Neuregulin-1 attenuates mortality associated with experimental cerebral malaria.

BackgroundCerebral Malaria (CM) is a diffuse encephalopathy caused by Plasmodium falciparum infection. Despite availability of antimalarial drugs, CM-associated mortality remains high at approximately 30% and a subset of survivors develop neurological and cognitive disabilities. While antimalarials are effective at clearing Plasmodium parasites they do little to protect against CM pathophysiology and parasite-induced brain inflammation that leads to seizures, coma and long-term neurological sequelae in CM patients. Thus, there is urgent need to explore therapeutics that can reduce or prevent CM pathogenesis and associated brain inflammation to improve survival. Neuregulin-1 (NRG-1) is a neurotrophic growth factor shown to protect against brain injury associated with acute ischemic stroke (AIS) and neurotoxin exposure. However, this drug has not been tested against CM-associated brain injury. Since CM-associated brain injuries and AIS share similar pathophysiological features, we hypothesized that NRG-1 will reduce or prevent neuroinflammation and brain damage as well as improve survival in mice with late-stage experimental cerebral malaria (ECM).MethodsWe tested the effects of NRG-1 on ECM-associated brain inflammation and mortality in P. berghei ANKA (PbA)-infected mice and compared to artemether (ARM) treatment; an antimalarial currently used in various combination therapies against malaria.ResultsTreatment with ARM (25 mg/kg/day) effectively cleared parasites and reduced mortality in PbA-infected mice by 82%. Remarkably, NRG-1 therapy (1.25 ng/kg/day) significantly improved survival against ECM by 73% despite increase in parasite burden within NRG-1-treated mice. Additionally, NRG-1 therapy reduced systemic and brain pro-inflammatory factors TNFalpha, IL-6, IL-1alpha and CXCL10 and enhanced anti-inflammatory factors, IL-5 and IL-13 while decreasing leukocyte accumulation in brain microvessels.ConclusionsThis study suggests that NRG-1 attenuates ECM-associated brain inflammation and injuries and may represent a novel supportive therapy for the management of CM

Heme Mediated STAT3 Activation in Severe Malaria

The mortality of severe malaria [cerebral malaria (CM), severe malaria anemia (SMA), acute lung injury (ALI) and acute respiratory distress syndrome (ARDS)] remains high despite the availability associated with adequate treatments. Recent studies in our laboratory and others have revealed a hitherto unknown correlation between chemokine CXCL10/CXCR3, Heme/HO-1 and STAT3 and cerebral malaria severity and mortality. Although Heme/HO-1 and CXCL10/CXCR3 interactions are directly involved in the pathogenesis of CM and fatal disease, the mechanism dictating how Heme/HO-1 and CXCL10/CXCR3 are expressed and regulated under these conditions is still unknown. We therefore tested the hypothesis that these factors share common signaling pathways and may be mutually regulated.We first clarified the roles of Heme/HO-1, CXCL10/CXCR3 and STAT3 in CM pathogenesis utilizing a well established experimental cerebral malaria mouse (ECM, P. berghei ANKA) model. Then, we further determined the mechanisms how STAT3 regulates HO-1 and CXCL10 as well as mutual regulation among them in CRL-2581, a murine endothelial cell line.The results demonstrate that (1) STAT3 is activated by P. berghei ANKA (PBA) infection in vivo and Heme in vitro. (2) Heme up-regulates HO-1 and CXCL10 production through STAT3 pathway, and regulates CXCL10 at the transcriptional level in vitro. (3) HO-1 transcription is positively regulated by CXCL10. (4) HO-1 regulates STAT3 signaling.Our data indicate that Heme/HO-1, CXCL10/CXCR3 and STAT3 molecules as well as related signaling pathways play very important roles in the pathogenesis of severe malaria. We conclude that these factors are mutually regulated and provide new opportunities to develop potential novel therapeutic targets that could be used to supplement traditional prophylactics and treatments for malaria and improve clinical outcomes while reducing malaria mortality. Our ultimate goal is to develop novel therapies targeting Heme or CXCL10-related biological signaling molecules associated with development of fatal malaria

Pharmacologic inhibition of CXCL10 in combination with anti-malarial therapy eliminates mortality associated with murine model of cerebral malaria.

Despite appropriate anti-malarial treatment, cerebral malaria (CM)-associated mortalities remain as high as 30%. Thus, adjunctive therapies are urgently needed to prevent or reduce such mortalities. Overproduction of CXCL10 in a subset of CM patients has been shown to be tightly associated with fatal human CM. Mice with deleted CXCL10 gene are partially protected against experimental cerebral malaria (ECM) mortality indicating the importance of CXCL10 in the pathogenesis of CM. However, the direct effect of increased CXCL10 production on brain cells is unknown. We assessed apoptotic effects of CXCL10 on human brain microvascular endothelial cells (HBVECs) and neuroglia cells in vitro. We tested the hypothesis that reducing overexpression of CXCL10 with a synthetic drug during CM pathogenesis will increase survival and reduce mortality. We utilized atorvastatin, a widely used synthetic blood cholesterol-lowering drug that specifically targets and reduces plasma CXCL10 levels in humans, to determine the effects of atorvastatin and artemether combination therapy on murine ECM outcome. We assessed effects of atorvastatin treatment on immune determinants of severity, survival, and parasitemia in ECM mice receiving a combination therapy from onset of ECM (day 6 through 9 post-infection) and compared results with controls. The results indicate that CXCL10 induces apoptosis in HBVECs and neuroglia cells in a dose-dependent manner suggesting that increased levels of CXCL10 in CM patients may play a role in vasculopathy, neuropathogenesis, and brain injury during CM pathogenesis. Treatment of ECM in mice with atorvastatin significantly reduced systemic and brain inflammation by reducing the levels of the anti-angiogenic and apoptotic factor (CXCL10) and increasing angiogenic factor (VEGF) production. Treatment with a combination of atorvastatin and artemether improved survival (100%) when compared with artemether monotherapy (70%), p<0.05. Thus, adjunctively reducing CXCL10 levels and inflammation by atorvastatin treatment during anti-malarial therapy may represent a novel approach to treating CM patients

Primer sequences used.

