Extracellular Vesicles and Cerebral Malaria.

Cerebral malaria (CM) remains a major problem of public health at the world level (Idro et al. 2010; WHO 2009), in spite of numerous efforts from various disciplines to improve our knowledge of disease mechanisms (Hunt and Grau 2003; Schofield and Grau 2005; van der Heyde et al. 2006). Our approach to a better understanding of CM pathogenesis has involved the dissection of immunopathological pathways which, in addition to direct changes caused by malaria parasite-infected erythrocytes (IE), lead to neurovascular lesions. We posited that immunopathology is important in CM because a role for cells and soluble mediators of the immune system has been widely recognised as contributing to the complications of viral, bacterial, fungal and many parasitic infections. As detailed earlier, it would be extraordinary if malaria did not conform to this general pattern. As a matter of fact, there now is strong evidence to support immune mechanisms in malarial pathogenesis (Grau and Hunt 2014).Extracellular vesicles (EV) and their subtypes have been described and reviewed by a number of investigators (Hosseini-Beheshti and Grau 2018, 2019; Raposo and Stahl 2019; Witwer et al. 2017; Zijlstra and Di Vizio 2018) and in others chapters of the present book.

Investigation of Plasma-Derived Lipidome Profiles in Experimental Cerebral Malaria in a Mouse Model Study.

Cerebral malaria (CM), a fatal complication of Plasmodium infection that affects children, especially under the age of five, in sub-Saharan Africa and adults in South-East Asia, results from incompletely understood pathogenetic mechanisms. Increased release of circulating miRNA, proteins, lipids and extracellular vesicles has been found in CM patients and experimental mouse models. We compared lipid profiles derived from the plasma of CBA mice infected with Plasmodium berghei ANKA (PbA), which causes CM, to those from Plasmodium yoelii (Py), which does not. We previously showed that platelet-free plasma (18k fractions enriched from plasma) contains a high number of extracellular vesicles (EVs). Here, we found that this fraction produced at the time of CM differed dramatically from those of non-CM mice, despite identical levels of parasitaemia. Using high-resolution liquid chromatography-mass spectrometry (LCMS), we identified over 300 lipid species within 12 lipid classes. We identified 45 and 75 lipid species, mostly including glycerolipids and phospholipids, with significantly altered concentrations in PbA-infected mice compared to Py-infected and uninfected mice, respectively. Total lysophosphatidylethanolamine (LPE) levels were significantly lower in PbA infection compared to Py infection and controls. These results suggest that experimental CM could be characterised by specific changes in the lipid composition of the 18k fraction containing circulating EVs and can be considered an appropriate model to study the role of lipids in the pathophysiology of CM.

Extracellular vesicles as mediators of immunopathology in infectious diseases.

In the last decades, extracellular vesicles have emerged as important elements in cell-cell communication and as key players in disease pathogenesis via transmission of their cargo between different cells. Various works have described different subpopulations of these membrane structures, based on their cell of origin, biogenesis, size, biophysical properties and cargo. In addition to their pathophysiological role in the development and progression of different diseases including infectious diseases, neurodegenerative disorders and cancer, extracellular vesicles are now recognized for their potential as novel therapeutic targets and intelligent drug delivery system. Here, we have reviewed the most recent data on different subtypes of extracellular vesicles, focusing on microvesicles and exosomes and their subpopulations, their involvement in immune-mediated pathogenesis of various infectious diseases and their role as potential therapeutic targets.

Host Resistance to Plasmodium-Induced Acute Immune Pathology Is Regulated by Interleukin-10 Receptor Signaling.

