Ferroptosis participates in neuron damage in experimental cerebral malaria and is partially induced by activated CD8+ T cells.

Cerebral malaria is the most serious complication of malaria infection, with 26% of surviving children having neurological sequelae, which may be caused by neuron damage, but the mechanism is not clear. Ferroptosis has been reported to play an important role in neuron damage in several nervous system diseases. However, the occurrence of ferroptosis in experimental cerebral malaria (ECM) pathogenesis is still unknown. In this study, we firstly detected increased levels of malondialdehyde (MDA) and iron, which are indicators of ferroptosis, in the cerebrum of ECM mice. Some important regulators of ferroptosis, including upregulated expression of transferrin receptor 1 (TfR1) and acyl-CoA synthetase long-chain family member 4 (ACSL4), and downregulation of glutathione peroxidase 4 (GPX4) levels, were also confirmed in ECM mice. Consistently, neuron damage, which was detected in the cerebrum of ECM mice, was positively correlated with reduced GPX4 expression and furtherly rescued by administration of the ferroptosis inhibitor ferrostatin-1 (Fer-1). In addition, primary neurons were damaged by activated CD8+ T cells, an effect that was also partially rescued by Fer-1 on amyloid precursor protein expression and mitochondrial membrane potential levels in vitro. Activated CD8+ T cells were also shown to infiltrate the cerebrum of ECM mice and upregulate TfR1 expression in primary neurons, which may be an important event for inducing ferroptosis in ECM. Altogether, we show that ferroptosis contributes to neuron damage in ECM pathogenesis, and activated CD8+ T cells may be important inducers of neuronal ferroptosis. Hence, targeting ferroptosis may be a promising adjuvant therapeutic strategy for neurological sequelae in patients with cerebral malaria.

L-arginine supplementation and thromboxane synthase inhibition increases cerebral blood flow in experimental cerebral malaria.

Cerebral malaria pathogenesis involves vascular dysfunction with low nitric oxide (NO) bioavailability, vasoconstriction and impaired vasodilation, leading to ischemia, tissue hypoxia and ultimately death. Cerebral blood flow (CBF) involves NO and other pathways, including arachidonic acid (AA)-derived metabolites. Here we show that mice with experimental cerebral malaria (ECM) by P. berghei ANKA showed marked decreases in CBF (as assessed by laser speckle contrast imaging - LSCI) and that administration of L-arginine supplementation (50 mg/kg) and/or of the thromboxane synthase inhibitor Ozagrel (100 mg/kg) induced immediate increases in CBF. L-arginine in combination with artesunate (32 mg/kg) induced immediate reversal of brain ischemia in the short-term (1 hour), but the effect subsided after 3 and 6 hours. Neither L-arginine nor Ozagrel reversed blood brain barrier breakdown. Mice with ECM showed brain levels of selected AA-derived metabolites with a vasoconstrictor profile, with increased levels of 8-isoprostanes, 20-HETE and 14,15-DHET, whereas mice infected with a non-ECM-inducing strain of P. berghei (NK65) showed a vasodilator profile, with normal levels of 20-HETE and 14,15-DHET and increased levels of PGE2. L-arginine is capable of partially reversing cerebral ischemia and AA metabolites may play a role in the cerebrovascular dysfunction in ECM.

Citrulline protects mice from experimental cerebral malaria by ameliorating hypoargininemia, urea cycle changes and vascular leak.

