is induced by IL-4 and IL-13. IL-4-induced IgE production is inhibited by IL-10, IL-12, IFN-α, IFN-γ and TGF-β. Production of IgM, IgG1, IgG3 and IgA is enhanced by IL-10. TGF-β induces isotype switching to IgA, and IgG2 production is induced by IFN-γ [19, 21]. In addition to the regulation of the innate and the adaptive immune responses, cytokines also regulate cell growth and differentiation, and influence embryo implantation and foetal development [27, 28]. Below, cytokines of interest in recent studies are described more in detail. IFN-γ IFN-γ is crucial in both innate and adaptive immune responses. It plays a critical role in immunity against intracellular pathogens and in tumor control. IFN-γ is mainly produced by NK, NKT, Th1 and CD8+ T lymphocytes. The constitutive expression of IFN-γ mRNA by NK- and NKT cells allows them to mount a rapid IFN-γ response against infections. CD4+- and CD8+ T cells produce little IFN-γ shortly after initial activation. IFN-γ induces expression of MHC class I and II molecules on APCs, and consequently increases their antigen-presentation capacity. IFN-γ enhances the phagocytic capacity of macrophages. IFN-γ also induces the production of pro-inflammatory cytokines and antimicrobial products such as nitric oxide (NO) in macrophages. In neutrophils and monocytes, IFN-γ stimulates the expression of high-affinity FcγRs. IFN-γ is a growth-promoting factor for T lymphocytes, whereas, IFN-γ inhibits the IL-4-induced B-cell growth. IFN-γ amplifies the recruitment of lymphocytes into tissues and prolongs their activation. The excessive production of IFN-γ has been associated with a number of autoimmune and inflammatory diseases. IFN-γ production by NK cells is inhibited by TGF-β [8, 29]. IL-4 IL-4 is mainly produced by activated Th2 cells [28]. Mast cells also secrete IL4, upon engagement of their FcεR or FcγR [30]. IL-4 induces proliferation and differentiation 12 of activated B cells, and enhances the expression of MHC class II and the IgE low affinity receptor (CD23) on resting B cells [31]. In activated B cells, IL-4 induces IgG4 production. IL-4 also induces isotype switching from IgM to IgE. IL-4-induced antibody-class switching is antagonized by IFN-γ [32, 33]. IL-4 stimulates NK cells, CD4+ T- and CD8+ T-cell proliferation [34]. IL-4 suppresses the production of IL-1β, TNF-α and IL-8 [28]. IL-10 A wide variety of cells, such as activated monocytes, macrophages, mast cells, epithelial cells, B cells, CD4+- and CD8+ T cells, produce IL-10. Also a variety of blood cells are affected by the inhibitory or stimulatory functions of IL-10. IL-10 promotes B-cell proliferation, survival and differentiation into Ig-secreting plasma cells. In addition, IL-10 displays inhibitory effects on various blood cells. The inhibitory effect of IL-10 is believed to be due to the induction of regulatory T cells. IL-10 inhibits IL-1, IL-6, TNF-α, IL-12 and subsequently IFN-γ production. IL-10 also down-regulates macrophages, NK cells and Th1 cells. IL-4- and IFN-γ-induced expression of MHC class II-antigen is suppressed by IL-10. Thus, IL-10 is an important regulatory cytokine [16, 19, 35-38]. Malaria The term malaria originates from Italian ‘mal aria’, which means ‘bad air’, for the incidence of the disease in marshy areas [39]. The malaria parasite has infected humans for 50,000-100,000 years [40] and remains a major cause of morbidity and mortality in the developing word, particularly in sub-Saharan Africa. About half a billion episodes of clinical malaria is estimated to occur each year, which results in deaths of more than one million children in sub-Saharan Africa alone [41]. Malaria is a vector-borne infectious disease caused by an intracellular protozoan parasite of the genus Plasmodium. More than 100 species of Plasmodium parasites are known, of which only four types can infect humans: P. falciparum, P. vivax, P. ovale and P. malariae. Among these four species, P. vivax and P. falciparum are the most prevalent Manijeh Vafa Homann 13 Human genetic factors involved in immunity to Plasmodium falciparum infection species and P. falciparum is the main cause of severe and fatal malaria [42]. Plasmodium parasites are transmitted to the vertebrate host by the female Anopheles mosquitoes. Life cycle of malaria parasite The life cycle of the malaria parasite is complex, includes sexual and asexual developmental stages and requires both mosquito and vertebrate hosts to be completed. The infected Anopheles mosquitoes carry Plasmodium sporozoites in their salivary glands. When an infected mosquito takes a blood meal, it injects 5 to 20 infective sporozoites into the bloodstream of its victim (Fig. 2). http://en.wikipedia.org/wiki/Image:MalariacycleBig.jpg Figure 2: Plasmodium falciparum life cycle Sporozoites migrate to the liver within 30 minutes and invade hepatocytes, where they multiply asexually for a period of 9-16 days. This division is called schizogony and generates merozoite forms of the parasite. Each sporozoite gives rise to thousands of merozoites which 14 are released into the bloodstream when the infected hepatocyte ruptures. Released merozoites invade erythrocytes. Once inside the erythrocyte, the merozoite enlarges to a uni-nucleated cell named trophozoite. Trophozoites undergo asexual divisions and produce schizonts with multiple nuclei. The schizonts then segment to generate 15 to 30 merozoites which are released into the blood, ready to invade new erythrocytes. Several such cycles occur and periodic, synchronized release of merozoites and toxins into the blood, results in malaria associated waves of fever (within 48–72 hours, depending on the species of Plasmodium). A small proportion of merozoites differentiate into micro- and macrogametocytes (male and female, respectively). A mosquito taking a blood meal on an infected individual may ingest these gametocytes. In the mosquito gut, the gametocytes form male and female gametes, which fertilize to form zygotes. These zygotes further differentiate, migrate through the mosquito gut wall, and undergo repeated divisions to produce large number of sporozoites. The infective sporozoites travel to the salivary gland and the life cycle is completed. Clinical features of malaria infection Infections with malaria parasites have a wide variety of symptoms, ranging from asymptomatic carriage of infection to severe malaria or even death. A large number of individuals living in malaria endemic areas carries parasites without presenting signs of clinical malaria. Clinical malaria can be categorized into mild (uncomplicated) or severe (complicated) malaria. In general, malaria is a curable disease if diagnosed and treated quickly and properly. A combination of fever, chills, sweats, headaches, nausea, malaise, cough, diarrhoea and muscular pain is presented in uncomplicated forms of malaria disease. Uncomplicated malaria can be caused by all four Plasmodium species, whereas P. falciparum is responsible for severe pathology and almost all deaths. Sequestration, a characteristic feature of P. falciparum infection, happens when P. falciparum infected red blood cells (iRBC) adhere to uninfected RBCs, endothelial cells and in some cases to placental cells (Fig. 1). Sequestration of iRBCs in various organs such as brain, kidney, placenta and lung may lead to serious organ failures or abnormalities in the patients’ blood and consequently to severe pathology [43]. Cerebral malaria (CM), severe Manijeh Vafa Homann 15 Human genetic factors involved in immunity to Plasmodium falciparum infection anaemia, hemoglobinuria, hypoglycaemia, renal failure, pulmonary oedema, abnormalities in blood coagulation and thrombocytopenia, convulsion and shock are some complications of severe malaria. Immune response to malaria The immune response to the malaria parasite, as the parasite’s life cycle, is complex and depends on the parasite species and on the stage of infection [44]. The infection starts when the sporozoites enter the human body. At this level protective immune responses to the parasite rely on antibodies against antigens on the surface of sporozoite, like the circumsporozoite protein (CSP). During the liver stage, when the parasite is inside the hepatocyte, a predominant cellular immune response is required. Later on, when the infection involves erythrocytes, protective immunity depends mainly on antibody responses. Merozoite-derived antigens expressed on the surface of the infected RBCs, merozoite surface proteins/antigens (MSP/MSA) and parasite antigens released into circulation, following erythrocyte rupture, are the major targets of antibody response in this stage [45]. Clinical immunity Individuals residing in malaria endemic areas experience repeated infections and acquire immunity to clinical malaria, termed “clinical immunity”, which prevents the disease despite the persistence of infection [46]. Clinical symptoms of malaria are related to the blood stage of infection. Thus, it is important to understand the immune response during this stage. The present study has mainly focused on the blood stage of the malaria infection. Hence, immune responses to asexual blood-stage of malaria parasite, specifically P. falciparum, are overviewed in this thesis. Innate immunity to blood stages Following the initial rise in parasitemia in blood, innate type of immune responses control parasite expansion, but this is usually not enough to eliminate the infection. 