The resolution of malaria infection is dependent on a balance between proinflammatory and regulatory immune responses. While early effector T cell responses are required for limiting parasitemia, these responses need to be switched off by regulatory mechanisms in a timely manner to avoid immune-mediated tissue damage. Interleukin-10 receptor (IL-10R) signaling is considered to be a vital component of regulatory responses, although its role in host resistance to severe immune pathology during acute malaria infections is not fully understood. In this study, we have determined the contribution of IL-10R signaling to the regulation of immune responses during Plasmodium berghei ANKA-induced experimental cerebral malaria (ECM). We show that antibody-mediated blockade of the IL-10R during P. berghei ANKA infection in ECM-resistant BALB/c mice leads to amplified T cell activation, higher serum gamma interferon (IFN-γ) concentrations, enhanced intravascular accumulation of both parasitized red blood cells and CD8+ T cells to the brain, and an increased incidence of ECM. Importantly, the pathogenic effects of IL-10R blockade during P. berghei ANKA infection were reversible by depletion of T cells and neutralization of IFN-γ. Our findings underscore the importance of IL-10R signaling in preventing T-cell- and cytokine-mediated pathology during potentially lethal malaria infections.

Potential efficacy of citicoline as adjunct therapy in treatment of cerebral malaria.

Cerebral malaria (CM) is characterized by a dysregulated immune response that results in endothelial membrane destabilization and increased microparticle (MP) production. Citicoline (CTC) is a membrane stabilizer used for the treatment of neurological disorders. We evaluated the efficacy of CTC as adjunct therapy to aid recovery from experimental CM. We show that CTC reduces MP production in vitro; in combination with artesunate in vivo, confers partial protection against CM; and prolongs survival.

The CTLA-4 and PD-1/PD-L1 inhibitory pathways independently regulate host resistance to Plasmodium-induced acute immune pathology.

The balance between pro-inflammatory and regulatory immune responses in determining optimal T cell activation is vital for the successful resolution of microbial infections. This balance is maintained in part by the negative regulators of T cell activation, CTLA-4 and PD-1/PD-L, which dampen effector responses during chronic infections. However, their role in acute infections, such as malaria, remains less clear. In this study, we determined the contribution of CTLA-4 and PD-1/PD-L to the regulation of T cell responses during Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM) in susceptible (C57BL/6) and resistant (BALB/c) mice. We found that the expression of CTLA-4 and PD-1 on T cells correlates with the extent of pro-inflammatory responses induced during PbA infection, being higher in C57BL/6 than in BALB/c mice. Thus, ECM develops despite high levels of expression of these inhibitory receptors. However, antibody-mediated blockade of either the CTLA-4 or PD-1/PD-L1, but not the PD-1/PD-L2, pathways during PbA-infection in ECM-resistant BALB/c mice resulted in higher levels of T cell activation, enhanced IFN-γ production, increased intravascular arrest of both parasitised erythrocytes and CD8(+) T cells to the brain, and augmented incidence of ECM. Thus, in ECM-resistant BALB/c mice, CTLA-4 and PD-1/PD-L1 represent essential, independent and non-redundant pathways for maintaining T cell homeostasis during a virulent malaria infection. Moreover, neutralisation of IFN-γ or depletion of CD8(+) T cells during PbA infection was shown to reverse the pathologic effects of regulatory pathway blockade, highlighting that the aetiology of ECM in the BALB/c mice is similar to that in C57BL/6 mice. In summary, our results underscore the differential and complex regulation that governs immune responses to malaria parasites.

Cellular communication via microparticles: role in transfer of multidrug resistance in cancer.

Multidrug resistance (MDR) continues to be a major impediment to the successful treatment of cancer. The two efflux transporters, P-glycoprotein (P-gp) and MRP1 are major contributors to cancer MDR clinically. The upregulation of P-gp leading to MDR was initially understood to occur via pre- and post-transcriptional mechanisms only. However, we demonstrated that microparticles mediate the intercellular exchange and trafficking of bioactive material, including functional P-gp and selected modulatory miRNAs. This exchange of P-gp leads to the dissemination of MDR within a cancer cell population. These findings have significant implications in understanding the cellular basis governing the intercellular acquisition of deleterious traits in cancers, serving to substantially advance our understanding of the molecular basis of the emergence of MDR in cancer clinically.