Clinical and model studies indicate that low nitric oxide (NO) bioavailability due in part to profound hypoargininemia contributes to cerebral malaria (CM) pathogenesis. Protection against CM pathogenesis may be achieved by altering the diet before infection with Plasmodium falciparum infection (nutraceutical) or by administering adjunctive therapy that decreases CM mortality (adjunctive therapy). This hypothesis was tested by administering citrulline or arginine in experimental CM (eCM). We report that citrulline injected as prophylaxis immediately post infection (PI) protected virtually all mice by ameliorating (i) hypoargininemia, (ii) urea cycle impairment, and (iii) disruption of blood brain barrier. Citrulline prophylaxis inhibited plasma arginase activity. Parasitemia was similar in citrulline- and vehicle control-groups, indicating that protection from pathogenesis was not due to decreased parasitemia. Both citrulline and arginine administered from day 1 PI in the drinking water significantly protected mice from eCM. These observations collectively indicate that increasing dietary citrulline or arginine decreases eCM mortality. Citrulline injected ip on day 4 PI with quinine-injected ip on day 6 PI partially protected mice from eCM; citrulline plus scavenging of superoxide with pegylated superoxide dismutase and pegylated catalase protected all recipients from eCM. These findings indicate that ameliorating hypoargininemia with citrulline plus superoxide scavenging decreases eCM mortality.

Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.

Cerebral malaria (CM) is a multifactorial syndrome involving an exacerbated proinflammatory status, endothelial cell activation, coagulopathy, hypoxia, and accumulation of leukocytes and parasites in the brain microvasculature. Despite significant improvements in malaria control, 15% of mortality is still observed in CM cases, and 25% of survivors develop neurologic sequelae for life-even after appropriate antimalarial therapy. A treatment that ameliorates CM clinical signs, resulting in complete healing, is urgently needed. Previously, we showed a hyperbaric oxygen (HBO)-protective effect against experimental CM. Here, we provide molecular evidence that HBO targets brain endothelial cells by decreasing their activation and inhibits parasite and leukocyte accumulation, thus improving cerebral microcirculatory blood flow. HBO treatment increased the expression of aryl hydrocarbon receptor over hypoxia-inducible factor 1-α (HIF-1α), an oxygen-sensitive cytosolic receptor, along with decreased indoleamine 2,3-dioxygenase 1 expression and kynurenine levels. Moreover, ablation of HIF-1α expression in endothelial cells in mice conferred protection against CM and improved survival. We propose that HBO should be pursued as an adjunctive therapy in CM patients to prolong survival and diminish deleterious proinflammatory reaction. Furthermore, our data support the use of HBO in therapeutic strategies to improve outcomes of non-CM disorders affecting the brain.-Bastos, M. F., Kayano, A. C. A. V., Silva-Filho, J. L., Dos-Santos, J. C. K., Judice, C., Blanco, Y. C., Shryock, N., Sercundes, M. K., Ortolan, L. S., Francelin, C., Leite, J. A., Oliveira, R., Elias, R. M., Câmara, N. O. S., Lopes, S. C. P., Albrecht, L., Farias, A. S., Vicente, C. P., Werneck, C. C., Giorgio, S., Verinaud, L., Epiphanio, S., Marinho, C. R. F., Lalwani, P., Amino, R., Aliberti, J., Costa, F. T. M. Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.

Antiplasmodial, anti-complement and anti-inflammatory in vitro effects of Biophytum umbraculum Welw. traditionally used against cerebral malaria in Mali.

ETHNOPHARMACOLOGICAL RELEVANCE: Biophytum umbraculum Welw. (Oxalidaceae) is a highly valued African medicinal plant used for treatment of cerebral malaria, a critical complication of falciparum malaria. AIM OF THE STUDY: To provide additional information about traditional use of B. umbraculum and to test plant extracts and isolated compounds for in vitro activities related to cerebral malaria. MATERIALS AND METHODS: The traditional practitioners were questioned about indication, mode of processing/application, dosage and local name of B. umbraculum. Organic extracts and some main constituents of the plant were investigated for anti-malaria, anti-complement activity and inhibition of NO secretion in a RAW 264.7 cell line. RESULTS: Treatment of cerebral malaria was the main use of B. umbraculum (fidelity level 56%). The ethyl acetate extract showed anti-complement activity (ICH50 5.7±1.6µg/ml), inhibition of macrophage activation (IC50 16.4±1.3µg/ml) and in vitro antiplasmodial activity (IC50 K1 5.6±0.13µg/ml, IC50 NF54 6.7±0.03µg/ml). The main constituents (flavone C-glycosides) did not contribute to the activity of the extract. CONCLUSION: Inhibition of complement activation and anti-inflammatory activity of B. umbraculum observed in this study might be possible targets for adjunctive therapy in cerebral malaria together with its antiplasmodial activity. However, clinical trials are necessary to evaluate the activity due to the complex pathogenesis of cerebral malaria.