16 Macrophages, DCs, NK cells and γδ T cells are effector cells involved in the innate immune responses to the malaria parasite [47]. PRRs mediate direct interaction between innate immune cells and the parasite. To date, TLR2, 4 and 9 have been shown to recognize malarial antigens in humans. The P. falciparum glycosyl-phosphatidylinositol (GPI) interacts with the TLR2/TLR1 complex, with a minor contribution of TLR4, and induces cytokine production in macrophages. Hemozoin, a product of haemoglobin digestion by P. falciparum in RBC, was surprisingly identified as a non-DNA ligand for TLR9. TLR9 was earlier described as a receptor for CpG-containing DNA. More recent studies have suggested that hemozoin presents DNA to TLR9, while hemozoin by itself, is not able to induce innate immune responses [5]. Plasmodium profilinlike proteins have been shown to activate the innate immune system in mice through TLR11 [48]. Of other PRRs involved in recognition of P. falciparum-derived antigens, the scavenger receptor CD36 can be mentioned. The P. falciparum erythrocyte membrane protein-1 (PfEMP-1) expressed on the surface of iRBC is the ligand for CD36 [4]. Binding of PfEMP-1 to CD36 enables macrophages to attach to, and engulf iRBCs in the absence of cytophilic or opsonizing malaria-specific antibodies. This suggests an important role for macrophages in innate immunity to malaria [49]. Besides ingesting iRBCs, macrophages can take the parasites out of iRBCs and let the parasite-free RBCs back to circulation [50, 51]. Macrophages can also kill/inhibit in vitro growth of the asexual blood stage through the release of NO and H2O2 and also mediate antibody-dependent cellular inhibition (ADCI) [52, 53]. Activation of DCs is one of the first events in the innate response to malaria. Malaria parasites interact with DCs to modulate their functions. Although studies have revealed a role of DCs in immune responses to many intracellular pathogens, not much is known about the role of DCs in malaria immunity. The first study on the role of DCs in malaria, showed that P. falciparum infected red blood cells (PfRBC) adhere to DCs, inhibit the maturation of DCs [54], and subsequently reduce their capacity to activate T cells [55]. PfRBCs were later shown to interact with the CD36 and CD51 molecules on DCs [54, 56]. Upon this interaction, DCs upregulate the expression of the co-stimulatory molecules CD40, CD80 and CD86, and instead of initiating a pro-inflammatory response, produce IL-10 and Manijeh Vafa Homann 17 Human genetic factors involved in immunity to Plasmodium falciparum infection elicit an anti-inflammatory response [57]. However, studies using experimental animals have shown that DCs from infected mice are fully functional APCs [58]. γδT-cells have been reported to expand during the first few days of P. falciparum infection [59]. P. falciparum–derived phosphorylated non-peptide antigens have been identified as ligands for human γδT-cells [60]. In vitro studies have shown that Pfactivated γδT-cells produce large amounts of IFN-γ [61, 62] and inhibit parasite growth [63]. Extracellular merozoites or late schizonts have been suggested to be targets for γδT-cells, since γδT-cells have been shown to inhibit growth of the asexual blood stages in vitro at the time of reinvasion [64]. γδT-cells kill the parasite by exocytosis of granulysin and perforin, following direct contact with iRBCs [65]. NK cells can lyse PfRBC without prior sensitization. Studies by ArtavanisTsakonas et al. have suggested that NK cells are the earliest source of pro-inflammatory cytokine IFN-γ [66], which is essential to control malaria parasitemia [67, 68], and that NK cells produce IFN-γ following a direct interaction with PfRBC [69]. However, recent studies have revealed that the innate IFN-γ production was mainly (in 93.3% of the analysed individuals) derived from γδT-cells. The majority of these γδT-cells expressed NK receptors (NKR). The overall IFN-γ levels produced by NK-like γδT-cells were positively associated with the expression of CD94 on these cells [70]. Acquired immunity to blood stages Role of antibodies in P. falciparum immunity Erythrocytes do not express the classical MHC class I and II molecules. Therefore, antibodies and B-cells are crucial in clinical immunity. Reduction of parasitaemia and clinical disease after passive transfer of gamma-globulins from immune donors to malaria patients first suggested a protective role for IgG antibodies [71]. Antibodies are generally accepted as indicators of infection. Malaria protective antibodies are mainly restricted to certain classes and subclasses. Analyses of anti-malarial IgG subclass profiles, in humans, have suggested that 18 IgG1 and IgG3 (called cytophilic antibodies) provide protection, whereas IgG2 and IgG4 do not. However, IgG2 has recently been demonstrated to be associated with malaria protection in individuals carrying a certain polymorphism in the FcγRII gene [72]. IgG4 is thought to counteract with anti-parasitic functions of IgG1 and IgG3 through inhibiting their binding to the antigens [73]. While IgG has been correlated to protection against malaria disease, antibodies of the IgE isotype have been linked to severe pathology. Higher levels of total and anti-malarial IgE were detected in patients suffering from cerebral malaria than individuals with uncomplicated malaria [74]. However, recent studies in Tanzania and Senegal have suggested a protective role for IgE against malaria, where elevated levels of P. falciparumspecific IgE were shown to reduce the risk of subsequent malaria attack [75, 76]. Thus, the role of IgE (total and malaria specific) in the pathogenesis/immunity to malaria is still unclear. Malaria-specific antibodies exert their protective roles through mediating a number of anti-parasitic effector functions including: inhibition of cytoadherence and thus preventing sequestration of iRBCs and instead letting them be removed by spleen [77]; inhibition of RBC invasion [78] and antibody-dependent cytotoxicity [79]. Anti-malarial antibodies are involved in the ADCI mechanism where the parasite growth is inhibited by antibodies bound to Fc receptors on phagocytes [80]. Antibodies may also facilitate parasite clearance by opsonization and phagocytosis by macrophages [81]. CD4+ T lymphocytes CD4+ T cells are important in the development and regulation of the immune response to malaria infection. Murine experimental systems have shown that Th1-produced cytokines, such as IFN-γ, initiate inflammatory responses needed to control infection at early stage. Cytokines produced by Th2 lymphocytes, e.g. IL-4 and IL-10, not only help B cells to produce antibodies, but also suppress Th1 responses, which if left uncontrolled, can lead to immunopathology [82, 83]. While Th1/Th2 type of responses seem to be of importance in certain murine experimental systems, the situation is less clear in humans. Another subset of CD4+ T lymphocytes involved in malaria blood stage immune response is Treg cells. A direct evidence for the role of Tregs in the outcome of malaria infection comes from studies in mice. The significant delay in parasite growth in CD4+ CD25+ depleted naïve mice, infected with P. berghei NK65, revealed that effector responses are Manijeh Vafa Homann 19 Human genetic