Antigen presentation by endothelial cells: what role in the pathophysiology of malaria?

Antigen presentation by endothelial cells: what role in the pathophysiology of malaria?

Review Antigen presentation by endothelial cells: what role in the pathophysiology of malaria? Romy Razakandrainibe1, Ste´phane Pelleau2, Georges E. ...

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Antigen presentation by endothelial cells: what role in the pathophysiology of malaria? Romy Razakandrainibe1, Ste´phane Pelleau2, Georges E. Grau3 and Ronan Jambou1 1

Institut Pasteur de Madagascar, BP 1274 Antananarivo 101, Madagascar Universite´ de la Me´diterrane´e, Unite´ Mixte de Recherche MD3, Marseille, France 3 Vascular Immunology Unit, University of Sydney, Camperdown, NSW, Australia 2

Disruption of the endothelial cell (EC) barrier leads to pathology via edema and inflammation. During infections, pathogens are known to invade the EC barrier and modulate vascular permeability. However, ECs are semiprofessional antigen-presenting cells, triggering T-cell costimulation and specific immune-cell activation. This in turn leads to the release of inflammatory mediators and the destruction of infected cells by effectors such as CD8+ T-cells. During malaria, transfer of parasite antigens to the EC surface is now established. At the same time, CD8 activation seems to play a major role in cerebral malaria. We summarize here some of the pathways leading to antigen presentation by ECs and address the involvement of these mechanisms in the pathophysiology of cerebral malaria. Endothelium: a crucial player during infection ECs, the major constituent of the microvasculature that line blood and lymphatic vessels, play a crucial role in the integrity of tissue. Intercellular junctions between ECs are tightly regulated and serve as selective filters to regulate the passage of various molecules and liquids across their membranes. Dysfunction and/or disruption of this barrier can lead to pathology through edema and inflammation. Dysfunction can result from (i) the dynamic opening of intercellular junctions, (ii) disorganization of the barrier, or (iii) destruction of ECs. During infection, barrier disruption can be induced by the liberation of active mediators, the interaction of microbial components with EC receptors (immunoglobulins; Toll-like receptor, TLR), or and interaction with pathogens and leukocytes which can be followed by activation of the transmigration pathway (Glossary). Disorganization of the barrier is a key step in the migration of leukocytes from the blood to the site of tissue infection. During this contact, ECs can also activate immune cells (ICs). Direct antigen presentation by ECs could be one of the mechanisms triggering this activation, but this mechanism has only been poorly investigated. It first requires that ECs can trap exoantigens and process them (Box 1). During malaria, recent findings suggest that transfer of parasite antigens to ECs can take place [1]. As with dengue virus, this paves the way to Corresponding author: Jambou, R. ([email protected]), ([email protected]). Keywords: endothelial cell; antigen presentation; dengue; malaria.

Glossary Antigen-presenting cells (APCs): cells that take up, process and present antigens to other immune cells to initiate and activate immune responses. Monocytes, macrophages, dendritic cells, B cells and endothelial cells are APCs. B7 family [B7-1 (CD80) and B7-2 (CD86)] molecules: these belong to the immunoglobulin superfamily and are homologous costimulatory ligands expressed on the surface of APCs. Binding of these molecules to the T-cell costimulatory receptors, CD28 and CTLA-4, is essential for the costimulation of T-cells. Coagulation cascade: a sequence of biochemical activities involving fibrin, platelets and ECs that halt bleeding by forming a clot. Two pathways (cascades of proteases) lead to the conversion of fibrinogen to fibrin and platelet activation. The damaged wall is then covered by a platelet and fibrincontaining clot to stop bleeding and begin repair of the damaged vessel. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4): following binding to B7.1 (CD80) or B7.2 (CD86) on APCs, CTLA4 signaling in activated T-cells induces cell cycle arrest, reduces cytokine production, and diminishes T-cell responses. T regulatory cells constitutively express CTLA4. Exoantigen: a toxin, protein or other antigen released by an abnormal cell or pathogen which recognized by a specific immune response. Fas/Fas ligand: Fas (also known as APO-1 or CD95) is a Type-I transmembrane protein, it is a widely expressed cell death receptor that has a critical role in the regulation of the immune system and tissue homeostasis. Fas ligand (FasL) is a Type-II Transmembrane protein of TNF. FasL is expressed predominantly in activated T lymphocytes and natural killer cells. In the immune system, engagement of the cell death surface receptor Fas by FasL results in apoptotic cell death, mediated by caspase activation. Fas and FasL are involved in downregulation of immune reactions. Malfunction of the Fas-FasL pathway causes lymphoproliferative disorders. Whereas its exacerbation may cause tissue destruction. Graft-versus-host disease (GVHD): a complication that occurs after stem cell or bone marrow transplant, especially in immune-compromised recipients, during which leukocytes contained in the graft attack tissues of the recipient. Intercellular adhesion molecule 1 (ICAM-1): a glycoprotein typically expressed on endothelial cells and cells of the immune system. It binds to integrins to stabilize cell–cell interaction. Invariant natural killer T (iNKT) cell: a T-cell that expresses specific poorly polymorphic variable gene segments of the T-cell receptor a chain (Va14 in mice and Va24 in human). Typically these cells coexpress cell-surface markers that are encoded by the NK locus and are activated by recognition of CD1d, particularly when a-galactosylceramide is bound in the groove of CD1d. Invasome: a cup generated on the membrane of a cell by a massive actin rearrangement and that mediates the phagocytosis and internalization of large bacterial aggregates. Ischemia-reperfusion injury: a process induced by restoration of the blood flow to an area that had previously experienced deficient blood supply. Multiple cell damage and survival pathways are activated during this process. MHC (major histocompatibility complex) class I heavy chain: MHC class I molecules are trimeric structures composed of a transmembrane heavy chain non-covalently associated with a light chain (b2-microglobulin) and an 8–11 amino acid peptide. The heavy chain consists of an extracellular part divided into three domains (a1, a2 and a3) followed by a transmembrane domain and a cytoplasmic domain containing conserved serine and tyrosine residues. The distal a1 and a2 domains are highly polymorphic and form a cavity where the 8–11 amino acid peptide binds. Microparticles (MPs): small (<0.1 mm) vesicles that are released from cells and circulate in the extracellular milieu. MPs consist of a plasma membrane surrounding a small amount of cytosol. The membrane of endothelial MPs

