Immunoglobulin M: Restrainer of Inflammation and Mediator of Immune Evasion by Plasmodium falciparum Malaria

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Review

Immunoglobulin M: Restrainer of Inflammation and Mediator of Immune Evasion by Plasmodium falciparum Malaria Richard J. Pleass,1,* Shona C. Moore,1,2 Liz Stevenson,3 and Lars Hviid3,* Immunoglobulin M (IgM) is an ancient antibody class that is found in all vertebrates, with the exception of coelacanths, and is indispensable in both innate and adaptive immunity. The equally ancient human malaria parasite, Plasmodium falciparum, formed an intimate relationship with IgM with which it coevolved. In this article, we discuss the association between IgM and human malaria parasites, building on several recent publications that implicate IgM as a crucial molecule that determines both host and parasite survival. Consequently, a better understanding of this association may lead to the development of improved intervention strategies.

Trends IgM is an important class of serum antibody that mediates the clearance of apoptotic and altered cells through complement-dependent and -independent mechanisms. IgM mediates protection against infection and may play an important role in controlling Plasmodium falciparum malaria. IgM provides new routes to more effective drugs and vaccines.

The Antibody Response to Malaria Antibodies are an important component of protective immunity to Plasmodium falciparum malaria [1]. Natural exposure to infection by these parasites drives strong antibody responses to several parasite asexual blood-stage antigens that are responsible for causing clinical disease. This is in contrast to antigens expressed by the pre-erythrocytic and gametocyte stages which do not generally induce protective antibodies following natural exposure to the parasites [2]. The asexual-stage antigens targeted by antibodies include merozoite surface molecules involved in erythrocyte invasion [3], and antigens expressed on the surface of infected erythrocytes (IEs) [4]. The latter antigens, and in particular members of the clonally variant PfEMP1 (P. falciparum erythrocyte membrane protein-1) family, mediate IE adhesion to host endothelial receptors and play a key role in parasite evasion of host immune responses [5]. Most studies have focused on how antibodies interfere with erythrocyte invasion or IE sequestration, or how they act as opsonins (see Opsonization in the Glossary) for phagocytosis, antibody-mediated cellular inhibition, and complement-mediated elimination of Plasmodium [6–8]. The great majority of these studies have focused almost exclusively on IgG, despite the fact that immunoglobulin M (IgM) is superior at triggering specific effector functions that are required for efficient pathogen elimination. For example, a single molecule of IgM can trigger complement-mediated lysis of an IE, something that would need 1000 IgG molecules [9,10]. We wish to highlight here recent research that is shedding light on the importance of IgM in malaria immunity, and point to outstanding research gaps that ought to be addressed. For a

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Fcm-binding proteins expressed on the surfaces of both infected erythrocytes and merozoites facilitate immune evasion through diverse mechanisms.

1 Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK 2 Warwick Systems Biology Centre, Senate House, University of Warwick, Coventry, CV4 7AL, UK 3 Centre for Medical Parasitology, Department of Immunology and Microbiology (ISIM), Faculty of Health and Medical Sciences, University of Copenhagen and Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark

*Correspondence: [email protected] (R.J. Pleass) and [email protected] (L. Hviid).

http://dx.doi.org/10.1016/j.pt.2015.09.007 © 2015 Elsevier Ltd. All rights reserved.

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review of the basic biology of IgM, we refer the reader to some of the several excellent reviews available [11–13].

IgM: The Basics IgM (Box 1) exists as membrane-bound monomeric receptors on B cells and as secreted molecules in plasma at concentrations of 1–2 mg/ml. Secreted IgM molecules are predominantly pentamers and hexamers, which provides soluble IgM with extraordinary functions, including multivalent binding to antigens, receptors, and complement [14,15]. Non-immune

