Dichloroacetate (DCA) reduces brain lactate but increases brain glutamine in experimental cerebral malaria: a 1H-NMR study

Recent findings that levels of brain lactate and alanine were elevated in murine cerebral malaria led us to investigate the effect of dichloroacetate (DCA; 60 mg/kg), an activator of pyruvate dehydrogenase, on the levels of brain metabolites, and on the survival of mice infected with Plasmodium berghei ANKA which normally causes lethal cerebral malaria. DCA significantly reduced brain lactate and alanine levels when administered to infected mice, had no effect on the TCA cycle-related metabolites glutamate, GABA and aspartate and was associated with increased brain glutamine levels: 40% of mice thus treated survived the normally lethal infection.     

Haptoglobin and malaria

Haptoglobin gene knockout mice and wild-type controls were infected with Plasmodium berghei ANKA or Plasmodium chabaudi. The peak parasitaemia and parasite burden were higher in Hp-/- mice than in Hp+/+ mice. The increase in spleen weight following malaria infection was smaller in Hp-/- mice than in Hp+/+ animals. The occurrence of cerebral malaria in P. berghei ANKA infection was not different in Hp gene knockout mice and their controls.     

Distinct placental malaria pathology caused by different Plasmodium berghei lines that fail to induce cerebral malaria in the C57Bl/6 mouse

ABSTRACT: BACKGROUND: Placental malaria (PM) is one major feature of malaria during pregnancy. A murine model of experimental PM using BALB/c mice infected with Plasmodium berghei ANKA was recently established, but there is need for additional PM models with different parasite/host combinations that allow to interrogate the involvement of specific host genetic factors in the placental inflammatory response to Plasmodium infection. METHODS: A mid-term infection protocol was used to test PM induction by three P. berghei parasite lines, derived from the K173, NK65 and ANKA strains of P. berghei that fail to induce cerebral malaria (CM) in the susceptible C57BL/6 mice. Parasitaemia course, pregnancy outcome and placenta pathology induced by the three parasite lines were compared. RESULTS: The three P. berghei lines were able to evoke severe PM pathology and poor pregnancy outcome features. The results indicate that parasite components required to induce PM are distinct from CM. Nevertheless, infection with parasites of the ANKADeltapm4 line, which lack expression of plasmepsin 4, displayed milder disease phenotypes associated with a strong innate immune response as compared to infections with NK65 and K173 parasites. CONCLUSIONS: Infection of pregnant C57BL/6 females with K173, NK65 and ANKADeltapm4 P. berghei parasites provide experimental systems to identify host molecular components involved in PM pathogenesis mechanisms.     

Concurrent gastro-intestinal nematode infection does not alter the development of experimental cerebral malaria

Concurrent helminth infections have been suggested to be associated with protection against cerebral malaria in humans, a condition characterised by systemic inflammation. Here we show that a concurrent chronic gastro-intestinal nematode infection does not alter the course of murine cerebral malaria. Mice infected with Heligmosomoides polygyrus, and co-infected with Plasmodium berghei ANKA 14 days later, developed malaria parasitemia, weight loss and anemia, at the same rate as mice without nematode infection. Both groups developed cerebral malaria around the same time point. The data suggest that a chronic helminth infection does not affect the development of cerebral malaria in a murine model.     

Oxidative Stress and Modification of Renal Vascular Permeability Are Associated with Acute Kidney Injury during P. berghei ANKA Infection

Malaria associated-acute kidney injury (AKI) is associated with 45% of mortality in adult patients hospitalized with severe form of the disease. However, the causes that lead to a framework of malaria-associated AKI are still poorly characterized. Some clinical studies speculate that oxidative stress products, a characteristic of Plasmodium infection, as well as proinflammatory response induced by the parasite are involved in its pathophysiology. Therefore, we aimed to investigate the development of malaria-associated AKI during infection by P. berghei ANKA, with special attention to the role played by the inflammatory response and the involvement of oxidative stress. For that, we took advantage of an experimental model of severe malaria that showed significant changes in the renal pathophysiology to investigate the role of malaria infection in the renal microvascular permeability and tissue injury. Therefore, BALB/c mice were infected with P. berghei ANKA. To assess renal function, creatinine, blood urea nitrogen, and ratio of proteinuria and creatininuria were evaluated. The products of oxidative stress, as well as cytokine profile were quantified in plasma and renal tissue. The change of renal microvascular permeability, tissue hypoxia and cellular apoptosis were also evaluated. Parasite infection resulted in renal dysfunction. Furthermore, we observed increased expression of adhesion molecule, proinflammatory cytokines and products of oxidative stress, associated with a decrease mRNA expression of HO-1 in kidney tissue of infected mice. The measurement of lipoprotein oxidizability also showed a significant increase in plasma of infected animals. Together, our findings support the idea that products of oxidative stress, as well as the immune response against the parasite are crucial to changes in kidney architecture and microvascular endothelial permeability of BALB/c mice infected with P. berghei ANKA.     