1471-4922/$ – see front matter ß 2012 Published by Elsevier Ltd. doi:10.1016/ Trends in Parasitology, April 2012, Vol. 28, No. 4


Review contains receptors and other cell-surface molecules. Under normal physiological conditions, low levels of MPs are constantly shed into the blood from ECs that line the blood vessels. The levels of endothelium-derived MPs circulating in the blood may increase in some vascular and infectious diseases. Mitogen-activated protein kinase (MAPK) pathway: a signal transduction pathway involving several kinases that is responsive to numerous external stimuli (growth factors, differentiation factors and other cellular conditions). Phagosome: a vesicle that forms in a cell as a result of phagocytosis and that contains the engulfed material. Phagolysosome: membrane-associated cytoplasmic vesicles formed by invagination of phagocytized material. They fuse with lysosomes to form phagolysosomes in which the hydrolytic enzymes of the lysosome digest the phagocytized material. Protein kinase Cd (PKCd): a member of the PKC family which is thought to play a crucial role in cell proliferation, differentiation and apoptosis. Rac-dependent pathway: Rac signaling pathways control cytoskeleton and transcription activity. Rac activation is responsible for stimulating cell spreading and migration. Semi-professional APC: APCs with facultative professional APC status; they are able to induce primary immune responses if properly activated. Toll-like receptor (TLR) pathways: TLR signaling plays an essential role in the innate immune response. Activation of TLR signaling through recognition of pathogen-associated molecular patterns leads to the transcriptional activation of genes encoding proinflammatory cytokines, chemokines and costimulatory molecules, which subsequently control the activation of the antigen-specific adaptive immune response. Transmigration pathway: a cellular process in which circulating lymphocytes bind to and then migrate across blood vessel walls to enter body tissues. Transporters associated with antigen processing (TAP1 and TAP2): ATPbinding cassette proteins involved in transporting short peptides from the cytosol into the lumen of the ER, where they associate with MHC class I molecules. Trogocytosis: a process whereby lymphocytes (B, T and NK cells) conjugated to antigen-presenting cells extract surface molecules from these cells and reexpress them on their own cell surface.

antigen presentation to the immune system, leading to damage to the endothelial barrier (Box 2). This review focuses on the key steps during EC–pathogen interactions that lead to IC–EC interaction and barrier dysfunction. Most data have been obtained from mouse models. In 2010, differences between mouse and human malaria were fully debated in these columns [2–8], but the mouse model can provide meaningful data on pathophysiology. Along this line, data are reviewed suggesting that the antigen-presentation mechanism is involved in the pathogenesis of cerebral malaria (CM).

