Cerebral Malaria Pathogenesis

Revisiting Parasite and Host Contributions

Georges Emile Raymond Grau; Alister Gordon Craig


Future Microbiol. 2012;7(2):291-302. 

In This Article

Host Contributions

For the sake of clarity, we will address findings in human CM (hCM) and those in experimental CM (eCM), separately.

Mediators: Cytokines & Coagulation Factors

More than half a century after Brian Maegraith's original suggestions[72,73] that endogenous mediators have a pathogenic role in severe malaria, a body of evidence suggests that cytokines are involved in CM pathogenesis: reviewed in.[74–76] In hCM, high levels of such proinflammatory cytokines have been reported for more than two decades,[77–79] and confirmed by numerous groups[80] even if some reports suggest that there is no association with severity and that endothelial molecules might be more reliable.[64]

In the human disease, at least in its in vitro modeling, attention has been drawn on the role of tissue factor[81] and more recently vWF.[47] The latter seems crucial in the early steps of Pf cytoadherence, in a dynamic flow chamber setting.[82] In addition to the release of vWF, the activation of the Weibel-Palade body also influences local EC activation through release of Ang2 via the Tie2 signaling system.[83] Ang1:Ang2 ratios have recently been identified as one of the best biomarkers for CM in African children.[84]

In eCM, using the Plasmodium berghei ANKA (PbA) mouse model,[85] TNF[86] and IFN-γ[87] have been identified as important mediators of disease, and lymphotoxin-αs being possibly the principal mediator responsible for the triggering of neuropathology.[88] Using an intervention approach, a recent confirmation of the role of cytokines in this model has been provided by the demonstration that CM can be induced in CM-resistant mice by inducing the endogenous secretion of proinflammatory cytokines. This was achieved by injections of CpG.[89] IFN-γ has clearly been shown to be central in eCM pathogenesis,[87] and several pathways can be triggered by this cytokine, notably IP-10/CXCL10.[90] A complication with many of these studies (in human and mouse systems) is the balance between protection and pathology elicited by the cytokine response. For example, experiments using the mouse model of eCM have determined that early production of this cytokine in the infection can be protective rather than deleterious.[91]

Other host mediators of importance in eCM pathogenesis include histamine,[92] via its H(3) receptor,[93] complement, particularly C5a,[94] endothelin[95] and HO-1 (reviewed in [96]). Aside from cytokines and noncytokine mediators, coagulation factors also participate in the triggering of pathology of eCM, as reviewed in [97].

Effector Cells

Leukocytes In hCM, only a few intravascular leukocytes are seen in brain sections of CM2 patients,[9,98] but the potential role of these cells remains unknown.

HIV-1 positive subjects are at risk of developing severe malaria (reviewed in [99]) but, while it has been proposed that AIDS and CM do not influence each other,[100] the relationship between these two syndromes is exceedingly complex, as discussed in.[101,102]

In eCM, the T-cell dependency of the neurological syndrome is clear, involving both CD4+ [103,104] and CD8+ [105,106] T cells (reviewed in [74,107]). Histopathologically, in eCM the presence of leukocytes is more conspicuous than in hCM, but no CD8 T-cells are visible locally, yet CD8 depletion prevents eCM.[97] The issue of Tregs in eCM has been addressed by Engwerda's team,[108] who showed that CD4+ CD25+ Tregs suppress CD4+ T-cell function and inhibit the development of P. berghei-specific Th1 responses involved in eCM pathogenesis.[109] More recently, however, in 2010, Haque et al. demonstrated that in vivo expansion of Tregs by using IL-2/anti-IL-2 complexes prevents eCM.[110] In African patients, Treg deficiency appears to be protective and Treg cells have been found to be increased in patients with hyperparasitemia; therefore, immune (or inflammation-based) pathology is clearly important in human severe malaria, including hCM.[111,112] The involvement of monocytes in eCM has been reviewed.[74]

