Euterpe oleracea fruit (Açai)-enriched diet suppresses the development of experimental cerebral malaria induced by Plasmodium berghei (ANKA) infection

Background Cerebral malaria is one of the most severe complications attributed to protozoal infection by Plasmodium falciparum, gaining prominence in children mortality rates in endemic areas. This condition has a complex pathogenesis associated with behavioral, cognitive and motor sequels in humans and current antimalarial therapies have shown little effect in those aspects. Natural products with antioxidant and anti-inflammatory properties have become a valuable alternative therapeutic option in the treatment of distinct conditions. In this context, this study investigated the neuroprotective effect of Euterpe oleracea (açai) enriched diet during the development of experimental cerebral malaria induced by the inoculation of Swiss albino mice with Plasmodium berghei ANKA strain. Methods After Plasmodium infection, animals were maintained on a feeding with Euterpe oleracea enriched ration and parameters such as survival curve, parasitemia and body weight were routinely monitored. The present study has also evaluated the effect of açai-enriched diet on the blood-brain barrier leakage, histological alterations and neurocognitive impairments in mice developing cerebral malaria. Results Our results demonstrate that between 7th–19th day post infection the survival rate of the group treated with açai enriched ration was higher when compared with Plasmodium-infected mice in which 100% of mice died until the 11th days post-infection, demonstrating that açai diet has a protective effect on the survival of infected treated animals. The same was observed in the brain vascular extravasation, where Evans blue dye assays showed significantly less dye extravasation in the brains of Plasmodium-infected mice treated with açai enriched ration, demonstrating more preserved blood-brain barrier integrity. Açai-enriched diet also attenuate the histopathological alterations elicited by Plasmodium berghei infection. We also showed a decrease of the neurological impairments arising from the exposure of cerebral parenchyma in the group treated with açai diet, ameliorating motor and neuropsychiatric changes, analyzed through the SHIRPA protocol. Conclusion With these results, we conclude that the treatment with açai enriched ration decreased the mortality of infected animals, as well as protected the blood-brain barrier and the neurocognitive deficits in Plasmodium-infected animals.

Intravenous whole blood transfusion results in faster recovery of vascular integrity and increased survival in experimental cerebral malaria

Transfusion of 10 mg/kg of whole blood via intraperitoneal route to mice with late-stage experimental cerebral malaria (ECM) along with artemether has been shown to result in markedly increased survival (75%) compared to artemether alone (51%). Intraperitoneal route was used to overcome the restrictions imposed by injection of large volumes of viscous fluid in small and deranged blood vessels of mice with ECM. In the present study, a method of intravenous transfusion was implemented by injecting 200mL of whole blood through the right jugular vein in mice with late-stage ECM, together with artemether given intraperitoneally, leading to a remarkable increase in survival, from 54% to 90%. On the contrary, mice receiving artemether plus plasma transfusion showed a worse outcome, with only 18% survival. Compared to the intraperitoneal route, intravascular transfusion led to faster and more pronounced recoveries of hematocrit, platelet counts, angiopoietins levels (ANG-1, ANG-2 and ANG-2/ANG-1) and blood brain barrier integrity. These findings indicate that whole blood transfusion when given intravenously show more efficacy over intraperitoneal transfusion, reinforcing evidence for benefit as an adjuvant therapy for cerebral malaria.

Reversible brain edema in experimental cerebral malaria is associated with transcellular blood-brain barrier disruption and delayed microhemorrhages

Brain swelling occurs in cerebral malaria (CM) and may either reverse or result in fatal outcome. It is currently unknown how brain swelling in CM reverses, as investigations have been hampered by inadequate animal models. In this study, we show that reversible brain swelling in experimental murine cerebral malaria (ECM) can be induced reliably after single vaccination with radiation-attenuated sporozoites as revealed by in vivo high-field (9.4T) magnetic resonance imaging. Our results provide evidence that parenchymal fluid increase and consecutive brain swelling results from transcellular blood-brain barrier disruption (BBBD), as revealed by electron microscopy. This mechanism enables reversal of brain swelling but does not prevent persistent focal brain damage, evidenced by microhemorrhages, in areas of most severe BBBD. In a cohort of 27 pediatric and adult CM patients (n=4 fatal, n=23 non-fatal) two out of four fatal CM patients (50%) and 8 out of 23 non-fatal CM patients (35%) showed microhemorrhages on MRI at clinical field strength of 1.5T, emphasizing the translational potential of the experimental model.