factors involved in immunity to Plasmodium falciparum infection enhanced in the absence of Tregs. Depletion of CD4+ CD25+ T cells in P. yoelii 17XL infected mice, was also shown to result in amplified T-cell responses to parasite antigens and control the normally lethal infection [84]. Moreover, studies in P. yoelii 17XL susceptible (BALB/c) and resistant (DBA/2) mice have indicated an increase in the Treg population early after infection, in BALB/c but not DBA/2 mice, suggesting that the suppression of the Th1 response during early infection probably results in enhanced parasitemias and fatal disease in BALB/c mice [85]. Similarly, in vivo studies in humans have shown that blood-stage infection rapidly induces Tregs which in turn results in a burst of TGF-β production. This leads to reduced pro-inflammatory cytokine production and antigen-specific immune responses and consequently gives rise to higher rates of parasite growth [86]. Optimally, it looks as if Treg activation and/or Treg-derived cytokine (IL-10 and TGF-β) production are needed to be triggered at the time that pro-inflammatory cytokines have brought parasite replication under control. Cytokines Besides the ability of individuals to control parasite multiplication, the term “clinical immunity” also refers to the ability of individuals to control the inflammatorycytokine response induced against the parasite [87]. In mice it seems as if an early pulse of pro-inflammatory cytokines is essential to limit the initial parasite replication [88]. An early IFN-γ response is crucial for the resolution of the primary infection [89]. Elevated plasma levels of IFN-γ have also been linked to resistance to re-infection by P. falciparum [90]. TNF-α synergises with IFN-γ to induce production of NO and thus parasite killing [91]. A pro-inflammatory response is essential for parasite clearance. However, excessive production of these cytokines may lead to malaria pathogenesis. Malaria patients have been shown to have higher plasma levels of IFN-γ as compared to asymptomatic individuals, suggesting a correlation between IFN-γ levels and malaria disease. Elevated IFNγ levels have also been associated with the onset of CM [92]. Circulating levels of TNF-α have been correlated with the risk of death from CM [93]. Anti-inflammatory cytokines have also been shown to play a critical role in malaria pathogenesis. An infection by P. chabaudi chabaudi AS has been shown to be more lethal in IL-10-deficient mice than in their wild type counterparts [94]. Similarly, in humans, lower IL-10 levels were found in fatal cases than survivors [95]. African children suffering 20 from anaemia have been reported to have low serum levels of IL-10 [96]. In anaemic children, the ratio of IL-10/TNF-α concentrations was linked to the severity of anaemia [97]. Thus, a delicate balance in levels and timing of pro- and anti-inflammatory responses may determine the degree of parasitemia, severity and outcome of the disease. Manijeh Vafa Homann 21 Human genetic factors involved in immunity to Plasmodium falciparum infection RELATED BACKGROUND Human genetic factors and malaria immunity Development of clinical immunity may be influenced by age, pregnancy, nutritional status, co-infections with other pathogens and host genetic factors [98]. The importance of human genetic factors in immunity to malaria has long been suggested by the high frequency of some genetic disorders in malaria endemic areas, which despite general disadvantages, confer protection against malaria. RBC variants RBCs are crucial in the life cycle of the malaria parasite. The parasite uses the RBC as shelter and food resources. Moreover, the interaction between infected RBCs and uninfected RBCs and other tissues results in pathogenesis of the disease. Therefore, changes in RBC structure or function may interfere with RBC invasion by the parasite and/or parasite growth and multiplication inside RBCs, and consequently affect the outcome of the disease. Following, some RBC variants important in malaria immunity and included in this study are listed. The ABO blood group system Polymorphisms in carbohydrate structure of glycoproteins and glycolipids on the surface of erythrocytes generate the ABO system, which is the most medically important RBC polymorphism. Studies on associations between the ABO system and malaria, have reported contradictory results. Some have shown a correlation between the ABO system and severity of the disease, while others reported no correlations between malaria disease and the ABO blood groups [99, 100]. However, blood groups A, B and AB have been shown to form rosettes (binding of iRBCs to uninfected ones and to endothelial cells, which is linked to severe pathology caused by P. falciparum), more readily than blood group O [101]. Thus, individuals with blood group A, B and AB are more likely to develop CM compared to 22 individuals with blood group O [102]. In malaria endemic areas, the ratio of blood group O to non-O is greater than 1, whereas this ratio is smaller than 1 in other populations. This has been suggested to be the result from the selection of blood group O by P. falciparum infections. However, no study has linked the ABO system to prevalence of infection, frequency of repeated malaria episodes or anti-malaria antibody levels [99, 100]. The haemoglobinopathies Haemoglobinopathies, disorders of haemoglobin (Hb) structure and production, are the most common human genetic disorders. Hb is the iron-containing protein found in RBCs that transports oxygen from the lungs to the rest of the body. Hb molecules consist of four polypeptide chains named globins (usually 2 alpha and 2 beta chains) and an ironcontaining molecule, termed haem, attached to each chain [103]. The haemoglobinopathies are classified into two categories. One caused by variations in Hb structure such as HbS, C and E, and the other results from variable production of normal α- or β-globin, causing α- and β- thalassemia, respectively [104]. The high prevalence of these disorders in malaria-endemic areas, are considered as being the result of selection under malaria pressure [105]. Sickle cell disease Sickle cell disease results from a point mutation in the β-globin subunit of haemoglobin. Haemoglobin S is formed when two wild-type α-globin subunits are associated with two mutant β-globins. Normal RBCs contain HbA and are quite elastic, which enables the cells to deform in order to pass through capillaries. HbS carrying RBCs are distorted, tend to lose their elasticity, and have shorter life time as compared to the normal ones. The homozygous form of HbS (HbSS) causes a severe disease and is lethal at early childhood, in the absence of adequate treatment. The heterozygous state (HbAS), also called sickle-cell trait, has long been linked to protection against severe malaria [106, 107]. A number of mechanisms, by which sickle-cell disease may mediate resistance to malaria, has been proposed in several studies [108]. In vitro studies have indicated: enhanced phagocytosis of ring-stage parasitized HbAS erythrocytes [109], reduced Manijeh Vafa Homann 23 Human genetic factors involved in immunity to Plasmodium falciparum infection erythrocyte invasion and impaired intra-erythrocytic parasite growth in AS-RBCs, as compared to infected normal erythrocytes [110, 111]. HbAS parasitized erythrocytes also tend to sickle [110], which may enhance removal of premature infected erythrocytes in the spleen [110, 112]. Enhanced immune responses to parasitized HbAS erythrocytes have also been reported [113, 114]. A recent study has shown significantly reduced binding of P. falciparum infected AS erythrocytes to micro-vascular-endothelial cells and blood monocytes as compared to infected normal RBCs [115]. This may take part in protection to severe malaria conferred by the sickle cell trait. HbAS has not been shown to influence asymptomatic parasitaemia, however, it protects against severe and fatal malaria [107]. Thus, the precise mechanism whereby sickle cell disease protects against malaria is yet unclear. Alpha-thalassemia The haemoglobin-alpha-chain gene is located on chromosome 16. Normally, there are 2 alpha-chain genes on each chromosome, 4 in total. Alpha-chain-protein production is evenly divided among the four genes. Alpha-thalassemia occurs when one or more genes are mutated or fail to work. The severity and type of disease depend on the number of affected genes, ranging from 4 genes deleted, to one deleted gene. The first defect is fatal prior to or shortly after birth, whereas the latter form is clinically silent (α+) [103]. Depending on the size of the deletion, α+ is categorized into different types [116]. In Africa, -α3.7 is the most prevalent α+–thalassemia [105]. Several studies have indicated that α-thalassemia confers significant protection against severe and fatal malaria [117, 118]. It has also been related to protection against mild malaria [118, 119]. However, α-thalassemia does not protect against asymptomatic parasiteamia [104], and does not influence parasite densities in individuals that go on to develop the disease [117-119]. Studies by Carlson et al. [120] proposed that α-thalassemic individuals are less likely to develop severe malaria, probably due to impaired ability of αthalassemic RBCs to form rosettes. It has been shown that α-thalassemia gives rise to reduced expression of the red-cell complement receptor 1 (CR1), an essential receptor for rosetting [121]. Therefore, impaired CR1-mediated rosetting may confer resistance to severe malaria seen in α-thalassemic individuals. 