Deletion of a malaria invasion gene reduces death and anemia, in model hosts

Malaria parasites induce complex cellular and clinical phenotypes, including anemia, cerebral malaria and death in a wide range of mammalian hosts. Host genes and parasite 'toxins' have been implicated in malarial disease, but the contribution of parasite genes remains to be fully defined. Here we assess disease in BALB/c mice and Wistar rats infected by the rodent malaria parasite Plasmodium berghei with a gene knock out for merozoite surface protein (MSP) 7. MSP7 is not essential for infection but in P. falciparum, it enhances erythrocyte invasion by 20%. In vivo, as compared to wild type, the P. berghei Δmsp7 mutant is associated with an abrogation of death and a decrease from 3% to 2% in peak, circulating parasitemia. The Δmsp7 mutant is also associated with less anemia and modest increase in the size of follicles in the spleen. Together these data show that deletion of a single parasite invasion ligand modulates blood stage disease, as measured by death and anemia. This work is the first to assess the contribution of a gene present in all plasmodial species in severe disease.     

IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection.     

Dichloroacetate (DCA) reduces brain lactate but increases brain glutamine in experimental cerebral malaria: a 1H-NMR study

Abstract

Recent findings that levels of brain lactate and alanine were elevated in murine cerebral malaria led us to investigate the effect of dichloroacetate (DCA; 60 mg/kg), an activator of pyruvate dehydrogenase, on the levels of brain metabolites, and on the survival of mice infected with Plasmodium berghei ANKA which normally causes lethal cerebral malaria. DCA significantly reduced brain lactate and alanine levels when administered to infected mice, had no effect on the TCA cycle-related metabolites glutamate, GABA and aspartate and was associated with increased brain glutamine levels: 40% of mice thus treated survived the normally lethal infection.     

Ligation of B and T lymphocyte attenuator prevents the genesis of experimental cerebral malaria

B and T lymphocyte attenuator (BTLA; CD272) is a coinhibitory receptor that is predominantly expressed on T and B cells and dampens T cell activation. In this study, we analyzed the function of BTLA during infection with Plasmodium berghei ANKA. Infection of C57BL/6 mice with this strain leads to sequestration of leukocytes in brain capillaries that is associated with a pathology resembling cerebral malaria in humans. During the course of infection, we found an induction of BTLA in several organs, which was either due to up-regulation of BTLA expression on T cells in the spleen or due to infiltration of BTLA-expressing T cells into the brain. In the brain, we observed a marked induction of BTLA and its ligand herpesvirus entry mediator during cerebral malaria, which was accompanied by an accumulation of predominantly CD8+ T cells, but also CD4+ T cells. Application of an agonistic anti-BTLA mAb caused a significantly reduced incidence of cerebral malaria compared with control mice. Treatment with this Ab also led to a decreased number of T cells that were sequestered in the brain of P. berghei ANKA-infected mice. Our findings indicate that BTLA-herpesvirus entry mediator interactions are functionally involved in T cell regulation during P. berghei ANKA infection of mice and that BTLA is a potential target for therapeutic interventions in severe malaria.     

CD4+ natural regulatory T cells prevent experimental cerebral malaria via CTLA-4 when expanded in vivo

Studies in malaria patients indicate that higher frequencies of peripheral blood CD4(+) Foxp3(+) CD25(+) regulatory T (Treg) cells correlate with increased blood parasitemia. This observation implies that Treg cells impair pathogen clearance and thus may be detrimental to the host during infection. In C57BL/6 mice infected with Plasmodium berghei ANKA, depletion of Foxp3(+) cells did not improve parasite control or disease outcome. In contrast, elevating frequencies of natural Treg cells in vivo using IL-2/anti-IL-2 complexes resulted in complete protection against severe disease. This protection was entirely dependent upon Foxp3(+) cells and resulted in lower parasite biomass, impaired antigen-specific CD4(+) T and CD8(+) T cell responses that would normally promote parasite tissue sequestration in this model, and reduced recruitment of conventional T cells to the brain. Furthermore, Foxp3(+) cell-mediated protection was dependent upon CTLA-4 but not IL-10. These data show that T cell-mediated parasite tissue sequestration can be reduced by regulatory T cells in a mouse model of malaria, thereby limiting malaria-induced immune pathology.     

Improved methods for haemozoin quantification in tissues yield organ- and parasite-specific information in malariainfected mice

ABSTRACT: BACKGROUND: Despite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against lifethreatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intraerythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivoimmunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence. METHODS: An improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann-Whitney U test and Pearsons correlation analysis. RESULTS: Most Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas subnanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels. CONCLUSIONS: The developed techniques were useful to quantify Hz in different organs with a high reproducibility and sensitivity. An organ-specific Hz deposition pattern was found and was independent of the parasite strain used. Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.     

