Neuregulin-1 attenuates experimental cerebral malaria (ECM) pathogenesis by regulating ErbB4/AKT/STAT3 signaling
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Human cerebral malaria (HCM) is a severe form of malaria characterized by sequestration of infected erythrocytes (IRBCs) in brain microvessels, increased levels of circulating free heme and pro-inflammatory cytokines and chemokines, brain swelling, vascular dysfunction, coma, and increased mortality. Neuregulin-1β (NRG-1) encoded by the gene NRG1, is a member of a family of polypeptide growth factors required for normal development of the nervous system and the heart. Utilizing an experimental cerebral malaria (ECM) model (Plasmodium berghei ANKA in C57BL/6), we reported that NRG-1 played a cytoprotective role in ECM and that circulating levels were inversely correlated with ECM severity. Intravenous infusion of NRG-1 reduced ECM mortality in mice by promoting a robust anti-inflammatory response coupled with reduction in accumulation of IRBCs in microvessels and reduced tissue damage.
In the current study, we examined how NRG-1 treatment attenuates pathogenesis and mortality associated with ECM. We examined whether NRG-1 protects against CXCL10- and heme-induced apoptosis using human brain microvascular endothelial (hCMEC/D3) cells and M059K neuroglial cells. hCMEC/D3 cells grown in a monolayer and a co-culture system with 30 μM heme and NRG-1 (100 ng/ml) were used to examine the role of NRG-1 on blood brain barrier (BBB) integrity. Using the in vivo ECM model, we examined whether the reduction of mortality was associated with the activation of ErbB4 and AKT and inactivation of STAT3 signaling pathways. For data analysis, unpaired t test or one-way ANOVA with Dunnett’s or Bonferroni’s post test was applied.
We determined that NRG-1 protects against cell death/apoptosis of human brain microvascular endothelial cells and neroglial cells, the two major components of BBB. NRG-1 treatment improved heme-induced disruption of the in vitro BBB model consisting of hCMEC/D3 and human M059K cells. In the ECM murine model, NRG-1 treatment stimulated ErbB4 phosphorylation (pErbB4) followed by activation of AKT and inactivation of STAT3, which attenuated ECM mortality.
Our results indicate a potential pathway by which NRG-1 treatment maintains BBB integrity in vitro, attenuates ECM-induced tissue injury, and reduces mortality. Furthermore, we postulate that augmenting NRG-1 during ECM therapy may be an effective adjunctive therapy to reduce CNS tissue injury and potentially increase the effectiveness of current anti-malaria therapy against human cerebral malaria (HCM).
KeywordsCerebral malaria (CM) Neuregulin-1 (NRG-1) ErbB4 STAT3 AKT P. berghei ANKA (PbA)
Blood brain barrier
Experimental cerebral malaria
Epidermal growth factor
Enzyme-linked immunosorbent assay
Ras/extracellular signal regulated kinase ½
Glial fibrillary acidic protein
Human cerebral malaria
human brain microvascular endothelial cell line
Nuclear factor of activated T cells
P. berghei ANKA
Protein kinase C
Real-time reverse transcription polymerase chain reaction
Sensory and motor neuron-derived factor
Signal transducer and activator of transcription 3
Von Willebrand factor VIII
Human cerebral malaria (HCM) is a severe form of malaria characterized by sequestration of infected erythrocytes (IRBCs) in brain microvessels, increased levels of circulating free heme and pro-inflammatory cytokines and chemokines, brain swelling, vascular dysfunction, coma, and increased mortality. The resulting leakiness of the blood brain barrier (BBB) caused by the decreased cerebralvascular integrity allows increased trafficking of toxic compounds into the brain parenchyma leading to exacerbation of neurological deficits [1, 2]. The BBB is a highly selective semipermeable membrane barrier consisting of cerebral vascular endothelial cells and astrocytes surrounding them. It separates the circulating blood from the brain and extracellular fluid  and protects neural tissues against various unfavorable compositions and toxins in the blood. Dysfunctional microvascular endothelial cells or astrocytes compromise the integrity of the BBB, a hallmark of HCM pathogenesis [4, 5]. We have reported that elevation of circulating CXCL10 and free heme induce apoptosis of human brain microvascular endothelial cells (hCMEC/D3) and astroglia/neuroglia (M059K) [6, 7], indicating the important roles played by circulating CXCL10 and free heme in mediating experimental cerebral malaria (ECM) and HCM pathogenesis, BBB integrity, and mortality [8, 9].
