Despite prompt administration of optimal antimalarial treatment, mortality associated with CM remains unacceptably high, thus, prompting the development of adjunct therapeutics that can reduce or prevent CM pathology and associated mortalities
[12, 13] Recent studies have shown that NRG-1 was effective in treating ABI such as AIS and acute neurotoxin exposure by preventing neuroinflammation and neuronal tissue death
[35, 36], which are similar to those observed in CM. Furthermore, NRG-1 stabilizes the BBB and mediates inflammatory pathways to prevent tissue damage associated with brain injury
[32, 33, 37]. Using a PbA ECM model that mimics significant features of human CM, we have demonstrated the effectiveness of NRG-1 therapy against ECM pathophysiology, and associated mortality.
Advances in drug therapies that eradicate malaria parasites are still unable to prevent mortality in up to 30% of CM patients. In humans, quinine and artemisinin derivatives (artesunate and artemether) are the mainstream drugs used to treat CM
[81, 82]. ARM was selected for use in this study as previous research has demonstrated that ARM was more effective against murine ECM than quinine, artemisinin and artesunate
. Despite anti-parasitic properties of ARM, mortality rates were as high as 18% in mice treated with ARM in the current study. However, no evidence of neurological dysfunction associated with ECM was observed in ARM-treated mice. This unacceptably high mortality in ARM-treated mice may be due to low efficacy of ARM against PbA that can lead to recrudescence and malarial anemia post treatment
. Furthermore, therapies targeting parasite eradication without addressing secondary effects of parasite infection, such as tissue damage and neurological complications, are inadequate for preventing mortalities. Thus, there is great need for adjunct therapeutics that target CM pathology that in conjunction with parasite-eradicating antimalarial agents can prevent mortality associated with CM.
Permanent or reversible neurological sequelae such as coma, residual epilepsy and cognitive deficits, are common clinical outcomes in CM patients. These neurological outcomes are associated with inflammatory cascades initiated by pathogen toxins that lead to widespread endothelial activation and brain damage (petechial hemorrhage and neuronal cell death) and involves inflammation-induced sequestration of infected RBCs
[83–85]. Similarly, accumulation of leukocytes occurs in brain microvessels of PbA-infected mice that leads to vascular congestion and contributes to brain damage.
[86, 87]. Although parasitemia levels were high in PbA-infected mice treated with NRG-1, there was significant reduction in leukocyte accumulation in brain microvessels after NRG-1 treatment. This indicates that NRG-1 therapy effectively reduces brain inflammation associated with ECM pathogenesis even in the presence of high parasitemia.
Human CM and murine ECM are characterized by a dysregulated immune response leading to overexpression of pro-inflammatory cytokines including TNFα, IL-1α, IL-6, and CXCL10
[67–69, 74, 75]. These cytokines are secreted by T-cells, macrophages and endothelial cells in response to infection and play several roles that include promotion of acute immune response, leukocyte recruitment, BBB disruption and negative hypothalamic mediation during febrile illness
[66, 70, 88–93]. Plasma and cerebrospinal fluid levels of TNFα, IL-1α and IL-6 are increased in children with CM
[20, 68] suggesting their role in human CM. We previously reported that increased plasma and cerebrospinal fluid levels of CXCL10 predict fatal CM
[72–74] and mice deficient in the CXCL10 gene are partially protected against murine ECM
. NRG-1 therapy significantly reduced serum TNFα, IL-1α, IL-6, and CXCL10 levels while improving survival against ECM. High serum levels of the growth factor G-CSF have been shown to correlate with fatal CM in humans
. However, NRG-1 reduced G-CSF, suggesting amelioration of pathogenic pathways that leads to induction of G-CSF observed in fatal CM
[20, 80]. Thus, further study is warranted to determine the role of G-CSF in severe disease and the NRG-1 protective effect in reducing G-CSF production. Furthermore, there is growing evidence of the role for anti-inflammatory factors, IL-5 and IL-13 in protection against malarial disease. In a population of south Asian malaria patients, increased levels of IL-5 was associated with reduced severity of disease
. Genetic studies in African and south-east Asian populations have linked IL-13 to protection against cerebral malaria and show that polymorphisms that alter IL-13 production may increase risk of severe malaria
[76–78]. In the present study, NRG-1 enhanced production of IL-5 and IL-13, and suggests NRG-1 promotes anti-inflammatory factors while dampening pro-inflammatory factors to ameliorate CM pathogenesis.