Malaria-specific and nonspecific activation of CD8+ T cells during blood stage of Plasmodium berghei infection

Cerebral malaria is one of the severe complications of Plasmodium falciparum infection. Studies using a rodent model of Plasmodium berghei ANKA infection established that CD8(+) T cells are involved in the pathogenesis of cerebral malaria. However, it is unclear whether and how Plasmodium-specific CD8(+) T cells can be activated during the erythrocyte stage of malaria infection. We generated recombinant Plasmodium berghei ANKA expressing OVA (OVA-PbA) to investigate the parasite-specific T cell responses during malaria infection. Using this model system, we demonstrate two types of CD8(+) T cell activations during the infection with malaria parasite. Ag (OVA)-specific CD8(+) T cells were activated by TAP-dependent cross-presentation during infection with OVA-PbA leading to their expression of an activation phenotype and granzyme B and the development to functional CTL. These highly activated CD8(+) T cells were preferentially sequestered in the brain, although it was unclear whether these cells were involved in the pathogenesis of cerebral malaria. Activation of OVA-specific CD8(+) T cells in RAG2 knockout TCR-transgenic mice during infection with OVA-PbA did not have a protective role but rather was pathogenic to the host as shown by their higher parasitemia and earlier death when compared with RAG2 knockout mice. The OVA-specific CD8(+) T cells, however, were also activated during infection with wild-type parasites in an Ag-nonspecific manner, although the levels of activation were much lower. This nonspecific activation occurred in a TAP-independent manner, appeared to require NK cells, and was not by itself pathogenic to the host.     

Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis

Experimental cerebral malaria (ECM) resulting from Plasmodium berghei ANKA infection involves T lymphocytes. However, the mechanisms of T cell-mediated pathogenesis remain unknown. We found that, in contrast to ECM-susceptible C57BL6 mice, perforin-deficient (PFP-KO) mice were resistant to ECM in the absence of brain lesions, whereas cytoadherence of parasitized erythrocytes and massive accumulation of activated/effector CD8 lymphocytes were observed in both groups of mice. ECM is induced in PFP-KO mice after adoptive transfer of cytotoxic CD8+ cells from infected C57BL6 mice, which were directed to the brain of PFP-KO mice. This specific recruitment might involve chemokine/chemokine receptors, since their expression was up-regulated on activated CD8 cells, and susceptibility to ECM was delayed in CCR5-KO mice. Thus, lymphocyte cytotoxicity and cell trafficking are key players in ECM pathogenesis.     

Vascular dysfunction as a target for adjuvant therapy in cerebral malaria

Cerebral malaria (CM) is a life-threatening complication of Plasmodium falciparum malaria that continues to be a major global health problem. Brain vascular dysfunction is a main factor underlying the pathogenesis of CM and can be a target for the development of adjuvant therapies for the disease. Vascular occlusion by parasitised red blood cells and vasoconstriction/vascular dysfunction results in impaired cerebral blood flow, ischaemia, hypoxia, acidosis and death. In this review, we discuss the mechanisms of vascular dysfunction in CM and the roles of low nitric oxide bioavailability, high levels of endothelin-1 and dysfunction of the angiopoietin-Tie2 axis. We also discuss the usefulness and relevance of the murine experimental model of CM by Plasmodium berghei ANKA to identify mechanisms of disease and to screen potential therapeutic interventions.     

Tissue distribution of indoleamine 2,3-dioxygenase in normal and malaria-infected tissue

An immunohistochemical method was developed, using a polyclonal antibody, to detect the enzyme indoleamine 2,3-dioxygenase (IDO) in normal and malaria-infected tissue. Plasmodium berghei ANKA, a cerebral malaria (CM) model, and P. berghei K173, a non-cerebral malaria (NCM) model, were used. It was found that vascular endothelial cells were the primary site of IDO expression in both models of malaria infection and that this response was systemic, with the vascular endothelium of brain, heart, lung, spleen and uterus all staining positive. These results suggest that IDO is part of a systemic host response to parasite infection. Although high levels of IDO production alone may not cause pathology, it is possible that when its production is combined with other features of CM, such as breakdown of the blood-brain barrier (BBB), metabolites of the kynurenine pathway may be able to influence the otherwise tightly regulated, immunologically privileged site of the CNS and cause some of the symptoms and pathology observed.     