The neuregulin family of ligands consist of four members, neuregulin 1β (NRG-1), NRG-2, NRG-3, and NRG-4. While little is known about the biological functions of NRG-2, NRG-3, and NRG-4 , NRG-1 has been widely studied in stroke [11, 12], cardiovascular diseases [13, 14], and tumors [15, 16]. NRG-1, a secreted trophic factor, is encoded by the neuregulin/NRG-1 gene located on the short arm of chromosome 8 [17, 18]. Alternative splicing produces at least 15 different NRG-1 isoforms, which are grouped as types I, II, and III [19, 20]. All four genes in the NRG-1 family (NRG1–4) share a common epidermal growth factor (EGF)-like domain. Type I NRGα and NRGβ isoforms are the predominant isoforms expressed in early embryogenesis, whereas types II and III NRG are not detectable until at mid-gestation stage . Type III, which is also called sensory and motor neuron-derived factor (SMDF), is the most dominant type of NRG-1 in the human adult brain, accounting for about 73% of total NRG-1 [21, 22]. The ErbB receptors are a family composed of receptors of ErbB1, ErbB2, ErbB3, and ErbB4. Any isoform of NRG1 is capable of directly binding and activating ErbB3 and ErbB4 receptors although the biological significance is incompletely understood . The ErbB3 receptor lacks an active kinase domain and is unable to form functional ErbB3 homodimers . ErbB4 undergoes tertiary structural changes in the juxtaembrane region when it binds to its ligand NRG-1 and forms functional homodimers or heterodimers with ErbB1, ErbB2, and ErbB3. Phosphorylation of the ErbB4 receptor results in the recruitment of adaptor/effecter molecules  and initiates/activates numerous downstream signaling pathways crucial to neuronal development, neuronal migration, axonal navigation, and synaptic function . We recently reported that intravenous infusion of NRG-1 significantly reduced mortality associated with ECM by promoting a robust anti-inflammatory response and reducing accumulation of intra vascular IRBC as well as tissue damage . In the current study, we explored a potential molecular mechanism by which NRG-1 regulates downstream pathways to improve/restore the integrity of the BBB and attenuate ECM pathogenesis.
NRG-1 protects against CXCL10- and heme-induced apoptosis in astrocytes and endothelial cells
NRG-1 attenuates heme-induced tight junction disruption in an in vitro BBB model
Effects of NRG-1 on permeability of endothelial cells
Regulation of brain NRG-1 levels following ECM
NRG-1 attenuated ECM mortality-associated ErbB4/STAT3/AKT signaling
NRG-1 is a member of a family of growth factors which are very important in the development of the heart, mammary glands, and the nervous system . In agreement with the known capacity of NRG-1 to attenuate neuronal apoptosis , we demonstrate that NRG-1 protects heme-induced endothelial and neuroglial cell death/apoptosis (Fig. 1). NRG-1 has been shown to be neuroprotective against CNS injury. For instance, in an in vivo experimental trauma model, NRG-1 improved endothelial biological features and reduced BBB permeability and then consequently strengthened BBB structure . In our in vitro BBB model, NRG-1 protected against heme-induced disruption of BBB integrity by strengthening tight junction proteins (Figs. 2 and 3). NRG-1, a growth factor distributes and accumulates in many regions of the brain, including frontal cortex, striatum, and ventral midbrain containing the substantia nigra after systemic application . It targets the entire neurovascular units/cell types such as neuronal cells, oligodendrocytes, endothelial cells, and macrophages to accomplish its potent neuroprotective roles. NRG-1 protects against neuronal death/apoptosis induced by oxygen-glucose deprivation (OGD) [45, 46, 47], ischemic reperfusion , and deep hypothermic circulatory arrest . It prevents apoptosis of oligodendrocyte progenitor cells caused by OGD  and involved in the repair process once the perinatal brain white matter is damaged . It inhibits inflammatory responses either by downregulating pro-inflammatory/inflammatory gene expression in macrophages and resident microglia or hampering macrophage infiltration . Thus, NRG-1 has the potential to be used as a novel therapeutic for treatment of CM since its pathway regulates endothelial cell activities and reduces BBB permeability.