Platelets In African patients with hCM, platelets have been found to accumulate in brain vessels, but not in cases of severe malarial anemia or coma of other (nonmalaria) causes.[98] This intravascular accumulation has been recently confirmed at the level of CM-associated retinopathy.[113] In eCM, platelets are found to accumulate in organs before these show lesions, as revealed by radiolabeled platelet localization experiments,[114] and intervention experiments have demonstrated that they are involved in pathology (reviewed in [115]). Furthermore, intravascular platelets are detectable by the laser-induced breakdown spectroscopy technology even before microvascular dysfunction can be detected by MRI.[116] These in vivo data have been analyzed in further detail in vitro, using mouse EC,[117] as well as human[39] EC, cocultures, notably disclosing the importance of the release of TGF-β1 by platelets.[118]

More recently, using an in vitro model of human CM, it was found that platelets can trigger a number of EC genes[119] involved in inflammation and apoptosis, such as genes involved in chemokine, TREM1, cytokine, IL-10, TGFβ, death-receptor and apoptosis signaling.

ECs: Both Targets & Effectors in CM Severe malaria is characterized by a pleomorphic EC activation. While studying various aspects of EC changes in relation to tissue damage, we showed that IE, together with host cells, leads to profound endothelial alterations.[120] In turn, EC can trigger immunopathological changes, as detailed in.[121] Among others, we identified miRNAs able to regulate ECs that are modulated in murine CM but not in uncomplicated malaria.[122]

A central feature of endothelial changes in CM is activation,[123–125] resulting from the direct contact with IE, as discussed in the 'Parasite contributions' section, or with leukocytes, as has been shown in vitro, using human cells.[126] The effect of these direct interactions is amplified by the cascade of inflammatory reactions discussed in the 'Mediators: cytokines & coagulation factors' and 'Effector cells' sections. Associated with this, a number of endothelial functions become altered,[34] notably permeability, leading to brain edema, which can be mild in adult CM[127] and more pronounced in pediatric CM.[128] In this respect, eCM reproduces the type of brain edema seen in pediatric rather than adult hCM, as evidenced by MRI,[129] including at the eye level[130] and by quantitative immunohistochemistry.[131] The latter study showed a marked upregulation of aquaporin 4 on astrocytic foot processes in eCM, a finding that has recently been found marginally modified in hCM, in adults where brain edema does not appear to be the cause of death.[132] However, even if brain swelling is found in adult patients with CM, simple therapeutic measures against brain edema, such as mannitol, do not protect against the neurological syndrome, and may even be harmful.[133] Therefore, fine mechanisms leading to brain edema need to be investigated in more depth.

Angiogenic dysregulation in severe malaria has been substantiated by the demonstration of altered levels of VEGF, soluble VEGFR-2,[134] and Ang2.[135] However, studies differ in recording an increase or decrease in VEGF, perhaps owing to differences in the clinical groups being considered. In specific comparisons of hCM from India, VEGF was reduced in plasma from patients with hCM compared with mild malaria cases and healthy controls, and further reduced in patients dying from hCM.[136] Yeo et al. have suggested that the increase in endothelial activation marked by increasing levels of Ang2 could be owing to reduced bioavailability of NO, which in turn may result from a reduction in its precursor, L-arginine. It is possible that the sequestered IE may cause this reduction through the release of arginases on schizont rupture. They showed that endothelial function, measuring reperfusion by reactive hyperemia-peripheral arterial tonometry,[137] was reduced in severe malaria and subsequently demonstrated that this deficiency could be partially rectified by the infusion of L-arginine.[138] Most likely is that a subtle balance between positive and negative regulation of endothelial functions contributes to the endothelial pathology of CM, as discussed elsewhere.[139]

Interestingly, an association has been found between susceptibility to CM development and endothelial reactivity to cytokines,[140] as had been suggested in the CM model.[141] The endothelial hyper-reactivity notably included the production of membrane microparticles, which are found in dramatically high numbers in patients with hCM[40,41,142] and could be novel players in disease pathogenesis, as reviewed extensively in.[120,143–145]

