Search

Cerebral malaria (CM) remains an important cause of morbidity and mortality. Risk for developing CM partially depends on host genetic factors, including variants encoded in the type I interferon (IFN) receptor 1 (IFNAR1). Type I IFNs bind to IFNAR1 resulting in increased expression of IFN responsive genes, which modulate innate and adaptive immune responses. To comprehensively study IFNAR1 genetic variant associations in Malawians with CM or uncomplicated malaria, we used a tag single nucleotide polymorphism approach, based on the HapMap Yoruba in Ibadan, Nigeria, population database. We identified three novel (rs914142, rs12626750, and rs1041867) and one previously published (Chr21:34696785 [C > G]) IFNAR1 variants to be associated with CM. Some of these variants are in gene regulatory regions. Chr21:34696785 (C > G) is in a region encoding histone modifications and transcription factor-binding sites, which suggests gene regulatory activity. Rs12626750 is predicted to bind embryonic lethal abnormal vision system-like RNA-binding protein 1, a RNA-binding protein which can increase the type I IFN response. Furthermore, we examined these variants in an expression quantitative trait loci database and found that a protective variant, rs914142, is associated with lower expression of IFNAR1, whereas the CM-associated variant rs12626750 was associated with increased IFNAR1 expression, suggesting that activation of the type I IFN pathway may contribute to pathogenesis of CM. Future functional studies of IFNAR1 variants are now needed to clarify the role of this pathway in severe malarial diseases.

Cerebral malaria (CM) presents as an encephalopathy and is due to infection with Plasmodium falciparum. Patients are comatose, often with fever, recurrent seizures and this condition is associated with a high mortality rate. The etiology of the coma and seizures are poorly understood. Circulating small molecules and lipids have bioactive functions and alterations in their concentrations have been implicated in seizure disorders and other forms of encephalopathy. We carried out a comprehensive analysis of blood metabolites during CM to explore a biochemical basis of this encephalopathy. A paired metabolomics analysis was performed on the plasma samples of Malawian children (n = 11) during CM and at convalescence thirty days later, to identify differentially abundant molecules associated with CM. We also report plasma molecules associated with CM mortality (n = 4) compared to survival (n = 19). Plasma metabolites were identified through ultra high performance liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry to maximize compound detection and accuracy and then compared to a library for identification. We detected a total of 432 small molecules in the plasma and 247 metabolites were significantly differentially abundant between CM and convalescence (p < 0.05, FDR < 0.10). These represented global changes across many classes of molecules including lipids, amino acids and hemoglobin metabolites. We observed significant changes in molecules that could impact neurologic function during CM; these include increased levels of kynurenate and decreased indolepropionate, glutamate, arginine and glutamine. Moreover, 1-methylimidazoleacetate, kyurenate, arachidonic acid and dimethylarginine were associated with mortality (p < 0.05, fold change > 1.2). These results highlight the broad changes in blood chemistry during CM. We have identified metabolites that may impact central nervous system physiology and disease outcomes and can be further explored for their mechanistic roles into the pathophysiology of CM.

