Background
Nearly 300 million persons each year are infected with
Plasmodium falciparum (
P. falciparum) infection, a subset of whom may develop severe anemia or a diffuse encephalopathy known as cerebral malaria (CM) [
1]. CM accounts for 110,000 deaths annually in children and one in four survivors develop neurological complications (cortical blindness, epilepsy, and monoparesis) and cognitive disability (speech deficits, working memory, and executive function disability) [
2‐
8]. Despite appropriate antimalarial treatment, mortality associated with CM may be as high as 30% in adults and 20% in children [
7,
9‐
11]. Thus, targeting parasite in acute disease is not sufficient to ameliorate persistent neurological sequelae and mortality associated with CM. Understanding immunopathogenic features such as brain inflammation and injury leading to fatal CM have led to the identification and development of small molecules or immunotherapeutics that may be used to stabilize the blood–brain barrier (BBB) and ameliorate CM-associated brain damage and mortality [
12‐
14]. However, most of these interventions administered as prophylactics to prevent development of neurological signs failed to reverse CM-associated brain injuries or resulted in minimal therapeutic benefit, whereas others were deleterious [
13,
14]. The use of prophylactic strategies may not be clinically relevant as most patients who present to clinics have neurological abnormalities or clinical signs of CM. It is therefore important for new therapeutic strategies to ameliorate complications associated with late stages of CM to improve clinical outcomes while reducing risk of neurological sequelae in surviving CM patients. Clinical studies in human CM and murine experimental CM (ECM) indicate an exaggerated activation and dysregulation of host inflammatory processes including brain endothelial activation, and disruption of the BBB during the pathogenesis of the disease [
15‐
18]. In fact, extensive research has linked strong host pro-inflammatory response to malaria disease states [
19‐
21] and genetic studies have identified several immune regulatory and effector loci that possess mutations associated with susceptibility and resistance to human severe (cerebral) malaria [
22‐
24]. Efforts underway to identify candidate therapeutics against CM have produced promising candidates including artovastatin, a statin with strong anti-inflammatory effects that effectively attenuates ECM [
25‐
27]. Thus, interventions aimed at modulating the deleterious hyper-inflammatory response to malaria infection while protecting against brain damage will potentially bolster therapeutics against severe malaria.
Neuregulin-1 (NRG-1) is a member of the neuregulin family of growth factors that promotes survival and function of neuronal cells [
28‐
31]. Studies have shown that NRG-1 attenuates tissue damage and immunopathology in animal models of acute brain injury (ABI) such as acute ischemic stroke (AIS), traumatic brain injury (TBI), and nerve agent poisoning [
32‐
37]. There are clear pathophysiological similarities between CM and AIS, including an exaggerated host expression of pro-inflammatory factors that lead to increased vascular endothelial activation with upregulation of adhesion molecules, glial activation, focal inflammation, activation of apoptotic pathways and eventually brain damage and death [
38‐
40]. Exogenous treatment with NRG-1 has been shown to significantly alter or inactivate inflammatory pathways associated with tissue damage during ischemic episodes [
36]. Furthermore, NRG-1 reduces brain inflammation via inhibition of immune and oxidative stress mediators involved in the pathogenesis of focal ischemic brain damage [
32]. Although NRG-1 has been studied extensively in AIS it has yet to be studied as a potential intervention against cerebral malaria. Using the
Plasmodium berghei (
P. berghei) ANKA (PbA) model of ECM, we tested the hypothesis that NRG-1 will reduce or prevent ECM-associated inflammation and improve survival in mice with late stage ECM. We show here that NRG-1 (1.25 ng/kg/day) significantly reduces ECM-associated brain and systemic inflammation and improves survival in mice with late-stage ECM.
Methods
Infection of mice with P. bergheiANKA
Six- to eight-week-old C57BL/6 J mice (Charles Rivers Laboratories, Wilmington, MA, USA) were housed in groups of four per cage on a 12 hr light/12 hr dark cycle with access to food ad libitum and water. Mice were allowed to acclimatize to their new environment for 3 days before experimentation. All experimental procedures were reviewed and approved by the Morehouse School of Medicine Institutional Animal Care and Use Committee (Permit Number 09–06). Procedures were performed with strict adherence to national regulations on animal care and experimentation with the use of Care of Laboratory Animal Resources (CLAR) guidelines to minimize pain. PbA was obtained from MR4, Manassas, VA, USA (BEI Resources Repository, NIAID, NIH; MR4 number MRA-311, deposited by TF McCutchan). Parasites were propagated in C57BL/6 J mice and a fresh blood sample from a passage mouse was used for each experiment. Experimental groups of mice were infected via intraperitoneal (i.p.) injection of 106 PbA-infected red blood cells (pRBCs). Mice were sham-injected with 106 non-infected red blood cells (RBCs).