Box 1. The Structure and Function of Human IgM Human IgM is a pentamer of a basic four-chain structure (monomeric unit) with a mass of
970 kDa (Figure I), although it can also exist as a hexamer without the J chain with a molecular mass of
1132 kDa. Hexameric IgM conveys increased avidity for the binding of both antigen and Fc receptors. As with all secreted antibodies, one part of the molecule (the Fab domains) binds to the pathogen, while the other part (the Fc portion) interacts with cells of the immune system via Fc and glycan receptors (see text for detail). A transmembrane monomeric form of IgM is also present as an antigen-specific receptor on mature B cells. Human IgM is present at
1–2 mg/ml in blood with a half-life of
5 days [11]. It has been reported that IgM levels are higher in females than in males [[1_TD$IF]114,115]. Numerous studies have identified a role for immune and non-immune IgM in the protection against numerous viral, bacterial, fungal, and parasitic infections [11]. The Cm4 domain appears to be particularly important in binding to DBL domains (see text, shown in dark red on the model of IgM in Figure I [14]). In the Cm4 domain there are three reported allotypes of human IgM (www.imgt.org). Two of these contain the insertion of an extra valine, and the other has an aspartic acid to glutamic acid substitution. Both these variants are found within the critical region proposed for binding to DBL domains [15]. This suggests inter-individual variation in IgM-mediated immune mechanisms against malaria parasites. The N-linked glycans on IgM contribute 12% of the molecular weight (as against 2–3% for IgG) but surprisingly their role in the interaction of IgM with either DBL domains or Fcm-receptors has not been investigated.

Asn-171

Asn-332 Asn-395 Asn-402

563 63 Asn-563

Key:

N-linked glycan Variable heavy domain Variable light domain Constant light domain Cμ4 constant heavy domains Cμ2-3 constant heavy domains Cμ1 constant heavy domain Fab domains

Cμ2 Cμ3

Fc domains J chain Inter-domain disulfide bridge

Figure I. Human IgM Structure.

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Glossary Apoptosis: the biochemical process of programmed cell death (PCD) that occurs in multicellular organisms, leading to characteristic cell changes and death. Apoptotic cells are cleared by IgM and complement activation. Autophagy: a process whereby cells degrade intracellular components to promote their own survival in response to cellular stress. Complement activation: biochemical pathways that have evolved to label pathogens and/or apoptotic cells for elimination. The classical pathway links to adaptive immune systems through IgM. The alternative and lectin pathways provide antibody-independent ‘innate’ immunity, and the alternative pathway is linked to and amplifies the classical pathway. Dendritic cells: leucocytes that sense tissue injury, capture antigens, and present those antigens to T lymphocytes to induce immunity to foreign antigens and enforce tolerance to self-antigens. Fragment crystalline (Fc) constant region: the portion of an antibody responsible for binding to immunoglobulin receptors on cells and the C1q component of complement. Fc receptor: a membrane-anchored or soluble glycoprotein that binds to the Fc constant region of antibodies. Fragment antigen (Fab): the part of an antibody molecule which contains the antigen-combining site, consisting of a light chain and part of the heavy chain; it is produced by enzymatic digestion. Galactose-a-1,3-galactose (aGal): a carbohydrate found in many organisms. It is not found in primates and humans whose immune systems recognize it as foreign and produce xenoreactive IgM antibodies to it. These can lead to organ rejection after transplantation. Immunoglobulin M (IgM): the first antibody produced in a primary immune response and that is largely confined to the intravascular pool. It is frequently associated with the immune response to antigenically complex, blood-borne infectious organisms. IgM is the most efficient activator of complement. Intravenous immunoglobulin (IVIg): a therapeutic preparation containing polyvalent IgG and/or IgM

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(or ‘natural’) IgM has low affinity for antigen, is broadly reactive, and is synthesized without prior exposure to antigens [11,13]. Immune (antigen-specific) IgM is produced in response to specific antigens from pathogens including malaria parasites [11,13]. IgM in Immune Defense Non-immune IgM is an important first-line defense against many pathogens [16,17] and can also recognize self-components [12,18]. Non-immune IgM is particularly adept at opsonizing and clearing small apoptotic particles, senescent erythrocytes, and even microbes [19–28]. It is not known whether it has such a role in malaria, where various products that are released at the time of IE rupture (pigment, erythrocyte ghosts, etc.) must be removed rapidly and safely from the circulation (see Outstanding Questions). Immune IgM has been shown to be crucial for protection from viruses [29], bacteria [30], protozoa [31,32], fungi [26], and helminths [33]. The cellular origin of protective IgM has shown distinct roles for the B1a, B1b, and B2 subsets of B cells [29,34,35] in the control of pathogens.

pooled from human donors. IVIg is used to treat primary immune deficiencies and is increasingly being used in the treatment of autoimmune disease. Lectin: carbohydrate-binding proteins that are highly specific for sugar moieties. Opsonization: a process by which phagocytosis is facilitated by the deposition of opsonins (e.g., antibody and complement) on the antigen. Phagocytosis: a process by which cells consume extracellular particles to form an internal vesicle containing those particles. Poly-reactivity: the ability of an antibody molecule to bind to more than one epitope or antigen.