Malaria remains a global health burden causing significant morbidity, yet the mechanisms underlying disease outcomes and protection are poorly understood. Herein, we analyzed the peripheral blood of a unique cohort of Malawian children with severe malaria, and performed a comprehensive overview of blood leukocytes and inflammatory mediators present in these patients. We reveal robust immune cell activation, notably of CD14+ inflammatory monocytes, NK cells and plasmacytoid dendritic cells (pDCs) that is associated with very high inflammation. Using the Plasmodium yoelii 17X YM surrogate mouse model of lethal malaria, we report a comparable pattern of immune cell activation and inflammation and found that type I IFN represents a key checkpoint for disease outcomes. Compared to wild type mice, mice lacking the type I interferon (IFN) receptor exhibited a significant decrease in immune cell activation and inflammatory response, ultimately surviving the infection. We demonstrate that pDCs were the major producers of systemic type I IFN in the bone marrow and the blood of infected mice, via TLR7/MyD88-mediated recognition of Plasmodium parasites. This robust type I IFN production required priming of pDCs by CD169+ macrophages undergoing activation upon STING-mediated sensing of parasites in the bone marrow. pDCs and macrophages displayed prolonged interactions in this compartment in infected mice as visualized by intravital microscopy. Altogether our findings describe a novel mechanism of pDC activation in vivo and precise stepwise cell/cell interactions taking place during severe malaria that contribute to immune cell activation and inflammation, and subsequent disease outcomes., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: Most patients with cerebral malaria (CM) sustain cerebral microvascular sequestration of Plasmodium falciparum-infected red blood cells (iRBCs). Although many young children are infected with P. falciparum, CM remains a rare outcome; thus, we hypothesized that specific host conditions facilitate iRBC cerebral sequestration. To identify these host factors, we compared the peripheral whole-blood transcriptomes of Malawian children with iRBC cerebral sequestration, identified as malarial-retinopathy-positive CM (Ret+CM), to the transcriptomes of children with CM and no cerebral iRBC sequestration, defined as malarial-retinopathy-negative CM (Ret-CM). Ret+CM was associated with upregulation of 103 gene set pathways, including cytokine, blood coagulation, and extracellular matrix (ECM) pathways (P < 0.01; false-discovery rate [FDR] of <0.05). Neutrophil transcripts were the most highly upregulated individual transcripts in Ret+CM patients. Activated neutrophils can modulate diverse host processes, including the ECM, inflammation, and platelet biology to potentially facilitate parasite sequestration. Therefore, we compared plasma neutrophil proteins and neutrophil chemotaxis between Ret+CM and Ret-CM patients. Plasma levels of human neutrophil elastase, myeloperoxidase, and proteinase 3, but not lactoferrin or lipocalin, were elevated in Ret+CM patients, and neutrophil chemotaxis was impaired, possibly related to increased plasma heme. Neutrophils were rarely seen in CM brain microvasculature autopsy samples, and no neutrophil extracellular traps were found, suggesting that a putative neutrophil effect on endothelial cell biology results from neutrophil soluble factors rather than direct neutrophil cellular tissue effects. Meanwhile, children with Ret-CM had lower levels of inflammation, higher levels of alpha interferon, and upregulation of Toll-like receptor pathways and other host transcriptional pathways, which may represent responses that do not favor cerebral iRBC sequestration., Importance: There were approximately 198 million cases of malaria worldwide in 2013, with an estimated 584,000 deaths occurring mostly in sub-Saharan African children. CM is a severe and rare form of Plasmodium falciparum infection and is associated with high rates of mortality and neurological morbidity, despite antimalarial treatment. A greater understanding of the pathophysiology of CM would allow the development of adjunctive therapies to improve clinical outcomes. A hallmark of CM is cerebral microvasculature sequestration of P. falciparum-infected red blood cells (iRBCs), which results in vasculopathy in some patients. Our data provide a global analysis of the host pathways associated with CM and newly identify an association of activated neutrophils with brain iRBC sequestration. Products of activated neutrophils could alter endothelial cell receptors and coagulation to facilitate iRBC adherence. Future studies can now examine the role of neutrophils in CM pathogenesis to improve health outcomes., (Copyright © 2016 Feintuch et al.)

Background: Cerebral malaria (CM) remains a significant cause of morbidity and mortality in children in sub-Saharan Africa. CM mortality has been associated with increased brain volume, seen on neuroimaging studies., Methods: To examine the potential role of blood metabolites and inflammatory mediators in increased brain volume in Malawian children with CM, an association study was performed between plasma metabolites, cytokine levels and phospholipase A2 (PLA2) activity with brain volume., Results: The metabolomics analysis demonstrated arachidonic acid and other lysophospholipids to be positively associated with brain swelling. These lipids are products of the PLA2 enzyme and an association of plasma PLA2 enzymatic activity with brain swelling was confirmed. TNFα, which can upregulate PLA2 activity, was associated with brain volume. In addition, CCL2 and IL-8 were also associated with brain volume. Some of these cytokines can alter endothelial cell tight junction proteins and increase blood brain barrier permeability., Conclusions: Taken together, paediatric CM brain volume was associated with products of the PLA2 pathway and inflammatory cytokines. Their role in causality is unknown. These molecules will need to undergo testing in vitro and in animal models to understand their role in processes of increased brain volume. These observations provide novel data on host physiology associated with paediatric CM brain swelling, and may both inform pathogenesis models and suggest adjunct therapies that could improve the morbidity and mortality associated with paediatric CM.

Plasmodium falciparum infection can result in severe disease that is associated with elevated inflammation and vital organ dysfunction; however, malaria-endemic residents gain protection from lethal outcomes and manifest only mild symptoms during infection. To characterize host responses associated with this more effective antimalarial response, we characterized whole-blood transcriptional profiles in Rwandan adults during a mild malaria episode and compared them with findings from a convalescence sample. We observed transcriptional up-regulation in many pathways, including type I interferon, interferon γ, complement activation, and nitric oxide during malaria infection, which provide benchmarks of mild disease physiology. Transcripts encoding negative regulators of T-cell activation, such as programmed death ligand 1 (PD-L1), programmed death 1 ligand 2 (PD-L2), and the butyrophilin family member butyrophilin-like 2 (BTNL2) were also increased. To support an important functional role for BTNL2 during malaria infection, we studied chimeric mice reconstituted with BTNL2(-/-) or wild-type hematopoietic cells that were inoculated with Plasmodium berghei ANKA, a murine model of cerebral malaria. We found that BTNL2(-/-) chimeric mice had a significant decrease in survival compared with wild-type counterparts. Collectively these data characterize the immune responses associated with mild malaria and uncover a novel role for BTNL2 in the host response to malaria., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