24 G6PD deficiency Glucose-6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme that functions as an antioxidant, and thus protects the cell from oxidative damage. Although the lack of this enzyme in RBCs is fatal, mutant forms with defects in function are common. The G6PD gene is located on the long arm of the X chromosome. Four hundred and forty two G6PD variants have been identified. More than 130 variants were found at the DNA level that lead to impaired enzyme activity [122]. Malaria parasites break down haemoglobin inside the RBC. This gives rise to products such as oxidized iron, which are toxic to the parasite. Any defects in G6PD function, which is important for cells to overcome the oxidative stress, is lethal for the parasite and can thus confer resistance to malaria infections [122]. The geographic distribution of G6PD variants matches with the historical distribution of malaria disease [123, 124]. The mutant G6PD A- with 10-50 percent of normal enzyme activity, is the most common G6PD deficiency allele in sub-Saharan Africa [125-127]. In two large case-control studies, Ruwende et al. have shown that G6PD A- causes a 46% reduction in the risk of developing severe malaria in heterozygous females, and 58% in hemizygous males [125]. Studies by Roth et al. revealed that parasitemia levels in cultures with G6PD-deficient RBCs (from hemizygous males and heterozygous females) were three times lower than those cultured in normal blood (blood from individuals with normal RBC) [128]. It has also been shown that ring-stage infected G6PD deficient RBCs are phagocytosed 2.3 times more intensely than infected normal RBCs [129]. However, the actual mechanism(s) whereby G6PD deficiencies protect against malaria is still not well understood. Others Duffy and Gerbich blood-group antigen deficiencies, HbC, HbE, β-thalassemia, CR1 polymorphism and Southeast Asian Ovalocytosis (SAO) are other examples of RBC variants, involved in malaria resistance [104]. Human leukocyte antigen (HLA) The MHC gene loci in humans are referred to the HLA complex and are highly polymorphic. There are evidences that specific HLA alleles might be correlated to Manijeh Vafa Homann 25 Human genetic factors involved in immunity to Plasmodium falciparum infection susceptibility/resistance to malaria disease. For instance, studies in The Gambia have correlated an HLA class I antigen, HLA-B*5301, and an HLA class II haplotype, HLADRB1*1302-DQB1*0501, with resistance to severe malaria [130]. HLA-DQB1*0501 has been linked to the reduced risk of re-infection and anaemia in Gabonese children [131], where DRB1*04 and DPB1*1701 were related to severe disease [132]. A case-control study in Ghana has revealed an association between HLA-DRB1*04 and severe malaria [133]. Ethnicity A recent study in Kenya has estimated that almost 25% of the variation in malaria incidence is a consequence of host genetic factors. This ratio was far beyond the effects expected for the haemoglobinopathies alone. The HbS gene, which is the strongest known resistance genetic factor, constitutes only 2% of the total variation. This suggests that a large number of protective genes exits in the population, each of which taking a small part in the protection [134]. Ethnic differences in susceptibility to malaria offer an important approach to identify immune genes behind the resistance or susceptibility to malaria. Studies in Mali [135], Burkina Faso [136]and Sudan [137] have revealed a significant inter-ethnic variation in susceptibility to malaria. In spite of exposure to the same parasite and living under the same transmission intensity, the Fulani ethnic group less frequently develop clinical malaria, as compared to their sympatric ethnic groups (the Dogon in Mali, the Mossi and Rimaibe in Burkina Faso and the Masaleit in Sudan). The Fulani have also been reported to have higher anti-malarial-immune reactivity and lower asymptomatic infection prevalence than their neighbours [135, 136, 138-142]. This relative resistance to malaria has been shown not to be correlated to classical malaria-resistance genes, since higher frequencies of α-thalassaemia, G6PD, HbC and HLA-B*5301 were found in the Mossi and Rimaibe as compared to the Fulani. Similar frequencies of HbS were observed amongst three sympatric groups [143]. Similarly in Mali, HbC was more frequent in the Dogon group than in the Fulani, and HbS showed a low similar frequency in both communities [135]. A recent study in Sudan has revealed a significantly higher frequency of HbAS in non-Fulani (Masaleit) compared to the Fulani [144]. Higher frequencies of spleen enlargement have been reported in the Fulani than non-Fulani groups [135, 137, 145-147]. 26 A recent study in Burkina Faso has revealed enhanced expression of both Th1(IL-18 and IFN-γ) and Th2 (IL-4 and GATA 3) related genes, in peripheral blood mononuclear cells (PBMC) from the Fulani compared to the Mossi. However, the Fulani had a reduced expression of Treg determinant genes (Foxp3 and CTLA4), suggesting that the defect in functional Treg in the Fulani could reflect their higher resistance to malaria [148]. These data are in agreement with previous reports from Mali, showing a more efficient Th1 and Th2 response towards P. falciparum antigens in the Fulani [141]. Thus, it is obvious that many different factors are involved in the protection against malaria, and these factors might differ in different populations. FcγR gene polymorphisms Three families of human FcγRs have been identified, FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Each FcγR family constitutes of several isoforms. A-, B- and C isoforms have been described for FcγR- I and II, whereas FcγRIII includes only two isoforms, A and B. The FcγR isoforms are expressed on a variety of cell types and vary in their binding affinity to different IgG subclasses. A number of studies has related some FcγRs gene polymorphisms (for instance FcγRIIA-G494A [149-151], FcγRIIB-T695C [152] and FcγRIIIB-NA1/NA2 [149]) to susceptibility to and/or severity of malaria disease. The G494A SNP in the FcγR IIA gene gives rise to amino acid exchange from arginine (R) to histidine (H) at position 131. FcγR IIA-131H is the only human FcγR which efficiently binds the IgG2 subclass [153]. The H allele has been correlated to both protection and susceptibility to malaria in different studies (reviwed by Braga et al. [154]). Studies in Sudan [144] and Mali [Israelsson et al., unpublished] have shown significantly higher frequencies of the H allele and the H/H genotype in the Fulani as compared to the non-Fulani. However, whether or not the FcγR IIA131H allele plays any role in reducing the susceptibility to malaria disease in the Fulani, is not clear yet. Manijeh Vafa Homann 27 Human genetic factors involved in immunity to Plasmodium falciparum infection Cytokine gene polymorphisms Accumulating evidence suggests that inter-individual variations in the production of cytokines involved in malaria immunity may influence the course of infection, and determine the outcome of the disease. It is also evident that the ability to produce variable amounts of cytokines can