IgM-Dependent Effector Functions Opsonization for phagocytosis and complement-mediated lysis (Figure 1) are important effector functions of IgM [21,23,36], and phagocytosis is markedly reduced in the absence of nonimmune IgM [23]. Recent studies have revealed several binding partners for IgM (see below), indicating that IgM may opsonize for phagocytosis directly [37–40]. Indeed, IgM can be superior to IgG as an opsonin for phagocytic clearance of very small particles and apoptotic microparticles (1–2 mm in size) [25], although the receptors involved remain uncharacterized. In addition to IgM and complement, mannose-binding lectin (MBL) is another important opsonin. Removal of MBL-opsonized cells can involve IgM because a small proportion of this antibody class possesses N-linked glycans in the fragment crystalline (Fc) constant region of the molecule that can bind to MBL already attached to pathogens or apoptotic cells [41,42]. Conversely, MBL may be recruited after IgM has attached to apoptotic cells via its fragment antigen (Fab) binding part [41,43,44]. The ingestion of IgM-opsonized apoptotic cells by immature dendritic cells (DCs) promotes an immunoregulatory milieu. This includes activation of IL-10-secreting B and T cells, that restrain the development of inflammation [44], and suppression of in vivo and in vitro inflammatory responses, including those induced by ligands for Toll-like receptors [43]. In fact, it is likely that an important function of non-immune IgM is to facilitate the clearance of aberrant self material and microbes without promoting inflammation [45] (Figure 1). For these reasons, IgM is increasingly being used for the diagnosis and therapy of malignancies and in neuronal repair [12,46–50]. However, IgM is also implicated in damage to organs and tissues following ischemia or reperfusion [51], and in multiple autoimmune diseases [52,53]. Cellular Receptors for the Fc Region of IgM Numerous membrane-bound receptors for IgM have been described. These include the polymeric immunoglobulin receptor [54], the IgM Fc receptor (FcmR) [38], the IgA/IgM Fc receptor (Fc/mR) [37], the tripartite motif-containing protein 21 [55], and the sialic acid-binding immunoglobulin-type lectin 2 (CD22) [39]. These receptors are not typically expressed on the surface of human monocytes, macrophages, or DCs found in blood, spleen, or bone marrow [56], although IgM does bind to these cells in humans [57] and mice [25]. Instead, they are most commonly expressed by B cells, T cells, and natural killer (NK) cells [56]. Human monocytes and DCs instead express alternative receptors that may also bind IgM [58]. Thus, the CD5-like molecule Sp/, which is an apoptosis inhibitor expressed by macrophages,

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(A)

Co m pl em en ta c v

(B)

Phagocyte and/or dendric cell

a

IL-10 TGFβ

on CR1/CR3

Ruptured RBC

? ? ? Key:

DC-SIGN CD36 Fcα/μR

MBL IgM

αGal on sporozoite

Cell debris

Merozoite

Spα

C5a

C1q

C5aR

Figure 1. Human Immunoglobulin M (IgM) and Immunity to Malaria. (A) Human IgM opsonizes sporozoites for destruction by complement-mediated lysis [67]. A byproduct of complement activation is the generation of the complement component C5a, which has been implicated in cerebral malaria [[1_TD$IF]116]. Overt pathology caused by C5a is controlled by the C5a receptor (C5aR) on monocytes and dendritic cells (DCs) through interleukin (IL)-10 production [[2_TD$IF]117,118]. (B) Human IgM may also direct merozoites, modified host molecules, and cell debris to macrophages and DCs through the recruitment of C1q and mannan-binding lectin (MBL), as is known to occur during removal of apoptotic cells in autoimmune disease [44]. Complement component C1q binds to the fragment crystalline (Fc) portion of IgM and, together with MBL, functions in concert to promote phagocytosis of released microparticles of cell debris through complement receptor type 1 (CR1) and complement receptor type 3 (CR3). What role the C-type lectin receptor present on the surface of both macrophages and DCs (DC-SIGN) and the Fc receptor Fc//mR play in this process is unclear, although both are found on DCs and are known to bind IgM. Plasma IgM is commonly bound to Sp/ [58], which can reduce inflammation through CD36 on DCs and the expression of anti-inflammatory cytokines, including IL-10 and transforming growth factor b (TGFb) [[3_TD$IF]60,119]. Sp/ also inhibits tumor necrosis factor (TNF) responses through CD36 interactions [53]. Abbreviations: DC-SIGN, DC-specific intercellular adhesion molecule-3-grabbing non-integrin; /Gal, galactose-/-1,3-galactose; RBC, red blood cell.