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Endothelial cells and antigen presentation: what is known? Interferon g (IFNg) was demonstrated in vitro to induce the expression of major histocompatibility complex (MHC) class II molecules on ECs (Box 1), resulting in T-cell activation and proliferation after contact [9]. Circulating T-cells were also demonstrated to recognize antigens on the surface of ECs, triggering EC activation and the release of mediators [10]. Similarly, EC expression of the human leukocyte antigen DR (HLA-DR) appears to be sufficient to stimulate purified CD4+ T-cell proliferation without the involvement of other leukocyte populations [11]. In vivo, constitutive expression of MHC class I and II molecules has been described in capillaries and coronary arteries, and the isoform of the proteasome that generates MHC class Icompatible peptides (Box 1) was characterized in brain ECs and astrocytes [12]. Last, it was demonstrated that ECs can acquire and cross-present exogenous antigen in association with MHC class I molecules [13]. However, to be rescued from the activated death program after stimulation by ECs, activated T-cells must receive a second signal of costimulation. It is potently delivered by CD80 (B7-1) and CD86 (B7-2) to (i) CD28 constitutively expressed on naı¨ve and activated T-cells, and (ii) cytotoxic T-lymphocyte-associated antigen (CTLA-4, also known as CD152) found on activated T-cells. B7-1 is the favored ligand for CTLA-4 whereas B7-2 is that of CD28 [14]. In 2001, in vitro treatment with tumor necrosis factor (TNF) was confirmed to upregulate B7-2 and induce the de novo expression of B7-1 on ECs [15]. Interactions of CTLA-4 and CD28 with the B7 family have fundamentally different effects on T-cells. Although CTLA4 is a crucial negative regulator of T-cell activation, CD28 is a positive signal for T-cell activation and proliferation, resulting in increased IL-2 gene expression [16]. They are thus semi-professional antigen-presenting cells (APCs). Professional APCs mainly present exogenous antigen to CD4+ helper T-cells which in turn can activate B cells, prime an anti-tumor response, maintain the function and

Box 1. How cells present molecules to lymphocytes T-cell antigen receptors recognize peptides bound to MHC molecules at the cell surface (Figure I). Most cells constitutively express MHC class I molecules that allow presentation of internal peptides to CD8+ T-cells and the destruction of altered cells. Some cells, termed antigen-presenting cells (APC), also express inducible MHC class II molecules which can present peptides from exogenous proteins to CD4+ helper T-cells. A third type, CD1 molecules, can present nonpeptidic antigens. MHC class I and CD1 are assembled in the endoplasmic reticulum (ER) and sent to the cell surface, whereas MHC class II molecules are synthesized and assembled in the ER, and are then transferred via the Golgi apparatus to the phagosome where the invariant chain is broken down, leaving a small fragment (the CLIP) inside the peptide-binding grove. For MHC class I presentation, endogenous proteins are first degraded into oligopeptides in the cytosol by the ubiquitin–proteasome pathway (or by aminopeptidases [71], and endo- or exopeptidases) and are transferred to the ER through a membrane-spanning heterodimer, the ‘transporter associated with antigen processing’ (TAP), consisting of two proteins: TAP1 and TAP2. In the ER, peptides associate with the MHC class I heavy chain and are transported to the cell surface. Both TAP1 and TAP2 are alternatively spliced to yield 152

different truncated non-functional proteins [72]. Fragmentation of exogenous antigens by proteases occurs in the phagolysosome. Peptides associate with the MHC class II molecules, and the complex is then shuttled to the cell surface. However, exogenous antigens can also be presented in association with MHC class I molecules [73,74], either after (i) lysosomal proteolysis {these are transferred from the phagosome into the cytosol, then cleaved into oligopeptides by the proteasome and transported to the rough ER (RER) by TAP [75]}, or (ii) after splitting into peptides by cathepsin S in the endocytic compartment where they bind to MHC class I molecules. MHC class I expression, antigen processing and presentation can be upregulated by IFNg, which induces: (i) an increase of the MHC class II transactivator (CIITA) [76,77], which in turn regulates the TAP system at the transcriptional level, or (ii) activation of Janus kinase/signal transducer and activator of transcription (JAK/STAT1) [78] and the production of proteasomal subunits [79]. This mechanism allows cytotoxic T-cell responses against pathogens which do not infect or replicate only poorly in professional APC. CD1 molecules are assembled in the ER and are sent to the cell surface. However, they are re-internalized into specific endocytic compartments where they can bind to lipid and glycolipid antigens


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which will be presented to T-cells. CD1-restricted T-cells are mainly Thelper type 1 (Th1) and are predominantly cytolytic. Another population can produce both Th1 and Th2 cytokines [80]. CD1 presentation is highly dependent on p38 and the ERK1/2 MAPK


pathway [81], as well as on the PKCd inhibition pathway. PKCd can control the MAP pathway; indeed, PKCd is an upstream regulatory molecule for p38 and ERK1/2 [82] and impairs CD1d-mediated antigen presentation [83].