be genetically regulated. A number of polymorphisms in cytokinerelated genes that modifies cytokine expression and/or function has been identified. For example, replacement of a C by a T at -590 position in the IL-4 gene promoter has been described to result in enhanced IL-4 production [155]. Similarly, a G to A substitution in the IL-10 gene promoter, position -1087, has been identified and related to elevated levels of IL10 production [156]. Quite a few studies have tried to relate polymorphisms in cytokine-related genes to various infections/diseases, including malaria. For instance, enhanced levels of TNF-α in cerebral malaria patients have been shown to be a consequence of a variation in the TNF-α promoter in these individuals [157-159]. In Thai malaria patients, the IL-13 -1055 T allele has been shown to protect against severe malaria [160]. The IL-4 -590 T allele has been correlated to elevated IgE levels in asthmatic children [155]. This allele has also been associated with elevated total IgE levels in children suffering from severe malaria. However, the frequency of the T allele did not differ between severe and mild malaria cases [161, 162]. In Burkina Faso, the IL-4 -590 T allele was associated with increased malaria-specific IgG levels within the Fulani individuals but not in non-Fulani [163]. Polymorphisms in interferon-regulatory factor (IRF)-1 have recently been linked to the control of P. falciparum infection in the same study area [164]. However, none of the studied polymorphisms has yet been suggested as a determinant of susceptibility/resistance to malaria infection/disease. Genetic diversity of malaria parasite The success of P. falciparum in evading host immunity, particularly in avoiding antibody-mediated immunity against parasite-derived surface proteins, is attributed to the antigenic diversity in the parasite population as a whole, as well as antigenic variation in the individual parasite. The malaria parasite is able to switch the expression of surface antigens and, thus, change the antigenicity during the course of infection [165]. The existence of phenotypically variable strains of the parasite in endemic areas enables the P. falciparum 28 parasite to re-infect previously exposed individuals. This leads to strain and variant specific immunity [166, 167]. Human experimental malaria studies have shown that a primary infection with a certain strain generates protective immunity against the infecting strain but not against infections by other strains [168]. This has been suggested to be the reason why long term exposure (years), to a pool of variable parasite strains, is required for the development of naturally acquired immunity to the Plasmodium parasite [169]. Thus, the acquired immunity has been suggested to develop after infection by multiple parasite strains. Multiplicity of malaria infection MOI is the number of various P. falciparum strains simultaneously infecting a single person. Individuals residing in malaria endemic areas usually carry multiple parasite strains. MOI can be an indicator of transmission intensity and immune status. MOI has been shown to be well related to transmission intensity [170, 171]. The presence of diverse infections in asymptomatic inhabitants in endemic areas is suggested to reflect acquired immunity [172], and thus may affect the outcome of the infection. Some studies have indicated that the increase in MOI decreases the risk of developing clinical malaria [173-175], whereas others indicated a positive link between malaria disease and MOI [176, 177]. Patients suffering from cerebral malaria have been shown to have higher MOI than mild malaria patients. However, a slight increase in clone numbers was found in healthy controls compared to patients with mild malaria [178]. In contrast to this, a recent study in Nigeria has revealed that children with single strain infection are at the higher risk to develop cerebral malaria as compared to those with multiple infections [179]. Thus, the impact of MOI on the development of malaria immunity is still unclear. MOI is suggested to vary over age, and this variation is highly influenced by transmission intensity [170, 180-182]. MOI seems to increase as immunity develops [172]. The age of acquiring natural immunity differs according to transmission intensity [183]. In areas with intensive malaria transmission, immunity is acquired at younger ages compared to less intense transmission areas [183]. However, the relation between infection by multiple parasite strains and malaria disease is still unclear. The number of msp2 variants is widely used as a marker to determine MOI [184]. Manijeh Vafa Homann 29 Human genetic factors involved in immunity to Plasmodium falciparum infection MSP2 MSP2, also called MSA2, is a 45 kDa polymorphic protein anchored in the merozoite membrane. The msp2 gene is a single-copy gene and consists of five blocks, encoding conserved, dimorphic and polymorphic sequences (Fig. 3). Blocks 1 and 5 encode highly conserved N (43 residues) and C (74 residues) termini, respectively. Blocks 2 and 4 are dimorphic and classified into two families, the 3D7 and FC27 (also known as type A and type B, respectively). The central region, block 3, consists of a series of variable repeat motifs, which vary in number, length and sequence among isolates. Individual MSP2 alleles are defined by the central repeats [185-187]. MSP-2 is involved in RBC invasion by merozoites [188]. FC27 3D7 Conserved regions Family specific non-repetetive regions Allelic specific repetetive regions Figure 3: Modified from Damon Eisen [189] 30 PRESENT STUDY Aim of study The overall aim of this study was to investigate possible associations between human genetic factors and malariometric indexes, with the following specific objectives: • To investigate correlations between IL-4 -590 C/T and IL-10 -1087 A/G SNPs, and inter-ethnic differences in malariometric indexes observed between the Fulani and the Dogon, living in Mali. • To correlate the levels of malaria-specific IgG, IgG1-4, IgE, as well as total IgE, to prevalence and multiplicity of asymptomatic infection and splenomegaly in the Fulani, and to compare the results to those observed in the Dogon. • To investigate the influence of IL-4 -590 C/T SNP on anti-malarial IgG, IgG1-4, IgE and total IgE levels in the Fulani and the Dogon. • To study: the impact of erythrocyte variants on MOI; correlation between MOI and the risk of clinical malaria; and associations between MOI and age, parasite density and seasonal variation, in asymptomatic children in Senegal. Manijeh Vafa Homann 31 Human genetic factors involved in immunity to Plasmodium falciparum infection Methodology Materials and methods, briefly mentioned below, are described in details in the respective papers (manuscripts). Paper I Age-matched asymptomatic individuals of the Fulani (214) and the Dogon (212), were included in this study. Finger-prick blood was collected on filter papers. Human and parasite DNA were extracted using the Chelex- and TE buffer-based methods, respectively. Polymerase-chain reaction (PCR) was performed for both human (IL-4 T/C and IL-10 A/G SNPs) and parasite (msp2/3D7 and msp2/FC27) genotyping. Paper II Parasite DNA was extracted from blood on filter papers using Chelex-100. Number of concurrent parasite clones was defined by msp2 genotyping using PCR. ELISA was performed to determine malaria-specific IgG, IgG1-4, IgE as well as total IgE antibody levels. Paper III Human genomic DNA was extracted from blood on filter papers using the Chelex-100 and genotyped for the IL-4 T/C SNP by PCR. Antibody levels were determined using ELISA. Paper IV Asymptomatic children (2 -10 years old), living in Senegal, were included in this study. Parasite densities were measured in June 2002 and January 2003. An active survey of malaria attacks was conducted during the transmission period in 2002 for a sub-group of 154 children. ABO blood groups were determined by serology, and the other red-blood-cell polymorphisms were detected using genomic DNA. Parasite DNA was extracted from blood on filter papers and analysed for the number of msp2 (FC27 and 3D7) clones per infection by PCR. 32 Results and discussions Paper I IL-4 and IL-10 are two crucial cytokines in immune responses in general and anti-malarial responses in particular [190-193]. IL-4-590 T/C and IL-10 -1087 A/G polymorphisms have been shown to result in variable production of these cytokines [155, 156]. We investigated the associations between the above mentioned SNPs and malariometric indexes amongst sympatric ethnic groups living in Mali, the Fulani and the Dogon. We observed that the allele and genotype frequencies of the IL-4 -590 T/C and IL-10 -1087 A/G SNPs varied significantly between the Fulani and the Dogon, indicating that these ethnic groups had distinct genetic backgrounds. This was consistent with studies in Burkina Faso, where HLA class I markers in the Fulani were found to be genetically very distant from their neighbours [194]. The Fulani from Mali (present study) and Burkina Faso [163] represented similar allele and genotype frequencies of IL-4 -590 T/C, supporting the same origin for the Fulani in the two countries [194]. In Burkina Faso, the Fulani T allele carriers had higher anti-malarial IgG levels compared to the CC genotype carriers. This was not seen in the non-Fulani groups living in the same area, suggesting that the IL-4 -590 polymorphism is not functional, but may be linked to other factor(s), which can be the real determinant of resistance to malaria [163]. We found that the IL-4 -590 T allele was correlated to the presence of P. falciparum infection in the Fulani group but not in the Dogon. This finding suggests that the persistence of infection in the T allele carriers gives rise to constitutive anti-malarial antibody production. Pronounced differences in the IL-4 -590 allele and genotype frequencies were observed between the non-Fulani groups living in the two countries (recent study and [163]). The Dogon carried a T/C allelic ratio of 82/18, which was very different from those reported for Mossi and Rimaibe (18/82 and 25/75, respectively). These data suggest that unknown factor(s) linked to this position, may be polymorphic in non-Fulani from different areas, and that those factors have been selected differently under variable selection pressures, including malaria. Within the Fulani, the T allele carriers had higher infection prevalence than those carrying the CC genotype. Although parasite densities did not vary significantly between the two groups, the Fulani CC genotype carriers had lower parasite loads compared Manijeh Vafa Homann 33 Human genetic factors involved in immunity to Plasmodium falciparum infection to the Dogon carrying the same genotype. This suggests that the Fulani CC genotype carriers may succeed better in clearing the infection, compared to the T allele carriers, and to the Dogon CC genotype carriers. However, to draw a firm conclusion regarding the correlations between IL-4 genotypes and parasitemia, parasite counts need to be more accurately determined in consecutive samplings. In line with former studies [135, 141, 145, 146], we noted lower frequencies of infection and higher spleen rates in the Fulani group than in the Dogon. Within the Fulani, spleen enlargement was associated with the presence of P. falciparum infection. This was not the case in the Dogon group. Our results contradict those reported by Bereczky et al. [195], where splenomegaly was associated with P. falciparum infections within the Dogon but not the Fulani. These data underline that more standardized analyses are needed to evaluate hostparasite interactions. No significant inter- and intra-ethnic differences, in the studied malariometric indexes, between different IL-10 genotypes were seen in this study. All together, these data suggest that unknown factor(s) is probably linked to the IL-4 -590 C and is polymorphic in the non-Fulani. This factor(s) might be involved in the control of infection and thus, avoiding the clinical presentation. However, further studies are required to better clarify the contribution of IL-4 and particularly the studied SNP in malaria immunity. Paper II Antibodies play important roles in clearance of blood-stage parasites [196]. In this study, we assessed correlations between anti-malarial antibody levels and malariometric characteristics in asymptomatic individuals of the Fulani and the Dogon. Higher anti-malarial IgG, IgG1-3, IgE and total IgE levels were detected within the Fulani as compared to the Dogon. However, no significant correlations between antibody levels and parasite prevalence, number of infective clones and spleen enlargement were noted. The higher spleen- and lower infection rates, seen in the Fulani compared to their neighbours, have been proposed to be the result from their enhanced anti-malarial reactivity. Interestingly, our results revealed no significant correlation between malariaspecific antibody levels and the presence of P. falciparum infection. This suggests that the relative resistance to malaria infection seen in the Fulani group is not mainly dependent on the 34 magnitude of anti-malarial humoral response. In support of this idea, studies in Sudan have shown that the Fulani had lower malaria-specific IgG1 and almost equal IgG and IgG3 levels compared to their neighbours, the Masalite (Nasr et al., unpublished). In this study, we noted that amongst the Fulani individuals, the increase in total IgE levels was weakly related to the presence of malaria parasites. In malaria patients, elevated total IgE levels have been associated with the development of CM [74]. Other studies have shown that unlike malaria-specific IgE [75, 76], total IgE levels did not influence the risk of subsequent clinical attacks [75]. Thus, the actual consequence of the increase in total IgE levels during malaria infection within the Fulani remains to be clarified. Correlations between the studied antibody levels and clone numbers were then analysed. An increase in Log10 values of malaria-specific IgG4 levels within the Fulani was linked to a decrease in clone numbers. ADCI, which is suggested to be the most efficient antimalarial mechanisms mediated by protective antibodies, is believed not to be mediated by IgG4 antibodies [72]. However, IgG4 might still display other anti-parasitic functions, such as inhibition of cytoadherence or inhibition of RBC invasion by the parasite. IgG4, raised against parasite antigens involved in cytoadherence, may help parasite clearance in the spleen through inhibiting sequestration of iRBCs. By binding to parasite antigens involved in parasite invasion, IgG4 by itself could interrupt invasion of new RBCs. Thus, the specificity, function and/or affinity of an antibody might be more important than the amount of the antibody. We observed no significant differences in IgG4 levels between the two studied groups. In the Fulani, IgG4 levels were negatively associated with the number of clones. Taken together, our results suggest that IgG4, in the two groups, may be raised against different parasite epitopes or have different affinities, and thus mediate various anti-parasitic functions. Spleen enlargement is mostly seen in individuals at the age of 15 and younger (children). Thus, the relation between antibody levels and splenomegaly was evaluated in the whole study population and separately in children. A weak correlation between elevated IgG and IgG3 levels and splenomegaly was noted in the Fulani group (in all ages included and in children only). Despite higher levels of IgG1 compared to IgG3, splenomegaly was not linked to IgG1 levels. This may be reflected by different specificities of IgG1 and IgG3. That is, IgG3 may identify antigens which may lead to less sequestration and thus, to more efficient parasite clearance by the spleen. However, our observation might also be the result from overestimation of significances due to not correcting the P values for multiple analyses. Manijeh Vafa Homann 35 Human genetic factors involved in immunity to Plasmodium falciparum infection Trends for correlations between IgG2 levels and splenomegaly, and multiplicity of infection within the Dogon were noted in this study. These results could not be explained by the frequency of the FcγRIIA-131H variant, which enables IgG2 to act as cytophilic antibody in parallel with IgG1 and IgG3 [73], since the H allele is less frequent than the R allele in the Dogon (Israelsson et al., unpublished). This emphasizes the need for further studies to investigate the actual role of IgG2 in responses to malaria. All together, our results suggest that the stronger anti-malarial humoral response