binds to all IgM fractions purified from plasma or serum [58]. Sp/ belongs to the scavenger receptor cysteine-rich superfamily of proteins. It binds to CD14+ human monocytes and macrophages via CD36, and induces autophagy and modulates inflammation in these cells [59,60]. Sp/ is also an inhibitor of apoptosis, which may explain recent controversies surrounding the physiological function and nomenclature of the FcmR, which has also been ascribed an antiapoptotic capability [61]. Indeed, the association of IgM with Sp/ has been shown to inhibit the binding of IgM to Fc//mR on splenic follicular DCs, thereby protecting IgM immune complexes from Fc//mR-mediated internalization and contributing to autoantibody production in obesity [59]. We recently reported that the DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a C-type lectin receptor present on the surface of human macrophages and DCs, binds to IgM in a glycan-dependent manner [40]. The murine homolog of DC-SIGN also binds to IgM and enhances apoptotic cell clearance [40]. The functional consequence of

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DC-SIGN-dependent IgM binding to DCs is unknown, although DC-SIGN signaling induces the production of interleukin (IL)-10, a cytokine involved in anti-inflammatory pathways.

The Role of IgM in Malaria IgM is particularly good at recognizing phylogenetically conserved structures such as phospholipids, nucleic acids, and carbohydrates [62]. Despite its low affinity for these epitopes compared with IgG, the multimeric conformation of soluble IgM endows it with a superior ability to agglutinate invading pathogens. Its poly-reactivity also allows binding to different structures on the same parasite, or to different structures on different pathogens, thereby enhancing neutralization [11]. Immune IgM Very little is known about the role of parasite-specific IgM in the immune response to malaria, apart from data from a few early studies in mouse models [63,64] and a couple of more recent observational studies in P. falciparum-exposed humans [65,66]. This is clearly an area where more research is needed. Interestingly, it was recently shown that galactose-a-1,3-galactose (/Gal)-specific IgM, presumably raised to commensal bacteria found in the human gut, crossreacts with an epitope also found on Plasmodium spp. sporozoites [67[12_TD$IF]], (reviewed in [68][13_TD$IF]) (Figure 1). Malaria parasites do not possess the glycosylation machinery necessary to generate the epitope de novo [[14_TD$IF]69], and the parasites most likely acquire the /Gal from mosquito-derived proteins [67]. Levels of /Gal-specific IgM correlate with protection from malaria infection in humans and provided sterile complement-dependent protection following vaccination in a mouse model [67]. Whether or not IgM specific for merozoite [[15_TD$IF]70] or IE antigens [[16_TD$IF]71] is cytotoxic via the classical pathway of complement activation (as it so clearly is for sporozoites) remains to be determined (see Outstanding Questions). Up to 5% of circulating IgM in healthy adults is /Gal-specific [[17_TD$IF]72]. Intravenous immunoglobulin preparations enriched for IgM (IVIgM), used in treatment of several conditions [[18_TD$IF]40,47,73–[19_TD$IF]77], may therefore be of prophylactic utility in malaria, particularly if used in the first 2 years of life when children are most at risk. These anti-/Gal antibodies might also protect children from Leishmania and Trypanosoma, which also express abundant /Gal [[20_TD$IF]78], and this approach may even be cost-beneficial in some resource-poor settings given recent developments allowing cost-effective, practical, and sustainable methods to deliver IVIgM [[21_TD$IF]79]. IVIgM can reduce inflammation [[2_TD$IF]80,81], and monoclonal IgM can repair neurons and can cross the blood–brain barrier [[23_TD$IF]47,82]. It would therefore be interesting to determine if IVIgM might have a role in the treatment of severe malaria, including cerebral malaria [[24_TD$IF]83] (see Outstanding Questions). However, vaccination against /Gal might be a more pragmatic long-term alternative to IVIgM prophylaxis against malaria, and strategies for vaccination with other candidate malaria antigens might also usefully incorporate adjuvants that drive IgM as well as IgG responses. Indeed, experiments with murine malaria parasites have shown that parasite-specific IgM can limit parasite growth and prime memory cell generation, and be a more potent adjuvant than Bordetella pertussis [63]. The ability of immune IgM to enhance antibody responses in vaccines is well known and is crucially dependent on its ability to activate complement [[25_TD$IF]84,85]. Whether this also applies to P. falciparum malaria now needs to be investigated. Several classes of antibodies are produced during infection of humans with P. falciparum malaria [[26_TD$IF]1,86,87]. Although protective activity has mostly been associated with IgG, several studies have implicated IgM as well [[27_TD$IF]65,88,89]. The study by Brown et al. [[28_TD$IF]88] is particularly interesting because it showed that the IgM fraction and mononuclear cells from infected children inhibited the growth of P. falciparum in vitro more efficiently than did IgG. Ethnic differences in resistance to malaria have also been attributed to immune IgM [65]. It is suspected that specific IgM