Repercussions on Brain Parenchyma

Glial Changes In hCM, alterations of glial cells have been studied in tissues from patients as well as in culture systems. As integral constituents of the BBB, astrocytes can show signs of dysfunction as a result of the IE sequestration. In patients with CM, several aspects of microglial activation have also been reviewed.[146]In vitro, soluble factors from Pf induce apoptosis in human brain vascular endothelial and neuroglia cells. More DNA fragmentation is found in brain EC than in neuroglia, suggesting that extended exposure to high levels of these soluble factors in circulation is critical.[147]

In eCM, similar glial changes, involving both astrocytes and microglial cells have been reported. The potential consequences of glial activation, notably glial-derived proteinases, in structural damage of the CNS have been reviewed in detail.[148] Among the inflammatory mediators, selected chemokines are dramatically upregulated in microvessels and adjacent glial cells.[149]

Neuronal In hCM, immunohistochemistry has been used to identify a variety of cellular stress and injury responses in the brains of patients with fatal Pf malaria. Oxidative stress predominated in the vicinity of vessels and hemorrhages. Some degree of DNA damage was found in the majority of malaria patients, but staining patterns suggest considerable stress response and reversible neuronal injury.[150]

Both axonal and astrocytic injury markers, the tau protein and S-100B, respectively, have been found in elevated concentrations in CSF samples from CM patients.[151] A disruption of axonal transport was further demonstrated by detection of µ- and m-calpain, the specific inhibitor calpastatin, in postmortem brain tissue from patients who died from severe malaria.[152]

The first evidence for a loss of axonal viability in eCM and the first demonstration of optic nerve involvement was provided by Ma et al..[153] Recently, MRI in this model revealed very early changes not only in the optic but also the trigeminal nerves. Cranial nerve injury was the earliest anatomic hallmark of the disease, visible even before brain edema became detectable.[130] The overproduction of IFN-γ, whose crucial role in eCM has been shown,[87] can increase expression of the heme enzyme indoleamine 2,3-dioxygenase in microvascular ECs. Raised indoleamine 2,3-dioxygenase levels leads to increased production of a range of biologically active metabolites that may be part of a tissue protective response. Damage to astrocytes may result in reduced production of the neuroprotectant kynurenic acid, leading to a decrease in its ratio relative to the neuroexcitotoxic quinolinic acid. This imbalance may contribute to some of the neurological signs of human and experimental CM, as reviewed in Hunt et al.[154]

Reactive astrocytes also can show expression of a molecule that was thought to be a neuron-specific, hypoxia-responsive and neuroprotective protein: neuroglobin was found to be present, aside from neurons, in reactive astroglia and in scar-forming astrocytes in various neuropathological conditions, including CM.[155] CM is a complex pathology that illustrates the impact and the role of endothelial, glial and neuronal cells in the process. The global role of the neurovascular unit, considered as a whole or as an organ is discussed elsewhere [GRAU ET AL. THE CROSSROADS OF NEUROINFLAMMATION IN INFECTIOUS DISEASES: ENDOTHELIAL CELLS AND ASTROCYTES (2012), MANUSCRIPT IN PREPARATION].

Behavioral/Cognitive Changes The development of neurological sequelae after hCM has long been recognized and have been recently characterized in detail. It was found that Kenyan children have neurocognitive impairments that are evident as long as 9 years later.[156,157] Potential cognitive rehabilitation solutions such as cognitive exercises, environmental enrichment, nutritional supplementation, physical therapy and speech therapy have been proposed.[158] In Ugandan children, a computerized cognitive training program 3 months after severe malaria had an immediate effect on cognitive outcomes but did not affect academic skills or behavior.[159] In addition to previously described neurological and cognitive sequelae, severe behavior problems may follow CM in children, including ADHD. In another study in Ugandan children, observed differences in patterns of sequelae may be owing to different pathogenic mechanisms, brain regions affected or extent of injury.[160]

In eCM, inflammatory changes in the CNS are also associated with behavioral impairment in P. berghei (strain ANKA)-infected mice.[161] As there is evidence that these behavioral changes may be due to – or at least associated with – oxidative stress, as assessed by elevated levels of malondialdehyde and conjugated dienes in the brains of PbA-infected C57BL/6 mice with CM, antioxidant treatment was evaluated as adjunct therapy. PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage.[162]


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