NRG-1 engages the ErbB receptor tyrosine kinases through its EGF-like domain, activation of ErbB receptors impacts cell proliferation, migration, differentiation, and apoptosis in a variety of cell types. The NRG-1/ErbB axis initiates and promotes Schwann cell development and myelination through mobilization of Ca2+, activation of nuclear factor of activated T cells (NFAT), activation of kinases such as Ca2+-dependent protein kinase C (PKC), ras/extracellular signal regulated kinase ½ (Erk1/2), and phophatidylinositol-3-kinase (PI3K/AKT) [50, 51]. Among these pathways, ErbB4-mediated PI3K/AKT signaling pathway is especially crucial against apoptosis . However, the mechanism by which NRG-1/ErbB interaction protects against cerebral malaria pathogenesis is unknown. For instance, it is unclear how NRG-1 activates its ErbB receptor to protect against cerebral malaria pathogenesis. The extent to which this protection occurs in any specific tissues and regions of the brain remains largely unknown. In the present study, we determined that the ErbB4 protein is inactivated through dephosphorylation in the cortex and hippocampus of mice with advanced stages of ECM (Fig. 6) indicating a diffuse distribution of brain lesions. In contrast, exogenous NRG-1 infusion increases phosphorylated ErbB4 levels in the cortex and hippocampus of infected mice, which subsequently reduces STAT3 activation—a typical pathogenic pathway in CM [7, 42] and increases AKT activation (Fig. 7). Our findings are consistent with Lok’s report which showed that NRG-1 increases pAKT to enhance survival of endothelial cells in in vitro cell cultures . Gene microarray data analysis revealed that NRG-1 downregulated hemeoxygenase-1 (HO-1), a heme scavenger , suggesting that NRG-1 may interrupt heme-induced pathogenic signaling pathways in brain endothelial cells. Our findings support the observation that NRG-1 reduces heme-induced STAT3 activation while increasing ErbB4/AKT activation when human endothelial cells are co-treated with heme and NRG-1 in cell culture (Fig. 8).
Although NRG-1 is initially synthesized as transmembrane protein, NRG-1 is cleaved by proteases and the soluble form of NRG-1 impacts significantly the function of the nervous system . Plasma soluble NRG-1 has been detected as a diagnostic biomarker for Alzheimer’s disease . The cause of increased expression of NRG-1 including blood NRG-1 levels after PbA infection is not clear. We speculate that STAT3 could be one of regulators since the promoter of NRG-1 contains a number of STAT3 binding sites. Our in vitro data strongly indicate that increased NRG-1 level reduced damage to the blood brain barrier. So far, no evidence indicates any functional differences between increased NRG-1 during infection and infused NRG-1. However, the bioavailability of infused NRG-1 is very low in circulation; Ford’s group (co-author) reported that the half-life of NRG-1 was short, the plasma t ½ was about 8 min after exogenous intraarterial injection of NRG-1, while it was present in brains of animals at 20 min post administration and remained at a constant level for up to 4 h post injection .
Our data demonstrates that circulating NRG-1 levels correlate with clinical severity of ECM. Interestingly, a previous report by Hama et al. in 2015  indicated that NRG-1 level in plasma did correlate with clinical severity of Parkinson’s disease. Surprisingly, infusion of higher doses of NRG-1 in our study did not produce exponential effects as expected compared to the optimum dose of NRG-1 utilized. The reason for this is unclear. However, Xu et al.  found that NRG-1 exerts a dual function in the myelination of oligodendrocytes. It appears that excessive stimulation of NRG-1β signaling may not be beneficial but detrimental for neural function. Low doses of NRG-1 stimulates myelination in vitro  while overexpression of NRG-1 in myelinating Schwann cells caused hind limb paralysis, which indicates that over-activation of NRG-1 signaling may generate aberrant Schwann cell activity . We are aware that the neuronal cell scenario is different from that of endothelial cells. However, both cell types are indirectly relevant to HCM and ECM and may explain why higher doses of NRG-1 may fail to produce exponential effects as observed for low doses. Taken together, our results indicate that judicious augmentation of NRG-1 during anti-malaria therapy against CM may be an effective therapeutic approach to increase effectiveness of current anti-malaria therapy against CM.
Neuregulin-1 (NRG-1) is encoded by the gene NRG1, a member of a family of polypeptide growth factors, which is essential for the normal development of the nervous system and the heart. Following our first report of its protective role in experimental cerebral malaria (ECM), we further examined how NRG-1 mechanistically attenuates ECM pathogenesis and reduces mortality. Our results indicate that NRG-1 protects against cell death/apoptosis of human brain microvascular endothelial and neuroglial cells and prevents BBB disruption. Our results also demonstrate that NRG-1 attenuated ECM mortality is associated the activation of ErbB4/AKT and inactivation of STAT3 signaling pathways.