Exogenous antigen




Phagosome Phagosomal proteases


Sorting endosome

Proteasome Peptides Golgi


TAP Endoplasmic reticulum β2m MHC class I

MHC class II



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Figure I. The exogenous pathway for antigen presentation. Exogenous antigens (proteins) are internalized into the phagosome and transferred to the cytosol. (a) The exogenous antigen is degraded by the proteasome, and the resulting peptides are transported to MHC class I in the ER by TAP. Within the ER, the MHC class I heavy chain, b2-microglobulin and peptides form a stable complex that is then transported to the cell surface. In non-hematopoietic cells, the pathway of MHC class I-restricted presentation of an endogenously synthesized antigen is crucial; otherwise, a specialized pathway allows display of exogenous antigens in the context of MHC class I molecules. This pathway is termed cross-presentation. (b) Exogenous antigens (proteins) are fragmented by proteases in an endosome. Like MHC class I, MHC class II molecules are synthesized in the ER where they are associated with the invariant chain which prevents them from binding to endogenous antigens. MHC class II are then transported through the Golgi and the trans-Golgi apparatus to reach the endosome, where the invariant chain is digested. Within the endosome, peptides bind to MHC class II molecules and are finally delivered to the plasma membrane. (c) The CD1 heavy chain is assembled with b2-microglobulin in the ER with the help of chaperones (calnexin, calreticulin and ERp57). The process of presentation of functional CD1 is not fully understood, but some components of the process have been identified. The first step is the trafficking of CD1 to the surface. The newly synthesized CD1 is delivered from the Golgi to the cell surface via the secretory pathway. During this trafficking, CD1 is thought to bind to self-derived lipids to block the binding of inappropriate antigens. At the cell surface, CD1 is re-internalized where it binds foreign lipids. CD1 either follows the endocytic recycling pathway and returns to the plasma membrane, or follows the recycling pathway from the lysosomal compartment (the same pathway that is used by MHC class II to traffic to the plasma membrane).

growth of CD8+ T-cells, and orchestrate innate immunity through cytokine release [17]. They can also exert cytotoxicity against cells expressing MHC class II during infection with Epstein–Barr Virus, human immunodeficiency virus (HIV) or cytomegalovirus (CMV), and also in malignancies [18–20]. ECs from some organs, such as the liver, can play an opposite role in the immune response. In addition to their professional scavenger function, uptake of antigens by liver sinusoidal ECs (LSEC) takes place by receptor-mediated endocytosis (mainly via mannose and scavenger receptors [21]); LSEC can then present antigen to CD4+ [22] and CD8+ T-cells [23], inducing antigen-specific CD8 tolerance and blocking T-helper type 1 (Th1) differentiation of CD4+ cells

[24]. This tolerance is important for food antigens, but such a mechanism also could be involved in regulating immune responses against malaria. Indeed CMV infection of mice causes functional maturation of antigen-presenting LSECs sufficient to promote antigen-specific differentiation of Tcells into effector CD8+ cells without any involvement of dendritic cells (DCs) or CD80/86 expression [25]. By quickly controlling infection, this rapid and local induction of T-cell immunity in the liver is likely to be advantageous to the host, and ECs can thus be considered as versatile nonmigratory APCs [26]. Although MHC-mediated presentation of antigens to ICs requires close contact between ICs and ECs, recent data suggest that microparticles (MPs) could be another pathway 153


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Box 2. Alteration of endothelium permeability by the immune response Several molecules which regulate inflammation and coagulation are expressed on the EC surface or are released by the endothelium. Activation or injury of ECs (during infection) can compromise these functions, leading to the activation of leukocytes which in turn adhere and migrate from the vessel lumen to the site of inflammation and finally enhance destruction of the ECs. In mice, early after inoculation (12–24 h), dengue virus invades the endothelium (Figure I). When a high viral titer is reached in ECs, infiltration of macrophages occurs in the vicinity of the endothelium that is associated with TNF production, changes in permeability, and hemorrhage. Both viral infection and macrophages are responsible for (i) disorganization of the actin cytoskeleton in infected cells, (ii) decreased expression and redistribution of both vascular endothelial cadherin and zona occludens-1, and (iii) increased actin stress fibers in adjacent non-infected cells, which modulate endothelial permeability [84]. Similarly, during hemorrhage, ECs overexpress NO synthase (iNOS) and nitrotyrosine which induce apoptosis of adjacent ECs and further increase hemorrhage. This is triggered by antibody binding to the viral non-structural protein 1 (NS1) expressed at the surface of ECs [85]. Monoclonal anti-NS1 can experimentally induce these alterations, but also cross-reacts with LYRIC (lysine-rich CEACAM1 co-isolated) protein on human umbilical vein endothelial cells (HUVECs), suggesting another potential mechanism of pathogenesis [86]. Anti-NSP1 can also induce apoptosis of infected cells, NO production by adjacent non-infected cells [87], activation of the NF-kB-regulated pathway, cytokine and chemokine production (IL-6, IL-8, RANTES, and MCP-1) [88], and ICAM-1 expression and adhesion