The mTOR Pathway in Platelets Contributes to the Pathophysiology of Experimental Cerebral Malaria

Background: Cerebral malaria is a highly prevalent infectious disease in Sub-Saharan Africa caused by the Plasmodium parasite. The pathogenesis of cerebral malaria results from damaged vascular endothelium induced by parasite sequestration, inflammatory cytokine production and vascular leakage, which results in increased brain permeability and death. While maladaptive responses from immune cells are thought to contribute, growing evidence suggests a crucial role of platelets in malaria pathophysiology. The mammalian target of rapamycin (mTOR) pathway is critical in regulating outcomes in malaria. Previous studies have demonstrated an mTOR specific inhibitor, rapamycin, is protective in a mouse model of experimental cerebral malaria (ECM). However, if the mTOR pathway in platelets specifically contributes to the pathogenesis of malaria is unknown. Methods: Platelet-specific mTOR-deficient (mTOR plt-/-) mice and littermate controls were subjected to a well-established model of ECM, using Plasmodium berghei ANKA. In addition, platelets isolated from human malaria patients were examined for differential regulation of the mTOR pathway using RNA-seq. Results: Platelet RNA-seq and Ingenuity Pathway Analysis from patients infected with P. vivax demonstrated enrichment of mTOR-associated pathways in platelets, such as mTOR signaling and p70S6K signaling, indicating mTOR associated genes are upregulated in human platelets during malaria infection. In mice infected with P. berghei ANKA, the mTOR pathway was activated in bone marrow-megakaryocytes and platelets based on phosphorylation of mTOR and its downstream effector, 4E-BP1. As the mTOR pathway regulates protein translation in platelets, we examined de novo protein synthesis and observed increased protein translation in platelets isolated from mice infected with P. berghei ANKA compared to uninfected controls. To study the specific role of platelet mTOR during ECM pathogenesis, mTOR plt-/- mice and wild-type controls (mTOR plt+/+), were infected with P. berghei ANKA. Platelet deficient-mTOR mice had significantly (p=0.0336) prolonged survival compared to wild-type mice. Increased survival was independent of parasitemia, suggesting platelets did not alter parasite reproduction. While thrombocytopenia and anemia were similar in both genotypes, mTOR plt-/- mice had significantly reduced brain (p=0.0067) and lung (p<0.0001) vascular permeability during late-stage ECM. Interestingly, flow cytometric assessment of leukocyte recruitment to the brain demonstrated a 1.7-fold (p=0.0442) reduction in inflammatory monocytes in platelet-deficient mTOR mice. However, mTOR plt-/- mice had significantly (1.4-fold, p=0.007) more inflammatory monocytes in the blood. Interestingly, circulating platelet-monocytes aggregates were significantly less in mTOR plt-/- compared to mTOR plt+/+ (p=0.0433). Taken together, these results suggest that platelets assist in the recruitment of leukocytes to the brain vasculature during ECM, which is impaired when mTOR is ablated. Conclusions: Our data demonstrates that the mTOR pathway in platelets plays a significant role in malaria pathogenesis. Deletion of platelet mTOR reduces vascular permeability and prolongs survival during ECM. We hypothesize that altered platelet-inflammatory monocyte interactions drive this phenotype. Disclosures Rondina: Platelet Transcriptomics: Patents & Royalties; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding.