Cytoadherence of Plasmodium berghei-infected red blood cells to murine brain and lung microvascular endothelial cells in vitro.

Sequestration of infected red blood cells (iRBC) within the cerebral and pulmonary microvasculature is a hallmark of human cerebral malaria (hCM). The interaction between iRBC and the endothelium in hCM has been studied extensively and is linked to the severity of malaria. Experimental CM (eCM) caused by Plasmodium berghei ANKA reproduces most features of hCM, although the sequestration of RBC infected by P. berghei ANKA (PbA-iRBC) has not been completely delineated. The role of PbA-iRBC sequestration in the severity of eCM is not well characterized. Using static and flow cytoadherence assays, we provide the first direct in vitro evidence for the binding of PbA-iRBC to murine brain and lung microvascular endothelial cells (MVEC). We found that basal PbA-iRBC cytoadherence to MVECs was significantly higher than that of normal red blood cells (NRBC) and of RBC infected with P. berghei K173 (PbK173-iRBC), a strain that causes noncerebral malaria (NCM). MVEC prestimulation with tumor necrosis factor (TNF) failed to promote any further significant increase in mixed-stage iRBC adherence. Interestingly, enrichment of the blood for mature parasites significantly increased PbA-iRBC binding to the MVECs prestimulated with TNF, while blockade of VCAM-1 reduced this adhesion. Our study provides evidence for the firm, flow-resistant binding to endothelial cells of iRBC from strain ANKA-infected mice, which develop CM, and for less binding of iRBC from strain K173-infected mice, which develop NCM. An understanding of P. berghei cytoadherence may help elucidate the importance of sequestration in the development of CM and aid the development of antibinding therapies to help reduce the burden of this syndrome.

Microparticle conferred microRNA profiles--implications in the transfer and dominance of cancer traits.

BACKGROUND: Microparticles (MPs) are membrane vesicles which are released from normal and malignant cells following a process of budding and detachment from donor cells. MPs contain surface antigens, proteins and genetic material and serve as vectors of intercellular communication. MPs comprise the major source of systemic RNA including microRNA (miRNA), the aberrant expression of which appears to be associated with stage, progression and spread of many cancers. Our previous study showed that MPs carry both transcripts and miRNAs associated with the acquisition of multidrug resistance in cancer. RESULTS: Herein, we expand on our previous finding and demonstrate that MPs carry the transcripts of the membrane vesiculation machinery (floppase and scramblase) as well as nucleic acids encoding the enzymes essential for microRNA biogenesis (Drosha, Dicer and Argonaute). We also demonstrate using microarray miRNA profiling analysis, the selective packaging of miRNAs (miR-1228*, miR-1246, miR-1308, miR-149*, miR-455-3p, miR-638 and miR-923) within the MP cargo upon release from the donor cells. CONCLUSIONS: These miRNAs are present in both haematological and non-haematological cancer cells and are involved in pathways implicated in cancer pathogenesis, membrane vesiculation and cascades regulated by ABC transporters. Our recent findings reinforce our earlier reports that MP transfer 're-templates' recipient cells so as to reflect donor cell traits. We now demonstrate that this process is likely to occur via a process of selective packaging of nucleic acid species, including regulatory nucleic acids upon MP vesiculation. These findings have significant implications in understanding the cellular basis governing the intercellular acquisition and dominance of deleterious traits in cancers.

CNS hypoxia is more pronounced in murine cerebral than noncerebral malaria and is reversed by erythropoietin.