of PBMC on ECs [89]. Cytokine overproduction results in EC activation and barrier damage through tight junction–cytoskeleton reorganization [90]. IL-8 triggers EC retraction and permeability increase in a CXCR2/Rac-dependent pathway [91]. It also induces actin stress-fiber formation through CXCR1-dependent Rho/Rho kinase activation and phospholipase D stimulation [92]. Dengue infection also induces apoptosis of ECs by increasing cell-surface Fas expression and the release of soluble Fas ligand [93]. Fas/FasL interaction triggers Fas trimerization, the recruitment of Fas-associated death-domain protein, and subsequent activation of procaspase-8 and caspase-3 pathways, leading to apoptosis. Autoimmunity can also induce barrier dysfunction [94]. For example, myeloperoxidase (MPO)-specific and proteinase 3 (PR3)specific anti-neutrophil cytoplasmic autoantibody (ANCA) can induce intravascular neutrophil activation and adhesion [95]. This results in the release of reactive oxygen species and lytic enzymes (including MPO and PR3) that damage vessel walls. In addition, ANCA-activated neutrophils and monocytes release interleukin 1b (IL-1b), IL-6, IL-8, TNF, monocyte chemoattractant protein 1 (CCL2) and leukotriene B4, which activate and recruit monocytes [96] and CD4+ T-cells [97]. Damage to the endothelium is also a prominent histological feature of allograft rejection. MHC molecules on donor cells are the targets of both humoral and cellular immune responses during graft-versus-host disease (GVHD), and graft rejection is mainly mediated by NK cells and complement [98,99]. CD8+ can also induce apoptosis through a proteinase-activated receptor 1 (PAR-1)-dependent modulation of the intrinsic apoptotic pathway and P38MAPK/caspase-3 activation.




NF-kB Endothelial cell pathway activation (2.3) Non- infected endothelial cell

Antibody anti-NS1 (2.1)


(4) Complement activation C5a; C3a



Increase vascular permeability Histamine release

Increase vascular permeability



Heparan sulfate (3.2) shedding




Mast cell

Increased neutrophil L-selectin binding

(1) Dengue virus infected endothelial cell




Active infection dengue virus


Heparan sulfate

Increase vascular permeability TRENDS in Parasitology

Figure I. Is dengue virus infection a model for malaria pathogenesis? During dengue infection, virus invades ECs where it replicates (0). Viral NS-1 proteins expressed on the surface of the cell are a key step in pathogenesis because they are recognized by antibodies. The same process of antigen transfer is described during malaria both through trogocytosis and iRBC engulfing. Malaria antibodies could thus induce the same alteration as for dengue virus where (i) anti-NS-1 reacts with infected cells (1), leading to apoptosis; (ii) anti-NS-1 (2) also reacts with non-infected cells, triggering (2.1) NO production via a NF-kB pathway, enhancing apoptosis; (2.2) production of cytokines and chemokines resulting in (2.3) immune cell activation and (2.4) binding through ICAM-1; (iii) conversely, vascular permeability alteration (3) as a result of neutrophil binding (3.1) is enhanced by heparan sulfate shedding (3.2); (iv) anti-NS-1 antibodies can promote an increase of vascular permeability (4) through complement activation leading to histamine release by mast cells (4.1). The same alterations are described during malaria but the role of antibodies has not yet been demonstrated.



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to antigen presentation that can act at long distances. Far from being inert, MPs and cell debris shed physiologically from the plasma membrane can activate CD4+ T-cells [27] in an allogenic-dependent pathway, and can also activate the coagulation cascade. Antigenic peptide-dependent MHC class II activation through MPs is still debated. ECs can release MPs during a variety of pathophysiological situations such as CM [28–30]. MPs are characterized by the presence of phosphatidylserine and proteins at their surface, including antigens from their originating cells [31]. MPs can thus carry surface antigens of the pathogen to activate T-cells far away from the primary site of the infection, without any contact between T-cells and APC, and without involving the TLR pathway. Transfer of a multidrug resistance pump between normal and resistant cancer lines has been demonstrated. Nevertheless, antigen presentation by MPs remains open to debate. Capture of malaria antigens by endothelial cells during malaria Transfer of antigens to ECs has mostly been described during viral infections. Related to cytokine release or EC infection, EC alterations vary with the type of virus and are key pathological factors (especially in bleeding) during


Cell death

viral hemorrhagic fevers (Box 2). Along the same line, transfer of malaria antigens to ECs is now demonstrated [1]. Adhesion of erythrocytes infected with mature stages of Plasmodium falciparum on ECs is a key step in the life cycle of the Plasmodium parasite, and this triggers CM pathology (Figure 1). Several EC proteins have been identified as receptors for infected erythrocytes, including thrombospondin, CD36, ICAM-1, VCAM-1, and endothelial leukocyte adhesion molecule-1 (ELAM-1) (reviewed in [32]). After adherence, the first step comprises rapid transfer of material from infected red blood cells (iRBCs) to human brain ECs via a mechanism closely related to trogocytosis [1]. This is followed shortly by tighter adhesion to ECs triggered by an ICAM actin-rich invasome-like structure which leads to P. falciparum iRBC internalization into ECs. Malaria antigens can be detected in the endosomal pathway of the cell up to 24 h later, indicative of antigen recycling. Another route for ECs to capture malaria antigen could be antibody-dependent. In areas of high P. falciparum malaria transmission, patients with CM were reported to have significantly higher plasma levels of both total and Plasmodium-specific IgE [33]. Moreover, as demonstrated in lung iRBC, adhesion to ECs induces overexpression of the


Direct cytotoxicity Internalization: trogocytose–loke mechanism

Antigen presentation?