Clinical assessment of ECM
All animals were checked several times daily for mortality and ECM symptoms. For better estimation of the overall clinical status of mice during infection, simple behavioral tests (transfer arousal, locomotor activity, tail elevation, wire maneuver, contact righting reflex, and righting in arena) adapted from the
SmithKline Beecham,
Harwell,
Imperial College,
Royal London Hospital,
phenotype
assessment (SHIRPA) protocol [
41‐
43] were used. Infected mice display signs of ECM by day 5 or 6 post infection [
41]. ECM is defined as the presentation of one or more signs of neurological deficit including ataxia, convulsions, limb paralysis, poor righting reflex, roll-over and coma [
41]. Presentation of these signs were evaluated and scored to better assess severity of ECM in mice [
44].
Assessment of NRG-1 and artemether treatment in mice infected with or without PbA
Mice were selected and randomized into treatment groups after diagnosis with ECM on day 5 to 6 post infection. For survival experiments, 11 mice per group were used to obtain significant statistical data. To determine the therapeutic benefit of NRG-1 on ECM-associated brain damage and mortality and to compare NRG-1 with artemether (ARM) treatment, PbA-infected mice were treated daily via i.p. injection with 50-μl doses of NRG-1 (1.25 ng/kg/day, EGF-like domain, R & D Systems, Minneapolis, MN, USA) [NP_039250] or artemether prepared in coconut oil (25 mg/kg/day, Sigma-Aldrich, St Louis, MO, USA), from day 6 to day 9 post infection. PbA-infected mice treated daily with 50 μl saline solution (i.p.) from day 6 to day 9 post infection were used as the control. Mice were checked several times daily for mortality and signs of ECM neurological symptoms such as ataxia, loss of reflex and hemiplegia. All murine ECM experiments were terminated 19 days after PbA infection with animals euthanized accordingly. Parasite load was monitored periodically (beginning on day 5 post infection) by Giemsa staining of thin blood smears and assessed by counting the number of pRBCs per 1,000 erythrocytes.
Assessment of leukocyte accumulation in brain parenchymal vessels during murine ECM in PbA-infected mice by H&E staining
To determine the effect of NRG-1 and ARM treatment on leukocyte accumulation in brain parenchymal vessels during murine ECM pathogenesis, PbA-infected C57BL/6 J mice were anesthetized with isoflurane inhalation and euthanized on day 5 and day 11 post infection. Mice were perfused with 10 ml of cold sterile phosphate-buffered saline to clear vessels of blood prior to collection of brain tissue (three mice per time point per treatment group). Whole brains were stored in formalin for fixation, embedded in paraffin, and sectioned at 10 μm. Sagittal sections of the brain (day 5 and day 11 post infection) were fixed in 4% paraformaldehyde and blocked with horse serum for 30 minutes at room temperature. Sections were stained with H&E and leukocytes in the blood vessels were quantified using an ocular grid calibrated with a × 400 magnification in an Axioskop 2 Plus microscope (Carl Zeiss Microscopy, Thornwood, NY, USA). The whole area of each section was similarly quantified with the ocular grid calibrated at × 40 magnification. Digital photos were captured by a high-resolution AxioCam HRc camera (Carl Zeiss Microscopy).
Determination of the effect of NRG-1 on mRNA expression of factors involved in vascular endothelial activation and BBB integrity
To determine the effect of NRG-1 on mRNA expression of factors involved in vascular endothelial activation and BBB integrity, whole brain tissue from PbA-infected mice treated with either saline, ARM or NRG-1 (three mice per group per time point) was collected and homogenized in Trizol reagent (Life Technologies, Gaithersburg, MD, USA) and total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Briefly, chloroform (0.2 ml) was added to the homogenate, and the lysate mixed thoroughly. After centrifuging at 12,000 × g for 20 minutes at 4°C, the aqueous layer was transferred to a new tube. RNA was precipitated with 500 μl of isopropanol and pelleted by centrifuging at 12,000 × g for 20 minutes at 4°C. RNase-Free DNase Set (Qiagen) was used according to the manufacturer’s instructions to remove contaminating genomic DNA. DNase-treated RNA samples were subsequently stored at -80°C until ready to use. Reverse transcription of RNA samples was performed prior to quantitative PCR. cDNA was synthesized from up to 2 μg of total RNA iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) using Multigene Gradient Thermal cycler (Labnet International, Inc. Edison, NJ, USA). The resulting cDNA was diluted 1:10 by addition of 180 μl of distilled water for quantitative PCR analysis. The primer sequences used for quantitative PCR are described in Table
1.