Angiopoietin-1 (a biomarker of endothelium quiescience and stability) and vascular permeability factor angiopoietin-2 (marker of vascular barrier breakdown) are potent modulators of vascular inflammation, endothelial activation and BBB function
[49, 94–96]. Angiopoietin-1 stabilizes the vascular endothelium barrier
 and regulates the activity of BBB permeability factors such as platelet-activating factor (PAF), vascular epithelial growth factor (VEGF), ELAM-1, bradykinin, thrombin and histamine
[98–101]. Increased activity of angiopoietin-1 promotes endothelial survival
[102, 103], modulates plasma leakage
[100, 104, 105] and reduces vascular inflammation by inhibiting ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression
. Conversely, pro-inflammatory cytokines TNF-α, IL-1β and vascular permeability factor, VEGF, mediate the release of angiopoietin-2, an antagonist to angiopoietin-1
[48, 107], that promotes increased vascular inflammation
, disruption of angiogenesis
, endothelial cell death
 and vascular regression
[110, 111]. Moreover, angiopoietin-2 expression is elevated in response to endothelial activation, hypoxia and ischemia
[107, 112–114]. In human CM, high levels of angiopoietin-2 and low levels of angiopoietin-1 are linked to CM severity and studies suggest these angiogenic factors function as prognostic biomarkers that can discriminate severe CM from mild malaria and predict fatal CM outcome
[53, 54]. Nakaoka et al. show that NRG-1 stimulates expression of angiopoietin-1
 and increased expression of angiopoietin-1 inhibits release or activity of angiopoietin-2
[107, 116]. In the present study, NRG-1 treatment increased expression of angiopoietin-1, thus promoting endothelial barrier function and integrity during ECM, while modulating angiopoietin-2 expression in the brains of PbA-infected mice.
Parasite sequestration and activation of endothelial cells by infected erythrocytes and pro-inflammatory cytokines are hallmark events in the brain pathology of pediatric CM patients
[64, 117, 118]. Parasite sequestration and endothelial activation correlate with an increase in adhesion molecules such as ICAM-1 and VCAM-1 that bind infected erythrocytes, influence leukocyte migration and promote further release of pro-inflammatory cytokines
[119–121]. ICAM-1 is a marker of endothelial activation whose expression is upregulated on the vascular endothelium in the brain in murine and human CM
[64, 122–124]. ICAM expression is induced by pro-inflammatory cytokines such as TNF-α, IFN-γ and VEGF
[47, 125, 126]. In human CM, increased ICAM-1 levels are associated with disease severity
[63, 119, 127]. Furthermore, murine ECM studies show increased expression of ICAM-1 contributes to the development of ECM
[47, 128, 129]. Previous studies indicate NRG-1 reduces the expression of ICAM-1 following ischemic stroke
. NRG-1 increases activity of PI3-kinase
[130, 131] which suppresses VEGF-mediated expression of ICAM-1 on endothelial cells
[106, 126]. Additionally, C/EBPβ is a critical regulator of acute host-response to infections and neuroinflammation
[55–58, 60] that stimulates release of inflammatory and adhesion factors such as IL-6, TNFα, CD40 and ICAM-1, thus contributing to ECM development
[56–59, 61]. In this study, NRG-1 treatment of murine ECM demonstrated inhibition of ICAM-1 and C/EBPβ in the ECM brain while reducing leukocyte adhesiveness and accumulation in brain microvessels.
NRG-1 was recently used as a treatment for heart failure and showed significant efficacy for improving cardiac function in a phase-II patient study
[132–134]. In this study, patients received placebo or NRG-1 at a dose of 0.3 to 1.2 μg/kg/day intravenously for 10 days, in addition to standard drug therapies. During a follow-up period 11 to 90 days after study initiation, NRG-1 significantly improved heart function in patients and the effective doses were shown to be safe and tolerable. Two additional clinical trials to determine the ability of NRG-1 to improve cardiac function after heart failure have been initiated in the US (ClinicalTrails.gov identifiers NCT01258387 and NCT01541202). During the period of study, no severe events were observed in either healthy or impaired patients.