Cerebral malaria (CM) is associated with high mortality and risk of sequelae, and development of adjunct therapies is hampered by limited knowledge of its pathogenesis. To assess the role of cerebral hypoxia, we used two experimental models of CM, Plasmodium berghei ANKA in CBA and C57BL/6 mice, and two models of malaria without neurologic signs, P. berghei K173 in CBA mice and P. berghei ANKA in BALB/c mice. Hypoxia was demonstrated in brain sections using intravenous pimonidazole and staining with hypoxia-inducible factor-1α-specific antibody. Cytopathic hypoxia was studied using poly (ADP-ribose) polymerase-1 (PARP-1) gene knockout mice. The effect of erythropoietin, an oxygen-sensitive cytokine that mediates protection against CM, on cerebral hypoxia was studied in C57BL/6 mice. Numerous hypoxic foci of neurons and glial cells were observed in mice with CM. Substantially fewer and smaller foci were observed in mice without CM, and hypoxia seemed to be confined to neuronal cell somas. PARP-1-deficient mice were not protected against CM, which argues against a role for cytopathic hypoxia. Erythropoietin therapy reversed the development of CM and substantially reduced the degree of neural hypoxia. These findings demonstrate cerebral hypoxia in malaria, strongly associated with cerebral dysfunction and a possible target for adjunctive therapy.

Vascular endothelial cells cultured from patients with cerebral or uncomplicated malaria exhibit differential reactivity to TNF.

Plasmodium falciparum malaria is a major cause of morbidity and mortality in African children, and factors that determine the development of uncomplicated (UM) versus cerebral malaria (CM) are not fully understood. We studied the ex vivo responsiveness of microvascular endothelial cells to pro-inflammatory stimulation and compared the findings between CM and UM patients. In patients with fatal disease we compared the properties of vascular endothelial cells cultured from brain tissue to those cultured from subcutaneous tissue, and found them to be very similar. We then isolated, purified and cultured primary endothelial cells from aspirated subcutaneous tissue of patients with CM (EC(CM) ) or UM (EC(UM) ) and confirmed the identity of the cells before analysis. Upon TNF stimulation in vitro, EC(CM) displayed a significantly higher capacity to upregulate ICAM-1, VCAM-1 and CD61 and to produce IL-6 and MCP-1 but not RANTES compared with EC(UM) . The shedding of endothelial microparticles, a recently described parameter of severity in CM, and the cellular level of activated caspase-3 were both significantly greater in EC(CM) than in EC(UM) . These data suggest that inter-individual differences in the endothelial inflammatory response to TNF may be an additional factor influencing the clinical course of malaria.

Bridging and Clumping: Investigating Platelet Interactions with P. falciparum-Infected Red Blood Cells and Endothelial Cells in Cerebral Malaria.

The methods presented in this chapter describe how to perform ex vivo clumping and in vitro bridging assays in the context of cerebral malaria. Both the protocols are detailed, and emphasis is made on how to prepare platelet suspensions suitable to each technique, including description of specific buffers and reagents to minimize the risk of aggregation while maintaining the platelet properties.

Dietary protein increases T-cell-independent sIgA production through changes in gut microbiota-derived extracellular vesicles.

Secretory IgA is a key mucosal component ensuring host-microbiota mutualism. Here we use nutritional geometry modelling in mice fed 10 different macronutrient-defined, isocaloric diets, and identify dietary protein as the major driver of secretory IgA production. Protein-driven secretory IgA induction is not mediated by T-cell-dependent pathways or changes in gut microbiota composition. Instead, the microbiota of high protein fed mice produces significantly higher quantities of extracellular vesicles, compared to those of mice fed high-carbohydrate or high-fat diets. These extracellular vesicles activate Toll-like receptor 4 to increase the epithelial expression of IgA-inducing cytokine, APRIL, B cell chemokine, CCL28, and the IgA transporter, PIGR. We show that succinate, produced in high concentrations by microbiota of high protein fed animals, increases generation of reactive oxygen species by bacteria, which in turn promotes extracellular vesicles production. Here we establish a link between dietary macronutrient composition, gut microbial extracellular vesicles release and host secretory IgA response.

Biochemical markers of nutritional status and childhood malaria severity in Cameroon.