Lymphocyte IL-10 Lymphocyte Lymphocyte


Platelets Infected –red blood cells


Red blood cell

CD-36 TGF-β1 IFN-γ

Increased expression of ICAM-1






Endothelial cell

Junction alteration

Basement membrane TRENDS in Parasitology

Figure 1. Plasmodium falciparum, immune cells and endothelial cells interact during blood–brain barrier breakdown. During malaria infection, Plasmodium undergoes complex interactions with ECs. Adhesion of erythrocytes infected with the mature stages of Plasmodium falciparum on ECs is a key step in the life cycle of the Plasmodium parasite, and this triggers CM pathology. Several proteins have been identified on ECs as receptors for infected erythrocytes, including thrombospondin, CD36, ICAM-1, VCAM-1 and endothelial leukocyte adhesion molecule-1 (ELAM-1). The PfEMP1–ICAM interaction induces NO production, thus leading to cell apoptosis. Platelet density also increases in the brain during severe malaria and enhances endothelial damage, fibrin deposition and micro-perfusion impairment. The release of tumor growth factor (TGF)-b1 by platelets induces apoptosis of ECs. Brain infiltration by CD8 T lymphocytes was also implicated in the development of CM pathogenesis by altering the barrier of ECs through direct cytotoxicity. In vivo disruption of the BBB is the key event in CM, and this occurs in areas of parasite sequestration. In vitro observations relate BBB disruption during CM to the accumulation of TNF-secreting monocytes in vivo in brain vessels and EC activation. Transfer of material from iRBC to human brain ECs is closely related to a trogocytosis-like mechanism that was also observed in vitro. The fate of internalized malarial antigens remains unknown, raising the question of how they are processed and presented.


Review low-affinity receptor FceRII/CD23 at the surface of ECs. Engagement of this receptor by IgE–malaria antigen complexes strongly enhances nitric oxide (NO) synthase expression and NO release [34]. This mediator plays a crucial role in neuronal dysfunction, leading to coma. This IgE-mediated alteration can be enhanced by FceRI-positive neutrophil activation, as recently demonstrated during experimental malaria. The deposition of malaria antigens in vivo in vessels was also reported during autopsies of patients dying from CM, and images of iRBC sequestered beneath ECs in infected vessels were reported [35]. In addition, intense deposition of P. falciparum antigens, IgG and fibrin in cerebral vessels associated with cerebral hemorrhages was reported [36]. Immunofluorescence studies revealed extravascular deposits of P. falciparum granular antigen in cerebral tissue that were associated with acute inflammatory lesions and with IgE deposition in the brain white matter [35] or beneath ECs. Similarly, Plasmodium-derived lactate dehydrogenase (pLDH) or pAldose were detected in a variety of organs during CM. These were most abundant in the blood vessels of brain, heart, and lung, and inside ECs [37]. This transfer of malaria antigens could be followed by their presentation to the immune system (which is not yet established), and activation of CD8+. Indeed, brain infiltration by CD8+ T lymphocytes has already been implicated in the alteration of EC barrier function and in the development of CM or lung alterations in children. However, it is not clear whether CD8+ T-cells are primarily activated in the brain or in lymphoid organs. During CM, another working hypothesis is that longdistance presentation of antigen could also take place via MP production as described above. A proinflammatory environment promotes the shedding of MPs and, during the acute phase of CM, a high level of MPs originating from ECs was described in malaria-infected children [38]. In addition to reflecting EC damage, MPs display potent proinflammatory activity and can induce the release of TNF and other mediators from mononuclear cells which consequently promote endothelial lesions [39]. MPs can also promote transfer of malaria antigens to ECs. They are released by several other cell types including platelets, monocytes, T lymphocytes, and RBCs, and can interact with and activate human brain ECs [40]. Binding of MPs to human brain ECs is followed by internalization and transfer of antigens to the ECs [33]. These findings strengthen the role of the EC as an APC. Finally, ECs can help other antigen-presenting cells such as DCs which can transmigrate into the CNS during degeneration and injury. Strong contacts can be established between DCs and brain microvascular ECs. Transfer of malaria antigens to ECs has now been demonstrated [1], and presentation of malaria antigens to cytotoxic cells could be another step towards explaining this pathology. However, analysis of the exact mechanism will require malariasensitized leukocytes from subjects living in tropical areas. Interaction of endothelial cells with immune cells during malaria Analysis of post-mortem brains of Malawian children with fatal malaria showed that a substantial number of macrophages, T-cells, neutrophils, B cells and natural killer (NK) 156