Table 1
Primer sequences used
HPRT
| GCTTTCCCTGGTTAAGCAGTACA | CAAACTTGTCTGGAATTTCAAATC |
ICAM-1
| GCCTCCGGACTTTCGATCTT | GTCAGGGGTGTCGAGCTTTG |
ANG-1
| ATGCTGTTCAAAACCACACG | TTTCAAGTCGGGATGTTTGAT |
ANG-2
| ATGTGGTGCAGAACCAGACA | GCAGCTCGAGTCTTGTCGTC |
C/EBPβ
| TCTACTACGAGCCCGACTGC | AGGTAGGGGCTGAAGTCGAT |
The quantitative real-time PCR assay was performed using Bio-Rad C1000 thermal cycler (Bio-Rad Laboratories). Approximately 20 ng of cDNA was used in each 25 μl PCR reaction using the Bio-Rad iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA) and 50 μM of each primer. After a 15-minute incubation at 95°C, amplification was achieved by 39 cycles of a 15-s denaturation incubation at 95°C, followed by a 30-s annealing incubation at 55°C and 30-s extension incubation at 72°C. The identity and purity of the PCR product was confirmed by using dissociation curves and by checking the melting temperature of the PCR product, independently of the PCR reaction. To determine the relative amount of target cDNA present, the cycles to threshold (Ct) values of the target genes were compared with the basal expression of the housekeeping gene, hypoxanthine guanine phosphoribosyltransferase (HPRT). The average amount of HPRT present in each mouse group was used to normalized the quantity of target mRNA sequence against total RNA in each reaction. The differences in Ct values between HPRT and target gene of day 11 after infection of each group were compared with day 5 after infection-untreated control samples to determine the relative change in mRNA expression.
Assessment of NRG-1 effects on expression of immune determinants of CM severity
To determine the effect of NRG-1 and ARM treatment on cytokine/chemokine levels, serum collected from blood harvested via cardiac puncture at pre-treatment (day 0 and day 5) and post treatment (day 11) from anesthetized mice (three to four mice per treatment group per day; pooled) was measured for levels of TNFα, IL-1α, IL-6, chemokine (C-X-C motif) ligand 10 (CXCL10), granulocyte colony stimulating factor (G-CSF), IL-5, and IL-13. Pooled serum samples were evaluated using Milliplex MAP mouse Cytokine/Chemokine bead-based immunoassay (Millipore, Billerica, MA, USA) coupled with the Luminex 200™ system (Austin, TX, USA) according to the manufacturer’s protocol. Samples were tested at a 1:2 dilution using optimal concentrations of standards and antibodies according to the manufacturer’s protocol.
Statistical analysis
Results were expressed as means ± SD from at least three separate experiments performed in triplicate unless otherwise stated. Differences between means among the treatment groups were analyzed by using the Student t-test or one-way analysis of variance (ANOVA) with Holm-Sidak post-test methods where appropriate. Differences in survival among treatment groups were analyzed with Mantel-Cox log rank test. A P-value less than 0.05 was considered significant. Statistical analysis was performed with SigmaPlot (Version 10.0) with SigmaStat (Version 3.5) software for windows.
Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Institutional Animal Care and Usage Committee (IACUC) of Morehouse School of Medicine (Permit Number 09–06) approved all protocols.
Discussion
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 [
41]. 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 [
41]. 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 [
75]. 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 [
80]. 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 [
79]. 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 [
97] 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 [
106]. 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 [
108], disruption of angiogenesis [
109], endothelial cell death [
110] 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 [
115] 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 [
32]. 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.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
WS designed and performed experiments, analyzed data and wrote the paper. NW performed experiments, analyzed data and wrote the paper. LA designed and performed experiments. SP designed and performed experiments. JP designed experiments and analyzed data. ML designed and performed experiments and analyzed data. BF designed experiments, analyzed data and assisted in the drafting this manuscript. JS conceived the study, designed and supervised experiments and assisted in the drafting this manuscript. All authors read and approved the final manuscript.