To investigate the part played by undernutrition in malaria severity, some biomarkers of nutritional status were assessed in children with severe malarial anaemia (MA) and cerebral malaria (CM) in comparison with healthy children or those with uncomplicated malaria. Undernutrition was assessed using the weight-for-age Z score (WAZ). Retinol was determined by HPLC; lipid profile, Ca, Mg and albumin were determined by spectrophotometry. Severe and moderate undernutritions were more prevalent in children with MA and those with the combined symptoms of CM and MA, but not in those with CM alone. Some perturbations were noticed in the lipid profile, but most of the values remained within the normal ranges. The risk of vitamin A deficiency, as assessed by plasma retinol concentration, was noteworthy in children with severe malaria: 0.48 × 10(-6) and 0.50 × 10(-6) mol/l, respectively, in children with MA and CM (reference value: >0.7 × 10(-6) mol/l). A significant difference was obtained for retinol values after an ANOVA of all the groups (P = 0.0029), with the value in the MA group being significantly low than that in the control group (P < 0.05); likewise, a significant difference was obtained after comparison of all the groups for Mg and albumin (P = 0.0064 and 0.0082, respectively). Despite their low number (n 6), fatal cases of CM had a normal mean WAZ on admission, but low values of retinol, albumin and HDL:LDL ratio. Despite these associations, undernutrition itself did not appear to be a primary factor associated with fatal outcome.

Severe malaria: what's new on the pathogenesis front?

Plasmodium falciparum causes the most severe and fatal form of malaria in humans with over half a million deaths each year. Cerebral malaria, a complex neurological syndrome of severe falciparum malaria, is often fatal and represents a major public health burden. Despite vigorous efforts, the pathophysiology of cerebral malaria remains to be elucidated, thereby hindering the development of adjunctive therapies. In recent years, multidisciplinary and collaborative approaches have led to groundbreaking progress both in the laboratory and in the field. Here we review the latest breakthroughs in severe malaria pathogenesis, with a specific focus on new pathogenetic mechanisms leading to cerebral malaria. The most recent findings point towards specific parasite phenotypes targeting brain microvasculature, endothelial dysfunction and subsequent oedema-induced brain swelling.

Cerebral malaria: role of microparticles and platelets in alterations of the blood-brain barrier.

Brain lesions of cerebral malaria (CM) are characterised by a sequestration of Plasmodium falciparum-parasitised red blood cells (PRBC), leucocytes and platelets within brain microvessels, by an excessive release of pro-inflammatory cytokines as well as by disruption of the blood-brain barrier (BBB). We evaluated the possibility that PRBC and platelets interact and induce functional alterations in brain endothelium. Using an in vitro model of endothelial lesion, we showed that platelets can act as bridges between PRBC and endothelial cells (EC) allowing the binding of PRBC to endothelium devoid of cytoadherence receptors. Furthermore, platelets potentiated the cytotoxicity of PRBC for brain EC by inducing an alteration of the integrity of their monolayer and increasing their apoptosis. These findings provide insights into the mechanisms by which platelets can be deleterious to the brain endothelium during CM. Another aspect of inflammatory and infectious diseases is that they often lead to activation of vascular and blood cells. Such activation results in an enhanced vesiculation, i.e. the release of circulating microparticles (MP). We thus explored plasma levels of endothelial MP in Malawian children with malaria. Plasma MP numbers were markedly increased on admission only in patients with severe malaria complicated with coma. Using the experimental mouse model of CM, we evaluated the pathogenic implications of MP using genetically deficient mice in which the capacity to vesiculate is impaired. Such mice, lacking the ABCA-1 gene, upon infection by Plasmodium berghei ANKA, showed complete resistance to CM. When purified from infected susceptible animals, MP were able to reduce normal plasma clotting time and to significantly enhance tumour necrosis factor release from naïve macrophages. Altogether these data provide a novel insight into the pathogenic mechanisms leading to the neurological syndrome. The finding that ABCA-1 gene deletion confers complete protection against cerebral pathology, linked to an impaired MP production, provides new potential targets for therapeutic amelioration of severe malaria.