<p>Primer sequences used.</p

Schematic model of signaling pathways ATV utilize to modulate the expression of CXCL10 identified by Ingenuity Pathway Analysis.

<p>In response to various stimulus, transcription factors, STAT1, NFκB, and RELA, are activated resulting in the transcription of CXCL10 gene to mRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Qiu1" target="_blank">[57]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Penafuerte1" target="_blank">[64]</a>. Atorvastatin inhibit activation of RELA (NFκB3/p65) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Moreno1" target="_blank">[56]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Qiu1" target="_blank">[57]</a>, NFκB complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Li1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Wagner1" target="_blank">[59]</a> and STAT1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Wagner1" target="_blank">[59]</a>. Atorvastatin increases expression of HO-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Gueler1" target="_blank">[68]</a> which inhibit activation of NFκB complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Drechsler1" target="_blank">[69]</a>, STAT1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Vareille1" target="_blank">[70]</a> as well as expression of CXCL10 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Mandal1" target="_blank">[71]</a>. Atorvastatin stimulates production of nitric oxide (NO) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Sasmazel1" target="_blank">[72]</a>, which inhibit active NFκB/p65 and NFκB complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Marshall1" target="_blank">[73]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Franek1" target="_blank">[76]</a>. ATV = atorvastatin; IRBC = infected red blood cells; HO-1 = heme oxygenase-1; NO = nitric oxide; RELA = v-rel reticuloendotheliosis viral oncogene homolog A (avian); NFκB = nuclear factor of kappa light polypeptide gene enhancer in B-cells; STAT1 = signal transducer and activator of transcription 1.</p

Dose-response apoptotic effect of recombinant human CXCL10 on HBVECs and neuroglia cells.

<p>(A) Shown are results of TUNEL assay of percentage of apoptotic HBVECs and neuroglia cells incubated with different concentrations of recombinant human CXCL10 for 24 hour at 37°C. (B) Homogeneous caspase assay showing HBVECs and Neuroglia cells exposed to different concentrations of recombinant human CXCL10 for 24 hours at 37°C. Subsequently, the cells were directly incubated with substrate solution for 2.5 hours at 37°C. The Relative Fluorescence Units (RFU) signal is converted to nM free Rhodamine via standard curve. The increase of the caspase activity was calculated as difference of the RFU signal of the induced cells to the RFU signal of non-induced cells. Bars represent standard deviations of three experiments.</p

Modulating CXCL10 levels improves survival in mice with late stage ECM.

<p>(A) Survival curves: The Kaplan-Meier curves are shown for efficacy of ARM at 25 mg/kg/day ( = 11), ATV at 25 mg/kg/day ( = 11) and ATV/ARM at 25 mg/kg/day ( = 11) in rescuing mice with late stage ECM. All treatments started no day 6 and ends on day 9 post infection. ARM-treated and ATV-treated mice showed survival rates of 75% and 50% respectively compared to controls (p<0.05). The survival rates improved with ATV/ARM-treated mice (100%) which are significantly superior to ARM-treated, ATV-treated or control mice (p<0.001). Survival was assessed twice daily. Significant differences in survival were assessed by Log rank test. (B) Parasitemia of <i>P. berghei</i> ANKA-infected mice after treatment. Parasitemia was monitored by Giemsa-stained blood smears using light microscopy at 100 magnifications with an oil immersion lens. Parasitemia was checked and quantified by counting the number of parasitized red blood cells in at least 1,000 red blood cells. The experiment is a representative of three independent infections. (C) Serum CXCL10 levels of <i>P. berghei</i> ANKA-infected mice after treatment. Serum CXCL10 was measured on samples collected on day 5 and day 11 post infection ( = 5 per group). The results in panels B and C are mean the standard deviation. Mean values were determined to be significantly different using the student <i>t</i>-test. A p value of<0.05 was considered significant. ARM = artemether; ATV = atorvastatin.</p

Inhibition of inflammation in the brain of P. berghei-infected mice with ECM after treatment.

<p>(A) The number of intravascular leukocytes per mm2 of brain area was markedly decreased after treatment. Parenchymal vessel of (B) untreated mice with ECM on day 5 and (C) saline-treated mice on day 11, plugged with leukocytes (black arrows). Parenchymal vessels of (D) ARM-treated mice, (E) ATV-treated mice and (F) ATV/ARM-treated mice showing remnant adherent leukocytes (black arrows) on day 11. The leukocytes counts are mean ± standard error. A p value of <0.05 was considered significant. Asterisks (*) denote statistically significant change compared with ctrl D5 and section sign (§) denote statistically significant change compared with ctrl D11. Ctrl = control; D = day; ARM = Artemether; ATV = Atorvastatin.</p

Atorvastatin regulated gene network of pro-inflammatory, anti-inflammatory and growth factors.

<p>Red-labeled genes were up-regulated and green-labeled genes were down-regulated in Atorvastatin-treated compared with saline-treated mice on day 11 of <i>P. berghei</i> ANKA infection. Color intensity signifies the degree of regulation. White labeled genes were not represented in the uploaded data set, but their connectivity was determined through network analysis.</p