Infection of mice with P. berghei ANKA was performed as described previously . Mice were selected and randomized into treatment groups after diagnosis with ECM  on days 5 to 6 post infection. 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 (5 or 25 μg/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 days 6 to day 11 post infection. The mice treated daily with 50 μl saline solution (i.p.) from days 6 to 11 post infection were used as the controls. 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 being euthanized accordingly .
Antibodies and reagents
Rabbit antibody against STAT3, phospho-STAT3, rabbit antibody against AKT, phospho-AKT, anti-NeuN, anti-Iba1, and anti-glial fibrillary acidic protein (GFAP) were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Polyclonal antibody to β-actin, lipopolysaccharide (LPS), and sodium fluorescein were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal antibodies against claudin5, ZO-1, and phosphorylated ErbB4 (monoclonal) antibody were purchased from Abcam (Cambridge, MA, USA). Rabbit polyclonal antibody against occludin and attachment factor (AF) was purchased from Invitrogen (Waltham, MA, USA). Rabbit antibody against VWF was purchased from Dako (Santa Clara, CA, USA). Anti-ErbB4 (polyclonal) and NRG-1 (polyclonal) antibodies were purchased from Santa Cruz (Dallas, TX, USA). All secondary antibodies used for Western blot were purchased from Calbiochem (La Jolla, CA, USA). Hemin (heme) was purchased from Frontier Scientific (Logan, UT, USA). Recombinant human NRG1β1/HRG1β EGF domain protein (γhNRG-1) and recombinant human CXCL10/IP-10 protein were purchased from R&D systems (Minneapolis, MN). Recombinant human interleukin-1β (IL-1β) was purchased from Millipore (Billerica, MA, USA).
The hCMEC/D3 a cell line (Cellutions Biosystems Inc., Ontario, Canada) was derived from human temporal lobe microvessels and were immortalized with hTERT and SV40 large T antigen. The cells have been extensively characterized for brain endothelial phenotype and are a model of human blood brain barrier (BBB) function. hCMEC/D3 cells were cultured at 37 °C with 5% CO2 in endothelial basal medium-2 (Lonza) supplemented with 5% fetal bovine serum (FBS; American Type Culture Collection (ATCC), Manassas, VA, USA), growth factors and other supplements including human recombinant epidermal growth factor (hEGF), hydrocortisone, GA-100 (Gentamicin, Amphotericin-B), human recombinant vascular endothelial growth factor (VEGF), recombinant human fibroblast growth factor-b (hFGF-b), recombinant long R insulin-like growth factor (R3-IGF-1), ascorbic acid, heparin, 100 U/ml of streptomycin, and 100 U/ml of penicillin (Lonza). The cells were harvested and passaged at about 70–90% confluence as described previously . The hCMEC/D3 cells (2 × 105 cells/ml) were seeded in 35-mm tissue culture dish and incubated at 37 °C in 5% CO2 for 24–48 h for future use. M059K cells purchased from ATCC were cultured at 37 °C with 5% CO2 in DMEM: K-12 medium.
Measurement of cell viability by MTT assay
The hCMEC/D3 or M059K cells were seeded at 1 × 104 cells in 100 μl of medium per well into 96-well plates and serum-starved for 24 h, incubating with 100 ng/ml γhNRG-1 for 30 min, followed by exposing to heme at 30 μM or 0.02 μg/ml CXCL10 for 24 h. MTT assay was performed in accordance to the manufacturer’s instructions. Ten microliters of MTT reagent (the ratio of MTT reagent to medium is 1:10) was added into each well and incubated in the dark at room temperature for 2 to 4 h. Absorbance at 570 nm was measured using 650 nm as reference filter by a CytoFluorTM 2300 plate reader and the software CytoFluorTM 2300 v. 3A1 (Millipore Co, Bedford, MA, USA).