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responses can ameliorate disease severity by inhibiting the tumor necrosis factor (TNF)-inducing activity of toxins released on erythrocyte rupture [[29_TD$IF]90]. Whether this involves Sp/ (see above) is presently unknown (see Outstanding Questions). Non-Immune IgM Given the importance of antibodies in acquired protective immunity to P. falciparum malaria [1], it is not surprising that the parasites have evolved various ways to evade this immunity. Indeed, they may even have found ways to use antibodies for their own benefit. Some of their tactics involve non-immune IgM (i.e., IgM that has no Fab-mediated specificity for malaria antigens), as outlined below. Several P. falciparum proteins can bind to the Fc region of IgM (Fcm) (Table 1), and the expression of such Fcm-binding proteins is linked to parasites causing severe malaria [15]. The binding has been localized to Duffy-binding-like (DBL) domains of the PfEMP1 adhesins, which mediate binding of infected erythrocytes to a variety of host receptors (Table 1) [5]. Each P. falciparum genome contains approximately 60 PfEMP1-encoding genes, and recent evidence shows that several PfEMP1 variants within a single genome are able to bind IgM [[6_TD$IF]91]. The function of Fcm-mediated binding of IgM to DBL domains is not fully understood, but parasite evasion of human immune effector mechanisms appears to be an important driver of

Table 1. Plasmodium falciparum Proteins Investigated for Binding of Non-Immune IgM Binds to Non-Immune IgM

Protein

DBL Domain Involved

Refs

Yes

FCR3var1csa

DBLe7

[[7_TD$IF]120]

FCR3var2csa

DBLe6

[[7_TD$IF]120]

HB3var06

DBLz2

[[5_TD$IF]109]

MAL6P1.4

DBLe2

[[6_TD$IF]91]

MAL6P1.4

DBLe3

[[6_TD$IF]91]

PFL0020w

DBLe4

[[6_TD$IF]91]

PFL0030c

DBLePAM5

[[6_TD$IF]91]

No

6

TM284var1

DBLz4

[121,122][8_TD$IF]

FCR3var1csa

DBLe4

[[7_TD$IF]120]

FCR3var1csa

DBLz6

[[7_TD$IF]120]

FCR3var2csa

DBLe4

[[7_TD$IF]120]

FCR3var2csa

DBLe5

[[7_TD$IF]120]

HB3var06

DBLe13

[[5_TD$IF]109]

HB3var06

DBLe14

[[5_TD$IF]109]

ITvar9/R29var1

DBLe3

[[5_TD$IF]109]

MAL6P.14

DBLe7

[[6_TD$IF]91]

PF07_0139

DBLe4

[[6_TD$IF]91]

PFL0020w

DBLz5

[[6_TD$IF]91]

PFL0030c

DBLePAM4

[[6_TD$IF]91]

PFL0030c

DBLePAM10

[[6_TD$IF]91]

TM284var1

DBLe3

[[9_TD$IF]121]

TM284var1

DBLe5

[[9_TD$IF]121]

Var0

DBLz

[[10_TD$IF]108]