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cells were stacked in the microvasculature [41]. Similarly, studies with Plasmodium berghei ANKA-infected mice showed that the number of abCD8+ cells [42], abCD4+ cells, as well as gd T-cells, increased at the same time as neurological signs, suggesting a pathologic role of these cells in the development of CM. Conversely, depletion of antibodies and/or knockout of CD4+/CD8+ in mice protects against experimental CM. CD8+ T-cell activation and IFNg release were also found to mediate iRBC accumulation in the brain and other organs [43]. CD1d-restricted NK T-cell (NKT) accumulation in the brain was also required for the induction of murine CM [44], and these cells can stimulate the recruitment of CXCR3+ T-cells to the brains of animals with the neurological syndrome. Antigen presentation by ECs was previously suggested to occur during CM [45]. However, to date the nature of the antigens presented and recognized by CD4+ or CD8+ during CM is unknown. Identifying these malaria antigens might contribute to the development of new therapeutic strategies to protect the blood–brain barrier (BBB). The dengue model provides further insight into the mechanisms that could be activated. In contrast to primary infection, which is generally not life-threatening, central nervous system damage during secondary infection with dengue virus can result in paralysis and/or death, mostly associated with CD8+ infiltration [46]. The dengue NS3 protein seems to play a key role in this pathology [47] because dengue virus-specific CD8+ T-cells recognize an HLA-B*-07 restricted T-cell epitope on this protein. The chemokine IFNg-inducible protein-10 kDa (IP-10, CXCL10), that is secreted during many inflammatory diseases, could be involved in the recruitment of these cells. It is the ligand for CXCR3 on activated/effector T-cells and NK cells that plays an essential role in T-cell recruitment and trafficking [48]. By contrast, the newly described invariant NKT cells (iNKT) could have a positive effect on the course of infection. In schistosomiasis, these accumulate in the liver and spleen of mice and promote Th1 responses by producing IFNg [49]. Similarly, during intravenous injection of Plasmodium yoelii and P. berghei sporozoites into mice, artificial activation of iNKT induces IFNg secretion which protects the liver from infection [50]. These iNKT populations expand in the liver, but not in the spleen, during P. yoelii infection [51]. EC dysfunction during malaria: the mechanisms EC activation and dysfunction after invasion and/or intracellular replication may be considered to be common determinants of microbial pathogenesis. Infected cells launch specific molecular defenses involving multiple adaptive signaling mechanisms, as demonstrated by numerous studies on dengue (Box 2). In vivo disruption of the BBB is the key event in CM. This occurs in areas of parasite sequestration and was related to the accumulation of TNF-secreting monocytes in brain vessels and adhesion of platelets to ECs [30,52]. Organ-specific pathogenesis mainly involved the sequestration of iRBCs on ECs, which then increase activation of extracellular signal-regulated kinase1/2 (ERK1/2), and P38 and JUN Nterminal kinase (JNK) mitogen-activated protein kinase (MAPK), through the binding of PfEMP-1 to ICAM-1 [53].


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GPI ICAM1 expression

Increased permeability

PfEMP-1-ICAM1 interaction

TLR-2 MyD88




PLCγ1 PKC PP60src Activation

IKKα Tyrosine kinase PKC

INOS induction




p130Cas activation NF-kB

Cortactin AJ


Peroxynitrite (ONOO-)

FAK ↑Cytokine and chemokines (e.g IL-12) GJ No

Cytoskeletal change



Dysfunction Key:

GJ: Gap junction

AJ: Adherens junction

TJ: Tight junction : Claudin

Zona occludens



: Occludin





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Figure 2. Opening of endothelial cell junctions during cerebral malaria. Activation of cell signaling during severe malaria contributes to pathogenicity. On the one hand, cytoadherence (PfEMP-1 and ICAM-1) of parasitized RBCs induces the production of NO and induces apoptosis, leading to BBB dysfunction. On the other hand, cytoadherence raises intracellular Ca2+, resulting in the activation of cortactin and then Rho, which in turn induces phosphorylation of P130cas. These ICAM-1-mediated pathways lead to the phosphorylation of cytoskeletal protein FAK and paxillin, thus inducing changes in junctional permeability. During CM, in addition to cytoadherence, BBB breakdown involves soluble proteins such as GPI. The major toxin of P. falciparum GPI interaction with TLR2 is responsible for NO production in ECs, leading to apoptosis. Moreover, GPI initiates tyrosine kinase and PKC pathways, promoting ICAM-1 expression, and amplifying cytoadherence. GPI–TLR2 stimulated MyD88 is crucial for IL-12 (associated with disease severity) production. Cell signaling leads to the activation of NF-kB, which is essential to the initiation of cytokine gene transcription. Abbreviations: AJ, adherens junction; FAK, focal adhesion kinase; GPI, glycosylphosphatidylinositol; GJ, gap junction; JAM, junctional adhesion molecule; PKC, protein kinase C; TJ, tight junction.