NK cells stimulate recruitment of CXCR3+ T cells to the brain during Plasmodium berghei-mediated cerebral malaria

NK cells are cytotoxic lymphocytes that also secrete regulatory cytokines and can therefore influence adaptive immune responses. NK cell function is largely controlled by genes present in a genomic region named the NK complex. It has been shown that the NK complex is a genetic determinant of murine cerebral malaria pathogenesis mediated by Plasmodium berghei ANKA. In this study, we show that NK cells are required for cerebral malaria disease induction and the control of parasitemia. NK cells were found infiltrating brains of cerebral malaria-affected mice. NK cell depletion resulted in inhibition of T cell recruitment to the brain of P. berghei-infected animals. NK cell-depleted mice displayed down-regulation of CXCR3 expression and a significant reduction of T cells migrating in response to IFN-gamma-inducible protein 10, indicating that this chemokine pathway plays an essential role in leukocyte trafficking leading to cerebral disease and fatalities.     

MDR1A (ABCB1)-deficient CF-1 mutant mice are susceptible to cerebral malaria induced by Plasmodium berghei ANKA

Under experimental conditions, Plasmodium berghei infection causes cerebral malaria (CM) in susceptible strains of mice such as C57BL/6 and CBA/Ca, whereas BALB/c or DBA/2J strains serve as a model for CM-resistant mice. The aim of the present study was to investigate the susceptibility of the CF1 mouse strain, carrying a spontaneous mutation of the mdr1a gene, to infection with Plasmodium berghei ANKA (PbA). The mdr1a gene codes for P-glycoprotein (P-gp/ABCB1), an efflux pump that is one of the major components of the blood-brain barrier. P-gp effluxes a broad range of xenobiotics from the brain to blood, preventing accumulation and toxicity in the central nervous system. CFI mdr1a (-/-) mice are used to investigate drug transport by efflux pumps. Because many antimalarial agents are effluxed by P-gp (mefloquine, quinine), it was important to determine whether CF1 mice can develop cerebral malaria to predict drug toxicity during cerebral malaria. Our work showed that CF1 mdr1a (-/-) mice are susceptible to PbA. CF1 and C57BL/6N mice (the reference strain) infected with PbA have similar profiles with regard to clinical signs, brain histological lesions, and brain macrophagic activation observed by immunohistological methods.     

Studies on the glycoprotein modification in erythrocyte membrane during experimental cerebral malaria

Plasmodium berghei ANKA (Pb ANKA) is a lethal strain of malaria that causes experimental cerebral malaria (ECM) in rodent models. Pathology of the disease is associated with the sequestration of the infected rbc (irbc) in the micro vessels of brain. In the present study, we analyzed the nature of the glycoprotein modification occurring in irbc membrane during erythrocytic stages of Pb ANKA infection. Titration of naturally occurring glycoproteins with concanavalin A (Con A) and wheat germ agglutinin (WGA) lectins revealed an enhanced lectin binding ability for the irbc membrane preparations. Partial characterization of the Con A specific determinants (alpha-d-methyl mannoside specificity) by lectin affinity chromatography followed by 2D electrophoresis and WGA specific determinants (sialic acid specificity) by Western analysis revealed the association of novel lectin specific determinants in irbc membrane. To correlate the biochemical changes with the morphological changes, SEM of irbc, and TEM of sequestered irbc were performed. These ultra structural studies revealed variable and irregular surface protrusions and deep surface indentations on irbc. These observations implicate that altered glycoprotein profiles may lead to cytoarchitectural changes in irbc membrane and such changes may be essential to establish contact with the host endothelial cells. These observations may be central to the microvascular sequestration and pathology of ECM.     

Human cerebral malaria and the blood-brain barrier

Malaria represents a continuing and major global health challenge and our understanding of how the Plasmodium parasite causes severe disease and death remains poor. One serious complication of the infection is cerebral malaria, a clinically complex syndrome of coma and potentially reversible encephalopathy, associated with a high mortality rate and increasingly recognised long-term sequelae in survivors. Research into the pathophysiology of cerebral malaria, using a combination of clinical and pathological studies, animal models and in vitro cell culture work, has focussed attention on the blood-brain barrier (BBB). This represents the key interface between the brain parenchyma and the parasite, which develops within an infected red cell but remains inside the vascular space. Studies of BBB function in cerebral malaria have provided some evidence for parasite-induced changes secondary to sequestration of parasitised red blood cells and host leukocytes within the cerebral microvasculature, such as redistribution of endothelial cell intercellular junction proteins and intracellular signaling. However, the evidence for a generalised increase in BBB permeability, leading to cerebral oedema, is conflicting. As well as direct cell adhesion-dependent effects, local adhesion-independent effects may activate and damage cerebral endothelial cells and perivascular cells, such as decreased blood flow, hypoxia or the effects of parasite toxins such as pigment. Finally, a number of systemic mechanisms could influence the BBB during malaria, such as the metabolic and inflammatory complications of severe disease acting 'at a distance'. This review will summarise evidence for these mechanisms from human studies of cerebral malaria and discuss the possible role for BBB dysfunction in this complex and challenging disease.