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this phenotype (Figure 2). It has been known for some time that IEs that can adhere to chondroitin sulfate A (CSA) – a property involved in the pathogenesis of placental malaria [[30_TD$IF]92] – can also bind to non-immune IgM [[31_TD$IF]93]. IE adhesion to CSA is mediated by a particular type of PfEMP1, called VAR2CSA [[32_TD$IF]94,95], and the function of non-immune IgM binding to VAR2CSA appears to be protection from phagocytic clearance of IgG-opsonized IEs without compromising the CSA-adhesive function of the antigen (Figure 2A) [[4_TD$IF]96]. Several other malaria parasite proteins involved in adhesion contain DBL domains [[3_TD$IF]97–[34_TD$IF]100], but so far there are no published data on their capacity for FcR-mediated IgM binding. Another important type of PfEMP1 mediates formation of rosettes, which occurs when several uninfected erythrocytes bind to and surround a central IE [[35_TD$IF]101,102]. Rosetting generally requires soluble plasma factors including IgM [[36_TD$IF]103,104], and rosetting and FcmR-dependent binding of IgM are IE phenotypes that often go together [[37_TD$IF]105]. This was originally assumed to be because non-immune IgM has a ‘bridging’ function similar to that described above [[38_TD$IF]104]. However, recent evidence points to an alternative explanation, in which IgM would augment the low-affinity (A)

(B)

(C)

Surface receptors on host cells

PfEMP1 on the IE surface

PfEMP1 on the IE surface

Key: IgM

IgG

Non-IgM binding DBL domain IgM binding DBL domain

PfEMP1 on the IE surface

α2Mac CIDR domain

Protein receptor

Glycoprotein receptor

Figure 2. The Binding of Non-Immune Human IgM to Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). (A) In the case of placental binding parasite isolates, IgM shields the antigen PfEMP1 from the destructive potential of immune IgG [[4_TD$IF]96]. (B) For rosetting parasite isolates, IgM binding appears to tether PfEMP1 for stronger interactions with carbohydrates found on surface receptors of host cells, such as uninfected erythrocytes [[5_TD$IF]109]. (C) IgM also binds to PfEMP1 on infected erythrocytes (IEs) that neither rosette nor sequester to the placenta [[6_TD$IF]91]. Note that, depending on the PfEMP1 in question, the structure is composed of multiple DBL (Duffy-binding-like) or cysteine-rich interdomain region (CIDR) domains of varying number and type. Abbreviation: /2Mac, /2 macroglobulin.

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interactions with the erythrocyte carbohydrate receptors involved in rosetting [[39_TD$IF]106–[10_TD$IF]108]. Pentameric IgM is necessary to support rosetting [[38_TD$IF]104], and each pentameric IgM molecule can bind two PfEMP1 proteins [[5_TD$IF]109]. This potentially allows IgM to coordinate the adhesive interaction of multiple PfEMP1 molecules (Figure 2B). This model is corroborated by the finding that another abundant serum protein, /2-macroglobulin (/2M), which has an even higher potential for crosslinking individual PfEMP1 molecules, appears to serve a similar function [[40_TD$IF]110]. Remarkably, /2M and IgM bind to the same DBL domain, and these two soluble factors synergize in their support of rosetting [[40_TD$IF]110]. Binding of non-immune IgM (and probably /2M) is not restricted to PfEMP1 types that bind CSA or mediate rosetting [[6_TD$IF]91]. Therefore, an important function of these molecules may be to expand the repertoire of host receptors that support sequestration, and thereby allow IEs to avoid being cleared by the spleen. Whether this is in fact the case remains to be determined. Despite significant sequence diversity among IgM-binding DBL domains, most appear to be DBLz or DBLe-type, such as those in MSPDBL1 and MSPDBL2 (Table 1). Based on the sevengenome analysis conducted by Rask et al. [[41_TD$IF]111], DBLe domains fall into two groups. The group containing DBLe1/11/13 is distinct from the majority of DBLe domains and contains no known IgM-binders (Table 1). However, no obvious binding motifs or essential amino acids in Fcmbinding DBLe domains have yet been identified, consistent with the lack of phylogenetic clustering of these domains (Figure 3). This might suggest that binding to IgM is highly dependent on the overall quaternary structure of these DBLe domains in the context of the