HIV infected individuals in malaria endemic areas experience more frequent and severe malaria episodes compared to non HIV infected. This clinical observation has been linked to a deficiency in antibody responses to Plasmodium falciparum antigens; however, prior studies have only focused on the antibody response to <0.5% of P. falciparum proteins. To obtain a broader and less-biased view of the effect of HIV on antibody responses to malaria we compared antibody profiles of HIV positive (HIV+) and negative (HIV-) Rwandan adults with symptomatic malaria using a microarray containing 824 P. falciparum proteins. We also investigated the cellular basis of the antibody response in the two groups by analyzing B and T cell subsets by flow cytometry. Although HIV malaria co-infected individuals generated antibodies to a large number of P. falciparum antigens, including potential vaccine candidates, the breadth and magnitude of their response was reduced compared to HIV- individuals. HIV malaria co-infection was also associated with a higher percentage of atypical memory B cells (MBC) (CD19+CD10-CD21-CD27-) compared to malaria infection alone. Among HIV+ individuals the CD4+ T cell count and HIV viral load only partially explained variability in the breadth of P. falciparum-specific antibody responses. Taken together, these data indicate that HIV malaria co-infection is associated with an expansion of atypical MBCs and a diminished antibody response to a diverse array of P. falciparum antigens, thus offering mechanistic insight into the higher risk of malaria in HIV+ individuals.

Serology data are limited for patients with sputum smear-negative HIV-associated active tuberculosis (TB). We evaluated the serum antibody responses against the mycobacterial proteins MPT51, MS, and echA1 and the 38-kDa protein via enzyme-linked immunosorbent assay (ELISA) in South African (S.A.) HIV-positive (HIV(+)) smear-negative TB patients (n = 56), U.S. HIV(+) controls with a positive tuberculin skin test (TST(+); n = 21), and S.A. HIV-negative (HIV(-)) (n = 18) and HIV(+) (n = 24) controls. TB patients had positive antibody reactivity against MPT51 (73%), echA1 (59%), MS (36%), and the 38-kDa protein (11%). Little reactivity against MPT51 and echA1 was observed in control groups at low risk for TB, i.e., S.A. HIV(-) (0% and 6%, respectively), and at moderate risk for TB development, i.e., U.S. HIV(+) TST(+) controls (14% and 10%, respectively). By contrast, more reactivity was detected in the S.A. HIV(+) control group at higher risk for TB (25% and 45%, respectively). Our data hold promise that antibody detection against MPT51 and echA1 might have adjunctive value in the detection of HIV(+) smear-negative TB and might reflect increasing Mycobacterium tuberculosis infection activity in asymptomatic HIV(+) individuals., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Infection with Plasmodium falciparum can lead to a range of severe to minimal symptoms, occasionally resulting in death in young children or nonimmune adults. In areas of high transmission, older children and adults generally suffer only mild or asymptomatic malaria infections and rarely develop severe disease. The immune features underlying this apparent immunity to severe disease remain elusive. To gain insight into host responses associated with severe and mild malaria, we conducted a longitudinal study of five children who first presented with severe malaria and, 1 month later, with mild malaria. Employing peripheral blood whole-genome profiling, we identified 68 genes that were associated with mild malaria compared to their expression in the severe malaria episode (paired Students t test, P < 0.05). These genes reflect the interferon (IFN) pathway and T cell biology and include IFN-induced protein transcripts 1 to 3, oligoadenylate synthetases 1 and 3, and the T cell markers cathepsin W and perforin. Gene set enrichment analysis identified Gene Ontology (GO) pathways associated with mild malaria to include the type I interferon-mediated signaling pathway (GO 0060337), T cell activation (GO 0042110), and other GO pathways representing many aspects of immune activation. In contrast, only six genes were associated with severe malaria, including thymidine kinase 1, which was recently found to be a biomarker of cerebral malaria susceptibility in the murine model, and carbonic anhydrase, reflecting the blood's abnormal acid base environment during severe disease. These data may provide potential insights to inform pathogenesis models and the development of therapeutics to reduce severe disease outcomes due to P. falciparum infection.