Bradykinin Is a Proximal Event in Experimental Cerebral Malaria

Human malaria is a complex disease and a leading cause of mortality in children under 5 years of age. Plasmodium falciparum (Pf) is the agent responsible for cerebral malaria. Parasite infected erythrocytes are sequestered in the brain vasculature, disrupting the blood-brain-barrier, and with systemic inflammation leading to progressive brain edema. The precise pathophysiologic mechanism(s) underlying brain swelling in CM is not known. Recent work from our laboratories indicates that there is a role for bradykinin (BK) in fluid transport in human brain microvascular endothelial cells (Front Med 6:75, 2019). We examined the role of bradykinin (BK) in pediatric CM. Initial studies showed recombinant falcipain-2, a cysteine protease contained in the parasite digestive vacuole, was inhibited by high molecular weight kininogen (HK), with an IC 50=36 nM. Further, falcipain-2, but not the related protease falcipain 3, hydrolyzed the chromogenic substrate S2302 (Pro-Phe-Arg-pNA) at pH 7.4 with an 88 nM K m. These results suggest that falcipain-2 has plasma kallikrein-like activity. HK is both an inhibitor and substrate of falcipain-2. Molar excess HK to falcipain-2 (ratio 8:1 to 2:1) blocked the proteolytic activity of the cysteine protease at pH 7.4. Equal molar falcipain-2 to HK (1:1) resulted in kallikrein-like cleavage of HK with stable BK liberation over 1 h. Molar excess falcipain-2 to HK (1:2 and greater) led to progressive HK cleavage into smaller proteins and peptides. The falcipain-2 major cleavages observed by N-terminal sequencing were in Domain 3 of the heavy chain of HK, the cysteine protease inhibitory region (I 292ASFSQNCDIYPGKDF 303, D 320IPTNSPELEETLT 334, and E 412KKIYPTVNCQPLG 425). P. falciparum trophozoite lysates completely hydrolyzed purified and plasma HK into a ~64 kDa heavy chain and ~46 kDa light chain in buffer containing EDTA, pepstatin, and PMSF. The cysteine proteinase inhibitor E64 blocked this cleavage, suggesting that the relevant activity was that of a cysteine protease. Plasma from Kenyan children presenting with CM (fever, parasitemia, coma) had evidence of circulating cHK, indicative of BK released from HK. Forty percent (8 of 20) of CM patients had no intact 120 kDa HK at hospital entry. In contrast, only 16% (3 of 8) of children with uncomplicated malaria had detectable cHK. In CM patients, the HK level before antimalarial treatment (58 ± 3.9 µg/ml) was significantly lower than the value after clinical recovery (69 ± 3.6 µg/ml; p<0.04) as measured by competitive ELISA. We also examined the roles of BK and HK in experimental cerebral malaria. 10 6 infected red blood cells with P. berghei ANKA were injected intraperitoneally into wild-type (C57BL/6) and total kininogen deficient (kgn1 -/-) C57BL/6 mice. The level of parasitemia on day 5 post-infection was ≥ 8% for both groups of mice (Figure 1). The kgn1 -/- mice had protected neuronal function measured by SHIRPA score relative to wild-type mice. Cerebral edema detected in wild- type mice by Evans Blue dye extravasation test was nearly completely attenuated in kgn1 -/- mice. Corroborative studies were performed in BK B2 receptor deleted (bdkrb2 -/-) mice. In mice with 15% parasitemia for both genotypes, there was significantly less neurologic function deterioration and a 30% reduction in cerebral Evans blue extravasation into brain parenchyma in the bdkrb2 -/- mice. These data strongly suggest that falcipain-2 liberates BK from HK by acting like plasma kallikrein and in high concentrations destroys HK's cysteine protease inhibitory region. Some children with CM have in vivo evidence of prior HK proteolysis. Total kininogen deficiency protects mice from lethal experimental CM. Taken together, these data suggest that bradykinin is a proximal mediator of cerebral malaria. Figure 1 Figure 1. Disclosures McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria.