seen in the Fulani is not responsible for the differences in malariometric indexes observed between the Fulani and the non-Fulani ethnic groups. A rapid, efficient and well-regulated innate immune response, which controls parasite counts before the acquired immune response has developed, may be the reason for the relative resistance to malaria seen in the Fulani. Paper III The impact of IL-4 -590 T/C SNP on the level of malaria-specific IgG, IgG1-4, IgE and total IgE levels, in asymptomatic individuals of the Fulani and the Dogon, was analysed in this study. We noted that amongst the Fulani, the increases in total and antimalarial IgE levels were associated with the IL-4 T allele after adjustment for age and sex. No correlation between IL-4 -590 variants and other studied antibodies were seen in any of the studied groups. IgE has been suggested to be involved in both pathogenesis and protection against malaria disease. Studies have shown higher levels of total and malaria-specific IgE in patients suffering from severe malaria compared to those with uncomplicated malaria [74, 197]. However, in asymptomatic individuals, enhanced levels of anti-malarial IgE decreased the risk of developing clinical symptoms [75, 76]. This suggests that the role of IgE in malaria is still unclear. In our study, we noted that total and malaria-specific IgE levels were higher in the T allele carriers compared to the CC genotype, only within the Fulani. Our former studies have shown a higher frequency of P. falciparum infection in the Fulani T allele carriers than those with the CC genotype (Paper I), and that the elevated levels of total IgE were correlated to the presence of infection (Paper II). Others have shown that the T allele is correlated to elevated levels of total IgE amongst children with severe malaria whereas the frequencies of the T allele did not differ significantly between severe and mild malaria cases [161, 162]. Therefore, whether the increase in the levels of IgE, seen in the T allele carriers in this study, 36 is a direct consequence of the impact of IL-4 -590 variants on the antibody-class switching, or if it is due to the higher infection frequencies observed in the T allele carriers, is not clear and remains to be investigated. All together, these data suggest that IL-4 and IgE might influence the outcome of the disease, in concert with other factors (genetic, epigenetic, epistatic or environmental). A recent study in Burkina Faso has revealed associations between a number of SNPs in the IRF1 gene and carriage of P. falciparum infection in both the Fulani and the Mossi. However, different patterns of polymorphisms were seen in the two groups, probably reflecting their different haplotypic architecture [164]. IRF-1 elicits the expression of IL-12p 40 [198, 199] and is critical for production of IL-12 [200], and subsequently for initiation of the Th1 type of responses [201, 202]. Thus, IRF-1 can modulate the balance between Th1 and Th2 type of responses, which is important for the IgE response in malaria [141]. Studies in Burkina Faso have revealed an association between the IL-4 -590 T allele and elevated levels of anti-malarial IgG, in the Fulani group [163]. In contrast to this, we noted that IgG levels did not vary significantly between IL-4 genotypes in any of the studied groups. Production of IL-4 and any other factor(s) involved in the regulation of antibody responses could also be affected by co-infections with other parasitic or bacterial pathogens or time after last exposure to malaria. This idea is supported by findings from other studies, where reverse or no relations between IL-4 -590 variants and IgG levels were seen [203, 204], indicating that the impact of IL-4 variants on IgG production, in turn, is influenced/regulated by other factors. Correlations between IL-4 -590 variants on the level of IgG subclasses have not been studied previously. IL-4 and IL-13 have been shown to promote IgG4 production [32, 33]. In this study, a trend for an increase in IgG4 levels in the T allele carriers was observed only within the Fulani individuals. However, analysis of IgG4 responses is difficult due to the generally low levels of these antibodies. Therefore, the impact of IL-4 -590 variants on IgG4 responses needs to be more analysed including larger study groups. No significant variations in levels of malaria-specific IgG1-3 between IL-4 -590 genotypes were seen in any of the studied groups. Taken together, our data suggest that IL-4 may play other roles in malaria immunity in addition to the regulation of antibody responses. The implication of the IL-4 -590 T allele in susceptibility and/or resistance to malaria infection/disease within the Fulani Manijeh Vafa Homann 37 Human genetic factors involved in immunity to Plasmodium falciparum infection remains to be addressed in follow-up and case-control studies. Furthermore, the actual role of IgE in protection and pathogenesis of malaria remains to be uncovered. Paper IV This study examined the impact of erythrocyte variants on MOI. Relations between MOI and age, seasonal variations as well as malaria morbidity were also analysed. MOI was measured before and after transmission season in asymptomatic children, in the age range of 2 to 10 years, living in a malaria mesoendemic area in Senegal. As expected, parasite density and MOI increased after the transmission season. MOI was positively correlated to parasite density, which is in concordance with data reported by others showing that with higher parasite counts, it is more probable to detect concurrent clones [205, 206]. The impact of different RBC variants on MOI has been limitedly studied. We noted that the general increase in MOI after the transmission season was not seen in αthalassaemic and G6PD mutated children. Alpha-thalassaemia alters the membrane properties of RBCs [207, 208], which may affect their invasion by merozoites. MSP2 is a polymorphic antigen involved in invasion [188]. Thus, the altered membrane of α-thalassaemic RBCs may impair the invasion by certain MSP2 variants and consequently protect against infection by certain parasite strains. However, other studies have shown no impact of α-thalassaemia on parasite prevalence, density and diversity in symptomatic individuals [209]. A reason for this contradiction might be due to that we studied asymptomatic subjects, whereas the former study included symptomatic cases. It is known that α -thalassaemia does not affect parasite loads in individuals who develop clinical disease [118, 119, 209]. The differences in sample sizes in the two studies could also be considered as a reason for this discrepancy. These data suggest that α-thalassaemia may protect against infection by certain parasite strains, but does not protect against clinical malaria in ongoing infections. Infected G6PD-deficient RBCs have been shown to be more efficiently phagocytosed than infected normal RBCs [129]. In this study, MOI did not increase after the transmission season in the G6PD-mutated children, which might be due to rapid parasite clearance in these children as compared to those carrying the normal G6PD gene. This effect was stronger in G6PD-mutated girls carrying the HbAS gene. However HbAS alone did not 38 affect MOI. G6PD deficiency did not influence MOI in male subjects, which might be due to the small size of this group. In the present study, the ABO blood system did not show any influence on MOI at any time points. The ABO system has been implicated in the outcome of the disease, where blood group O is less likely to develop CM as compared to A, B and AB carriers [101]. However, the ABO system did not seem to affect parasite density and diversity in asymptomatic individuals. Sickle cell trait did not influence MOI in our study. Former studies have reported none [210] or reverse [211] relations between HbAS and MOI. The reason for these discrepancies is not known, but they might be due to differences in the ages of the studied populations and/or variations in transmission intensities between the study areas. MOI has been shown to be age-dependent in areas with intense transmission, and to be independent of age in a malaria mesoendemic area [210, 212]. In line with the latter report, MOI was not influenced by age in the present study. However, these findings need to be confirmed in a study population with a broader