In pulmonary microvascular ECs, for example, ICAM-1 binding induces (i) phosphorylation of P38–MAPK-dependant heat-shock protein 27 (HSP-27), which in turn modulates cytoskeletal reorganization [54], and (ii) P38 and ERK1/2 MAPK-dependent IL-6 expression [55]. In cerebral ECs, ICAM-1 binding leads to a number of functional changes because ICAM-1 interacts with Ezrin/Radixin/ Moesin family molecules which activate Rho GTPases, protooncogene tyrosine-protein kinase (Src), protein kinase C (PKC), and MAPK [56]. Concomitantly, ICAM-1 activation triggers tyrosine phosphorylation of phosphatidylinositol-phospholipase C (PLC), inducing a rise in intracellular calcium. The Rho-dependent pathway induces p130 phosphorylation and JNK activation, whereas PLC via calcium signaling and the PKC-dependent pathway is responsible for cortactin phosphorylation and Src activation. Both pathways allow phosphorylation of focal adhesion kinase (FAK) and paxillin, resulting in cytoskeletal rearrangement [57], thus increasing permeability. These data were strengthened by a study showing decreased expression of tight-junction proteins [zonula

occludens-1 (ZO), occludin and vinculin] in ECs when cocultured with iRBCs from patients with CM [58]. Downregulation of expression of tight-junction molecules decreases the integrity of the endothelial barrier and leads to edema, particularly in the brain (Figure 2). Significant downregulation of tight junction-associated proteins (occludin, claudin5 and ZO-1) was shown to be associated with tight-junction disassembly in ECs and the opening of intercellular junctions [59]. This opening of junctions is not strictly related to cytoadherence because (i) P. falciparum can modulate EC signaling cascades through soluble proteins [60,61], and (ii) glycophosphatidylinositol (GPI) anchors derived from P. falciparum merozoites and hemozoin can trigger TLR2 and TLR9 activation, respectively [62,63]. This MyD88-dependent TLR signaling can mediate the brain pathogenesis of severe malaria [64]. It can also modulate the ability of DCs to prime T-cell responses and to activate acquired immunity by increasing antigen uptake capacity and presentation by MHC molecules [65,66]. Interactions of TLRs with their agonists initiate different signaling pathways that result in the 157

Review activation of MAPK and ERK, and transcription of nuclear factor kB (NF-kB) and the interferon regulatory factor (IRF) responsive genes that are pivotal for immunity [67], by recruiting MyD88, TRIF, TIRAP/MAL and TRAM [68,69]. The involvement of MyD88 and TLR 2/4 pathways in the development of experimental CM was recently confirmed [70] but is subject to ongoing debate. Concluding remarks: the missing points Understanding IC/EC interactions during infection could be a major breakthrough in the treatment of patients suffering from complications of severe infectious diseases. Treatment of the pathogen by a specific drug is not enough to decrease mortality. Even with efficient antiplasmodial treatment, alteration of the BBB during CM is followed by edema and inflammation, leading to a poor outcome even in a resuscitation unit. Indeed, permeability varies in normal conditions and between organs. Some endothelial beds are leaky with apparent fenestrae, allowing rapid passage of large molecules and fluid, as in the kidney and liver, whereas others are very tightly bound, as in the brain. During infectious disease, mechanisms involved in the destruction of the endothelial wall are complex, resembling ischemia–reperfusion processes, and need to be fully investigated. In this context, the mechanisms required for antigen presentation to T-cells have already been described in ECs, and these are associated with the activation of immune cells and destruction of the endothelial wall. Similar invasion of the endothelium by bacteria or viruses has already been confirmed, and invasion is also likely to take place for both yeast and P. falciparum. The involvement of these mechanisms in the pathophysiology of malaria (as for hemorrhagic fevers) is further supported by epidemiological features: (i) severe malaria attacks occur predominantly in older children or in young adults who have already developed malaria, and (ii) attacks are more frequent during the second part of the transmission season. Overall, all the elements summarized here argue that transfer of malaria antigens to ECs together with antigenspecific immune activation play a crucial role in the destruction of the endothelial barrier. These new data may provide new opportunities to strengthen the treatment of CM. More studies should now be conducted with immune cells from patients who have recovered from disease to explore these mechanisms. Acknowledgments This work has been supported by grants from the Institut Pasteur de Madagascar, the Association pour la Recherche sur le Cancer (ARC), the National Health and Medical Research Council (NHMRC), the Sydney Medical School’s A.L. Kerr Foundation, the Cooper Foundation, and the Sixth Framework Program of the European Commission (project MPCM: Pathogenic role of micro-vesiculation in cerebral Malaria).

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