Observation of the Gut Microbiota Profile in C57BL/6 Mice Induced by Plasmodium berghei ANKA Infection

The genus of Plasmodium parasites can cause malaria, which is a prevalent infectious disease worldwide, especially in tropical and subtropical regions. C57BL/6 mice infected with P. berghei ANKA (PbA) will suffer from experimental cerebral malaria (ECM). However, the gut microbiota in C57BL/6 mice has rarely been investigated, especially regarding changes in the intestinal environment caused by infectious parasites. P. berghei ANKA-infected (PbA group) and uninfected C57BL/6 (Ctrl group) mice were used in this study. C57BL/6 mice were infected with PbA via intraperitoneal injection of 1 × 106 infected red blood cells. Fecal samples of two groups were collected. The microbiota of feces obtained from both uninfected and infected mice was characterized by targeting the V4 region of the 16S rRNA through the Illumina MiSeq platform. The variations in the total gut microbiota composition were determined based on alpha and beta diversity analyses of 16S rRNA sequencing. The raw sequences from all samples were generated and clustered using ≥ 97% sequence identity into many microbial operational taxonomic units (OTUs). The typical microbiota composition in the gut was dominated by Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia at the phylum level. Bacteroidetes and Verrucomicrobia were considerably decreased after PbA infection compared with the control group (Ctrl), while Firmicutes and Proteobacteria were increased substantially after PbA infection compared with Ctrl. The alpha diversity index showed that the observed OTU number was increased in the PbA group compared with the Ctrl group. Moreover, the discreteness of the beta diversity revealed that the PbA group samples had a higher number of OTUs than the Ctrl group. LEfSe analysis revealed that several potential bacterial biomarkers were clearly related to the PbA-infected mice at the phylogenetic level. Several bacterial genera, such as Acinetobacter, Lactobacillus, and Lachnospiraceae_NK4A136_group, were overrepresented in the PbA-infected fecal microbiota. Meanwhile, a method similar to gene coexpression network construction was used to generate the OTU co-abundance units. These results indicated that P. berghei ANKA infection could alter the gut microbiota composition of C57BL/6 mice. In addition, potential biomarkers should offer insight into malaria pathogenesis and antimalarial drug and malaria vaccine studies.

Eosinophils Suppress the Migration of T Cells Into the Brain of Plasmodium berghei-Infected Ifnar1-/- Mice and Protect Them From Experimental Cerebral Malaria

Cerebral malaria is a potentially lethal disease, which is caused by excessive inflammatory responses to Plasmodium parasites. Here we use a newly developed transgenic Plasmodium berghei ANKA (PbAAma1OVA) parasite that can be used to study parasite-specific T cell responses. Our present study demonstrates that Ifnar1-/- mice, which lack type I interferon receptor-dependent signaling, are protected from experimental cerebral malaria (ECM) when infected with this novel parasite. Although CD8+ T cell responses generated in the spleen are essential for the development of ECM, we measured comparable parasite-specific cytotoxic T cell responses in ECM-protected Ifnar1-/- mice and wild type mice suffering from ECM. Importantly, CD8+ T cells were increased in the spleens of ECM-protected Ifnar1-/- mice and the blood-brain-barrier remained intact. This was associated with elevated splenic levels of CCL5, a T cell and eosinophil chemotactic chemokine, which was mainly produced by eosinophils, and an increase in eosinophil numbers. Depletion of eosinophils enhanced CD8+ T cell infiltration into the brain and increased ECM induction in PbAAma1OVA-infected Ifnar1-/- mice. However, eosinophil-depletion did not reduce the CD8+ T cell population in the spleen or reduce splenic CCL5 concentrations. Our study demonstrates that eosinophils impact CD8+ T cell migration and proliferation during PbAAma1OVA-infection in Ifnar1-/- mice and thereby are contributing to the protection from ECM.