Cells grown in monolayer cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100 and blocked with 10% goat serum prior to antibody staining. Specific primary antibodies were added at 1:100 dilution and incubated at 4 °C overnight. Fluorescent staining was developed using the FITC or Cy3 fluorescence system (Sigma-Aldrich, St. Louis, MO, USA). TUNEL assay, in situ cell death detection kit (TMR red; Boehringer-Mannheim, Mannheim, Germany) was used. The sections were incubated with the TUNEL reaction solution for 60 min at 37 °C in the dark. Cover slips were mounted onto slides with Vectashield mounting medium with DAPI (H-1200; Vector Laboratories Inc.). Fluorescent images were collected by using a Zeiss laser scanning microscope (LSM) 510 confocal microscope, and images were captured with LSM software, version 2.3 (Carl Zeiss, Wetzlar, Germany). Apoptotic cells and total cells were counted in ten randomly chosen microscopic fields, and the percentage of apoptotic cells was calculated and compared between the experimental and control groups.
Enzyme-linked immunosorbent assay (ELISA)
Peripheral blood NRG-1 quantification was assayed by ELISA (MyBioSource, San Diego, CA, USA). At indicated days after PbA infection, a subset of mice (n = 5/group) was anesthetized, then 500 μL of peripheral blood was acquired through cardiac puncture. Blood was immediately placed in a biopsy tube with EDTA to prevent clotting. Plasma was obtained by centrifuging the whole blood at 1200 g for 15 min at room temperature. ELISA was performed on all samples to quantify plasma levels of mouse NRG-1. The samples were read on a microplate reader (TECAN) at 450 nm.
Endothelial cell permeability assay
Endothelial cell permeability assay was performed as previously described [28, 43, 55]. In brief, the hCMEC/D3 cells were cultured in complete growth media EGM-2 MV (Lonza, Walkersville, MD, USA). The hCMEC/D3 cells were seeded into the inner surface of attachment factor (AF)/ collagen-coated transwell inserts (24 mm diameter, 0.4-μm pore size polyester filter; Corning, Corning, NY, USA), which were placed in wells of a 6-well plate with human neuroglial/astrocytes (A172 glioma cells). About 7 days after seeding, when hCMEC/D3 cells grown on the inner surface of the insert were confluent (confirmed by testing permeability to media), the cells were serum starved overnight with EBM media without growth supplement, then incubated with IL-1β (10 ng/ml), LPS (1 μg/ml), and heme (30 μM) for 24 h. Thirty minutes prior to addition of IL-1β, LPS and heme, NRG1 (100 ng/ml = 12.5 nM), or vehicle (PBS) was added. After 24 h, media in both upper and lower chambers were removed and replaced with fresh media. Permeability was measured by adding 0.1 mg/ml of Sodium Fluorescein (MW, 376 kDa, Sigma, St. Louis, MO, USA) to the upper chamber. After incubation for 0.5 h, the sample from the lower compartment was collected and 100 μl of sample was measured for fluorescence at excitation 485 nm and emission 535 nm. All three independent experiments were performed in triplicate.
Real-time RT-PCR analysis
Primers for real-time polymerase chain reaction
Forward primer (5′→3′)
Reverse primer (5′→3′)
Cells were lysed with lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Nonidet P-40, 100 mM NaF, 10 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). Samples were separated by SDS/PAGE, and separated proteins were transferred to nitrocellulose membranes and identified by immunoblotting. Primary antibodies were obtained from commercial sources; these antibodies were diluted at the ratio of 1:1000 according to the manufacturer’s instruction. Blots were developed with Supersignal Pico or Femto substrate (Pierce). A densitometric analysis of the bands was performed with the ImageQuant program (Bio-Rad).
Statistically significant differences were determined using Prism statistical software (Graph Prism 4.03, San Diego, CA, USA). All data were presented as mean ± SEM of at least three independent experiments. For data analysis, unpaired t test or one-way ANOVA with Dunnett’s or Bonferroni’s post test was applied. All P values resulted from two-sided statistical tests and statistical significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001.
JKS is funded by the National Institute of Neurological Disorders and Stroke NIH/NINDS 1 R56, NS091616-01 NIH/NINDS 1 R01NS091616-01, and NIH/RCMI RR033062 (G12); the funding body has no involvement in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
Data sharing not applicable to this article. The authors confirm that all relevant data are included in the article.
ML, WS, JCC, and NOW carried out the experiments. ML and JCC performed the statistical analysis. ML, BF, and JKS conceived of the study and participated in the design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
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 protocol was approved by the Institutional Animal Care and Usage Committee (IACUC) of Morehouse School of Medicine (Permit Number 09-06).
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