Iron prevents the development of experimental cerebral malaria by attenuating CXCR3-mediated T cell chemotaxis.

Cerebral malaria is a severe neurological complication of Plasmodium falciparum infection. Previous studies have suggested that iron overload can suppress the generation of a cytotoxic immune response; however, the effect of iron on experimental cerebral malaria (ECM) is yet unknown. Here we determined that the incidence of ECM was markedly reduced in mice treated with iron dextran. Protection was concomitant with a significant decrease in the sequestration of CD4+ and CD8+ T cells within the brain. CD4+ T cells demonstrated markedly decreased CXCR3 expression and had reduced IFNγ-responsiveness, as indicated by mitigated expression of IFNγR2 and T-bet. Additional analysis of the splenic cell populations indicated that parenteral iron supplementation was also associated with a decrease in NK cells and increase in regulatory T cells. Altogether, these results suggest that iron is able to inhibit ECM pathology by attenuating the capacity of T cells to migrate to the brain.

Pro-stimulatory role of methemoglobin in inflammation through hemin oxidation and polymerization.

Inflammation or vascular occlusion by parasitized red blood cell contributes to the pathogenesis of cerebral malaria. The current study aimed to characterize the role of major pro-oxidant factor methemoglobin present in the malaria culture supernatant contributing in inflammation during malaria. Heme and heme polymer stimulate macrophage to secrete large amount of reactive oxygen species into the external micro-environment. The addition of methemoglobin along with heme or heme polymer amplifies production of ROS from macrophages several folds. Methemoglobin mediated stimulatory effect is not due to release of iron, enhanced production of H2O2 or mutual interaction of reaction components. Spectroscopic studies show that methemoglobin accepts heme as a substrate and oxidizes it through a single electron transfer mechanism. Heme oxidation product is a heme polymer with similar chemical and structural properties to synthetic ß-hematin. Phenyl N-t-butylnitrone inhibits heme polymerization (IC50=30 nM) and indicates the absolute necessity of heme oxidation and heme free radical generation for heme polymerization. Methemoglobin produced heme polymer is a potent pro-inflammatory factor to release ROS into external microenvironment. Interestingly, methemoglobin not only produces pro-inflammatory heme polymer, but it also amplifies the potential of heme or preformed heme polymer (haemozoin or ß-hematin) to produce several folds high ROS production from macrophages. This study illustrates the pro-inflammatory effect of methemoglobin, the underlying novel mechanism by which this occurs and a possible clinical intervention. Based on the results, we recommend methemoglobin directed peroxidase inhibitors as an adjuvant therapy during malaria.

Brain gene expression, metabolism, and bioenergetics: interrelationships in murine models of cerebral and noncerebral malaria.

Malaria infection can cause cerebral symptoms without parasite invasion of brain tissue. We examined the relationships between brain biochemistry, bioenergetics, and gene expression in murine models of cerebral (Plasmodium berghei ANKA) and noncerebral (P. berghei K173) malaria using multinuclear NMR spectroscopy, neuropharmacological approaches, and real-time RT-PCR. In cerebral malaria caused by P. berghei ANKA infection, we found biochemical changes consistent with increased glutamatergic activity and decreased flux through the Krebs cycle, followed by increased production of the hypoxia markers lactate and alanine. This was accompanied by compromised brain bioenergetics. There were few significant changes in expression of mRNA for metabolic enzymes or transporters or in the rate of transport of glutamate or glucose. However, in keeping with a role for endogenous cytokines in malaria cerebral pathology, there was significant up-regulation of mRNAs for TNF-alpha, interferon-gamma, and lymphotoxin. These changes are consistent with a state of cytopathic hypoxia. By contrast, in P. berghei K173 infection the brain showed increased metabolic rate, with no deleterious effect on bioenergetics. This was accompanied by mild up-regulation of expression of metabolic enzymes. These changes are consistent with benign hypermetabolism whose cause remains a subject of speculation.