age range. A clinical survey was performed including 154 children to evaluate correlations between MOI and clinical attacks. Forty five percent of these children had at least one clinical attack during the survey. No significant association between clinical malaria and MOI was noted, at any time points. Both reverse [173-175] and positive [176, 177] correlations between MOI and malaria morbidity have been reported. Our finding does not agree with any of those reports, which may partly be due to different genotyping methods used in these studies. Differences in sensitivity and accuracy of genotyping protocols have been shown to give rise to variations in results from different laboratories [206, 210, 213, 214]. Moreover, in the present study, sampling was not done at the time of clinical presentation. This may also be a reason why MOI was not related to morbidity. In any case, these results emphasize that malaria morbidity is not only affected by parasitological indexes, but could also be influenced by host genetic factors and/or transmission levels. Taken together, methodology should be considered when comparing results from different studies on MOI. The results from PCR may vary if different protocols and reagents are used or even may differ from hand to hand [206]. PCR may underestimate the MOI. Importantly, PCR is unable to differenciate between virulent and avirulent strains. Thus, to draw a firm conclusion from studies on MOI in different areas, a more standardized methodology is required. Manijeh Vafa Homann 39 Human genetic factors involved in immunity to Plasmodium falciparum infection CONCLUDING REMARKS AND FUTURE PERSPECTIVES We conclude from our studies that the Fulani and the Dogon are genetically markedly different, using IL-4 -590 and IL-10 -1087 variants, as markers. The IL-4 genotype pattern was similar in the Fulani from different areas, whereas it varied significantly between non-Fulani groups living in different countries. The IL-4 T allele was previously suggested to be linked to unknown factor(s), which could be the real determinant of resistance to malaria [163]. Our results suggest that the unknown factor is probably linked to the C allele and is polymorphic in non-Fulani. This factor might be involved in a better parasite clearance. This study concludes that the magnitude of anti-malarial IgG response may not be as important as it is thought to be. Instead, other features of antibodies such as specificity, function and/or affinity may be more crucial in protective response to malaria. The implications of IgE antibodies in malaria protection and pathogenesis remain to be better clarified. IgG4 may not mediate ADCI, but might still have some roles in malaria protection, through mediating anti-parasitic functions different from those mediated by IgG1 and IgG3. Our results also indicate that the relative resistance to malaria infection seen in the Fulani does not rely mainly on their augmented anti-malarial antibody response, as it has been believed to be. Rather, other factors such as a rapid and well-regulated innate response, which can control the infection and consequently avoid the clinical presentations, might also take part in this relative resistance to malaria. Studies on the associations between IL-4 -590 variants and IgE have indicated that relationship between IL-4 and IgE might be affected by other genetic/epigenetic/epistatic or environmental factors. Therefore, the outcome of the disease could be reflected by the action of these factors together with IL-4 and IgE. The production of IL-4 and other cytokines/factors, in turn, could be affected by the time after last exposure to malaria or by co-infection with other pathogens which might explain the contradictory results of the impact of IL-4 -590 variants on IgG levels observed by a number of studies. All together, studies on IL-4 (genotype/levels/-producing cells) in Mali and Burkina Faso suggest that the contribution of IL-4 in inter-ethnic differences in malaria susceptibility is probably not through its role in antibody regulation, rather owing to its implication in other aspects of immune responses, e.g. induction of NK cell proliferation which help in parasite killing and/or suppression of anti-inflammatory cytokine productions which avoids immunopathogenesis. 40 The last study concludes that in asymptomatic children; α-thalassaemic RBCs may resist against infection by certain parasite strains; protection conferred by G6PD deficiency might be due to rapid parasite clearance at early stage of infection; the ABO blood system and HbAS do not affect the MOI. The multiplicity of infection was correlated to parasite density in a malaria mesoendemic area, but did not vary over the age, in the range of 2 to 10 years. The risk of clinical attack was not affected by MOI. However, these data need to be confirmed using different genotyping methods and including larger size of the study group with broader age range. To address the actual implications of IL-4 -590 variants in malaria (especially inter-ethnic variations in malaria susceptibility), a follow-up study of a population with known IL-4 -590 genotypes of the Fulani and the Dogon, joined with a case-control study, is required. Consecutive determination of antibody levels together with parasitological data, during these studies, might help to clarify the impact of IL-4 -590 variants on antibody responses. Most importantly, studying other genetic factors adjacent to IL-4 gene on chromosome 5q may help in identifying the actual factor(s), involved in resistance/protection to malaria infection. Sequencing of this region in individuals of the two groups would be of a great help in this regard. Manijeh Vafa Homann 41 Human genetic factors involved in immunity to Plasmodium falciparum infection ACKNOWLEDGMENT I left my family and home with the wish to continue my studies and become a researcher. It was a long, tough journey to bring the wish close to reality; a long way that I would never have been able to make on my own. I would, therefore, like to express my sincere gratitude to all those who have helped and supported me during these years. In particular, I would like to thank: Professor Marita Troye-Blomberg; for accepting me into your group, for your support and guidance, for being patient with my endless questions, and for always being calm and caring. I learnt so much from you. Present and former seniors at the Immunology Department; Professor Klavs Berzins, Professor Carmen Fernandez, Professor Eva Severinson, Docent Eva Sverremark Ekström and Docent Manuchehr Abedi-Valugerdi; for being excellent teachers, for your critique and advice, and above all for providing a family-like environment at the department. Margareta Hagstedt (Maggan) and Ann Sjölund; for your excellent technical support and for your incredible speed in responding to any call for help. Gelana Yadeta and Anna-Leena Jarva; for excellent administration, always having a smile, and for your readiness to assist. Present and former students at the Immunology Department, for making the department a nice and friendly place to work, coauthors, for your contributions, friends, in and out of university, for the very beautiful time we shared and the Nadjafi family in Stockholm, for giving me the warmth and feelings of home and family whilst in cold Stockholm. I would also like to thank my dear siblings, Aboutaleb, Mahmood, Kobra, Azra, Mehri and Monireh, and their families; your love and prayers have been my best company all these years. What would my life mean without you guys?! Mehri, you are the best of friends and sister, ever. You were available anytime I needed you, and filled every moment of my loneliness after I left home; despite miles of physical distance our hearts and minds have been tightly tied together moment by moment, I love you and miss you very much. My love, my friend, my husband, Peter Homann; where would I have been if not for you going through the whole emotional rollercoaster with me during the past months; for your 42 morning encouraging cuddles and evening relaxing hugs, for believing in me and bringing my self-confidence back when I had none left. And for my incomparable mother and father, Mohammad E. Vafa and Sara Madadi; words are not enough to express my love and gratitude to you. 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