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

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

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

mRNA expression of HO-1 (A), CXCL10 (B), and CXCR3 (C) in whole brain homogenates of control, ARM, ATV, ATV/ARM-treated<i> P. berghei</i>ANKA-infected mice ( = 5 per group).

<p>Expression, measured by real-time PCR on day 11 post infection, is relative to the mean expression value in untreated mice day 5 post-infection brain samples and normalized on HPRT values. Treatment with ATV or ATV/ARM increased HO-1 expression and decreased CXCL10 expression in <i>P. berghei</i> ANKA-infection mice brain tissue. Columns and bars represent means standard error. Mean expression values were determined to be significantly different using one-way ANOVA with Holm-Sidak post-tests method for all pairwise multiple comparison. 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. Note the different scales used on each graph. Ctrl = control; D = day; ARM = artemether; ATV = atorvastatin.</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

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

Assessment of transcriptome associated with endothelium activation and blood-brain barrier integrity.

<p>mRNA expression of angiopoietin-1 (A), angiopoietin-2 (B), ICAM-1 (C), perforin (D), IFN-γ (E), and FasL (F) in whole brain homogenates of control, ARM, ATV, and ATV/ARM-treated <i>P. berghei</i>-infected mice ( = 5 per group). Expression, measured by real-time PCR on day 11 post-infection, is relative to the mean expression value in control mice day 5 post-infection brain samples and normalized with HPRT values. Treatment with ATV or ATV/ARM decreases endothelium activation and improves blood-brain barrier integrity. Columns and bars represent means standard error. Mean expression values were determined to be significantly different using one-way ANOVA with Holm-Sidak post-tests method for all pairwise multiple comparison. 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. Note the different scales used in each graph. Ctrl = control; D = day; ARM = artemether; ATV = atorvastatin.</p

Heme-Mediated Induction of CXCL10 and Depletion of CD34+ Progenitor Cells Is Toll-Like Receptor 4 Dependent

<div><p><i>Plasmodium falciparum</i> infection can cause microvascular dysfunction, cerebral encephalopathy and death if untreated. We have previously shown that high concentrations of free heme, and C-X-C motif chemokine 10 (CXCL10) in sera of malaria patients induce apoptosis in microvascular endothelial and neuronal cells contributing to vascular dysfunction, blood-brain barrier (BBB) damage and mortality. Endothelial progenitor cells (EPC) are microvascular endothelial cell precursors partly responsible for repair and regeneration of damaged BBB endothelium. Studies have shown that EPC’s are depleted in severe malaria patients, but the mechanisms mediating this phenomenon are unknown. Toll-like receptors recognize a wide variety of pathogen-associated molecular patterns generated by pathogens such as bacteria and parasites. We tested the hypothesis that EPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by CXCL10 induction and toll-like receptor (TLR) activation. Heme and CXCL10 concentrations in plasma obtained from malaria patients were elevated compared with non-malaria subjects. EPC numbers were significantly decreased in malaria patients (<i>P</i> < 0.02) and TLR4 expression was significantly elevated <i>in vivo</i>. These findings were confirmed in EPC precursors <i>in vitro</i>; where it was determined that heme-induced apoptosis and CXCL10 expression was TLR4-mediated. We conclude that increased serum heme mediates depletion of EPC during malaria pathogenesis.</p></div

Heme induces apoptosis in HBVEC and CD34⁺-HSPC <i>in vitro</i>.

<p>Apoptosis was quantified using Guava TUNEL assay and analyzed by fluorescence-activated cell sorting (FACS). Apoptosis was analyzed using analysis of variance followed by Tukey’s multiple comparisons test; In HBVEC <i>p <</i> 0.0001 in heme-treated versus NaOH vehicle, and in CD34⁺-HSPC <i>p</i> = 0.0004 in heme-treated versus NaOH vehicle. CPT is a potent inducer of apoptosis used as positive control (<i>p</i> < 0.0001 in both cell types).</p