Modulatory potential of iron chelation therapy on nitric oxide formation in cerebral malaria.

To determine whether iron chelation modulates nitric oxide (NO) formation and cell-mediated immune effector function in children with cerebral malaria, serum concentrations were measured of the stable end products of NO, nitrite and nitrate (NO2-/NO3-), interleukin (IL)-4, -6, and -10, and neopterin in 39 Zambian children enrolled in a placebo-controlled trial of desferrioxamine B and quinine therapy. Mean concentrations of NO2-/NO3- increased significantly over 3 days in children receiving desferrioxamine plus quinine but not in those given placebo and quinine. Neopterin levels declined significantly with placebo but not with desferrioxamine. IL-4 levels increased progressively in the placebo group and ultimately decreased in the desferrioxamine group, but the trends were not statistically significant. IL-6 and IL-10 levels were elevated initially and decreased significantly in both groups over 3 days. These data are consistent with the hypothesis that iron chelation therapy in children with cerebral malaria strengthens Th1-mediated immune effector function involving increased production of NO.

Upregulation of reactive oxygen and nitrogen intermediates in Plasmodium berghei infected mice after rescue therapy with chloroquine or artemether.

Plasmodium berghei ANKA infected C57B1/6 mice develop cerebral malaria at a parasitaemia of 15-25%. When parasitaemia reached 10%, P. berghei infected mice were treated with artemether, chloroquine or clindamycin in order to prevent the occurrence of cerebral malaria. Artemether and chloroquine were highly efficient. Functional tests revealed that zymosan stimulated spleen cells from untreated mice with cerebral malaria showed a slight decrease in their capacity to produce reactive oxygen intermediates (ROI) when compared with naive mice. After artemether or chloroquine treatment, the ROI production was significantly enhanced. The interferon-gamma induced production of reactive nitrogen intermediates (RNI) was slightly elevated in mice with cerebral malaria, but markedly elevated in artemether or chloroquine treated mice when compared with naive mice. Moreover, high levels of inducible nitric oxide synthase gene expression could be detected by in-situ hybridization in spleen sections of mice which had been treated with artemether or chloroquine. These findings suggest that increased production of ROI and RNI after chemotherapy may play a protective role for the host during malaria.

Protection against murine cerebral malaria by dietary-induced oxidative stress.

Feeding 20% (w/w) menhaden-fish oil in a standard laboratory chow diet for 4 wk partially protected CBA/CaJ mice from the central nervous system consequences of infection with Plasmodium berghei (ANKA). Full protection (complete survival for 14 days postinfection) could be obtained by feeding a purified pro-oxidant vitamin E-deficient diet containing 4% (w/w) menhaden oil (MO - VE diet). The purified pro-oxidant MO - VE diet also exerted a pronounced suppressive effect against the parasite (depressed 6-day parasitemias). The anitmalarial effect of the MO - VE diet could be prevented by supplementing the diet with vitamin E or with either of 2 synthetic antioxidants, N,N'-diphenyl-p-phenylenediamine or probucol. These results suggest that the fish oil exerts its antimalarial effect by imposing a dietary-induced oxidative stress on the infected host erythrocyte, the parasite, or both. Nutritional manipulation of host oxidative stress status may be a useful adjunct therapy in patients undergoing treatment with pro-oxidant antimalarials such as drugs of the qinghaosu family.