Lε5

M MA AL6P1.4 DBL L6 ε7 P1 .4 DB Lε2

Lε4

PA M 4

DBLε PAM A

Lε

DB

DB

030C

0C

sa

03

PFL0

r2c

L0

DB ar2csa FCR3v

3va

FCR

PF

0.1

M

1.0

BL SPD

1D

BLε

1.0

FCR

1.0

3va

MSPDBL2

r2c

DBLε

sa

DB Lε6 DBLεPAM 10

0.15 0.14 0.19 0.32 1.0 0.50 0.17 0.82 0.28 0.43

0.95

.4 DBLε3

MAL6P1

0.62

TM284var1 DBLε3

a DBL

BLε D PF07_0139

BLε sa D 3va r1c

HB3 ITv var0 ar 6D 9/ BLε R2 13 9v ar 1D BL ε3

ar1cs

FCR

0.99

FCR3v

W DBLε4

7

DB

Lε 14

1.0

HB 3v ar 06

5 BLε 1D r a v 284 TM

PFL0020

PFL0030C

ε4

Figure 3. Phylogenetic Relationships between IgM-Binding and Non-Binding Duffy Binding-Like (DBL)-e Domains. A phylogenetic tree of IgM-binding and non-binding DBLe domains. Maximum-likelihood phylogram of eight IgM-binding DBLe domains (in red) and 12 non-binding DBLe domains (in blue). Edge numbers indicate the bipartition bootstrap support (%). Scale bar indicates amino acid substitutions per site.

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native protein. In any case, a more comprehensive analysis must await the publication of sequence data from the thousands of P. falciparum genomes that are currently being analyzed. Finally, non-immune IgM binding to parasite proteins may directly compromise the development of protective immunity. P. falciparum parasites appear to be able to undermine the crucial antigen-presenting function of DCs. They do so by expressing PfEMP1 proteins that can bind to CD36 on immature DCs [[42_TD$IF]112,113]. Similar impairment of DC function may result from parasitebound IgM binding directly to the DCs via DC-SIGN, or indirectly via SP/ and CD36 (see above). DC-SIGN binds to mannose-type carbohydrates that are absent from P. falciparum [[14_TD$IF]69], and the parasites may therefore exploit IgM as a glycan bridge to compromise DC function through DC-SIGN and/or CD36.

Concluding Remarks IgM is of central importance in the immune defense against many infections. Malaria is unlikely to be an exception to this rule, and recent evidence points to an unanticipated importance of IgM in resistance to the sporozoite stages of the infection. Conversely, the malaria parasites have evolved a range of mechanisms to evade host immunity, several of which involve IgM. These features have the potential to frustrate anti-malarial interventions. However, they also hold the promise of new ways to interfere with parasite survival in the human host. Therefore, we believe that research aimed at a deeper understanding of the known and suspected IgM-dependent mechanisms we have reviewed here should be given priority in coming years. New tools to combat this devastating health problem are desperately needed, and we need to think out of the box.

Outstanding Questions Is non-immune IgM involved in clearing up debris after erythrocyte rupture during a malaria infection? How do P. falciparum parasites expressing PfEMP1 that binds to non-immune IgM avoid complementmediated destruction of the IgMopsonized infected erythrocytes? Are immune IgM responses made to PfEMP1? Does complement-mediated parasite clearance involving antigen-specific IgM play a role in immune control of malaria? Is there a role for IVIgM in malaria therapy or vaccine development? Do sporozoites and gametocytes also bind host IgM at their surfaces via Fcmbinding proteins, and are these also mediated by DBL domains?

Acknowledgments We thank the Engineering and Physical Sciences Research Council (EPSRC) and the Commission of the European Communities (FP7/2007-2013, grant 242095 – EVIMalaR) for PhD studentships to Shona Moore and Liz Stevenson, respectively. We thank Dr Jiabin Wang for providing the molecular model of IgM used in Figure I in Box 1. R.P.J. thanks the Research Centre for Drugs and Diagnostics and is supported by a Wellcome Trust Institutional Strategic Support Fund (ISSF) award to the Liverpool School of Tropical Medicine. We thank the reviewers for their constructive and useful suggestions.

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Hviid, L. and Jensen, A.T. (2015) PfEMP1 – a parasite protein family of key importance in Plasmodium falciparum malaria immunity and pathogenesis. Adv. Parasitol. 88, 51–84

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