Breast cancer-derived microparticles display tissue selectivity in the transfer of resistance proteins to cells.

Microparticles (MPs) play a vital role in cell communication by facilitating the horizontal transfer of cargo between cells. Recently, we described a novel "non-genetic" mechanism for the acquisition of multidrug resistance (MDR) in cancer cells by intercellular transfer of functional P-gp, via MPs. MDR is caused by the overexpression of the efflux transporters P-glycoprotein (P-gp) and Multidrug Resistance-Associated Protein 1 (MRP1). These transporters efflux anticancer drugs from resistant cancer cells and maintain sublethal intracellular drug concentrations. By conducting MP transfer experiments, we show that MPs derived from DX breast cancer cells selectively transfer P-gp to malignant MCF-7 breast cells only, in contrast to VLB100 leukaemic cell-derived MPs that transfer P-gp and MRP1 to both malignant and non-malignant cells. The observed transfer selectivity is not the result of membrane restrictions for intercellular exchange, limitations in MP binding to recipient cells or the differential expression of the cytoskeletal protein, Ezrin. CD44 (isoform 10) was found to be selectively present on the breast cancer-derived MPs and not on leukaemic MPs and may contribute to the observed selective transfer of P-gp to malignant breast cells observed. Using the MCF-7 murine tumour xenograft model we demonstrated the stable transfer of P-gp by MPs in vivo, which was found to localize to the tumour core as early as 24 hours post MP exposure and to remain stable for at least 2 weeks. These findings demonstrate a remarkable capacity by MPs to disseminate a stable resistant trait in the absence of any selective pressure.

Microparticle drug sequestration provides a parallel pathway in the acquisition of cancer drug resistance.

Expanding on our previous findings demonstrating that microparticles (MPs) spread cancer multidrug resistance, we now show that MPs sequester drugs, reducing the free drug concentration available to cells. MPs were isolated from drug-sensitive and drug-resistant sub-clones of a human breast adenocarcinoma cell line and from human acute lymphoblastic leukemia cells. MPs were assessed for size, mitochondria, RNA and phospholipid content, P-glycoprotein (P-gp) expression and orientation and ATPase activity relative to drug sequestration capacity. Of the drug classes examined, MPs sequestered the anthracycline class to a significant degree. The degree of sequestration was likely due to the size of MPs and thus the amount of cargo they contain, to which the anthracyclines bind. Moreover, a proportion of the P-gp present on MPs was inside-out in orientation, enabling it to influx drugs rather than its typical efflux function. This was confirmed by surface immunofluorescence and by assessment of drug-stimulated ATPase activity following MP permeabilization. Thus we determined that breast cancer MPs carried a proportion of their P-gp oriented inside-out, providing active sequestration within the microvesicular compartment. These results demonstrate a capacity for MPs to sequester chemotherapeutic drugs, which has a predominantly active sequestration component for MPs derived from drug-resistant cells and a predominantly passive component for MPs derived from drug-sensitive cells. This reduction in available drug concentration has potential to contribute to a parallel pathway and complements that of the intercellular transfer of P-gp. These findings lend further support to the role of MPs in limiting the successful management of cancer.

Cerebral malaria pathogenesis: revisiting parasite and host contributions.

Cerebral malaria is one of a number of clinical syndromes associated with infection by human malaria parasites of the genus Plasmodium. The etiology of cerebral malaria derives from sequestration of parasitized red cells in brain microvasculature and is thought to be enhanced by the proinflammatory status of the host and virulence characteristics of the infecting parasite variant. In this article we examine the range of factors thought to influence the development of Plasmodium falciparum cerebral malaria in humans and review the evidence to support their role.