Increased S1P₃-mediated ERK-1/2, but not Akt signaling by LPS.

<p>Astrocytes were serum-deprived during the 12 h treatment with LPS and were then stimulated for 20 min with 10 µM S1P₃ agonist (Compound 20) or 1 µM AUY954. The results show the relative ERK-1/2 phosphorylation (A, C ) and Akt phosphorylation (B, D) ± SEM normalized against actin for three (A, B) and two (C, D) independent experiments. One-Way ANOVA followed by Bonferroni's multiple comparison test: *p<0.05, **p<0.01, ***p<0.001.</p

SphK1 is activated in response to LPS.

<p>Serum-deprived astrocytes were incubated for 30 min with 100 ng/ml LPS in the presence or absence of SKI (10 µg/ml). 100 µg of cell extracts were used to determine SphK1 activity by thin layer chromatography. Data represents the mean ± SEM of three independent experiments. One-Way ANOVA followed by Bonferroni's multiple comparison test: *p<0.05, **p<0.01.</p

S1P₃ contributes to the S1P-induced CXCL1 release by primary astrocytes.

<p>Astrocytes were pre-treated with or without LPS (100 ng/ml) for 12 h in serum-free medium and then stimulated or not with S1P (1 µM), a S1P₃ agonist (Compound 20, 10 µM) or a S1P₁ agonist (AUY954, 10 µM) for 5 h. The graph shows the effect of LPS pre-treatment on the release of CXCL1 by S1P, S1P₃ or S1P₁ agonists. Data are a pool of two independent experiments each performed in six replicates ± SEM. One-Way ANOVA followed by Bonferroni's multiple comparison test: **p<0.01, ***p<0.001.</p

S1P₃ and SphK1 are expressed by reactive astrocytes and macrophages in MS lesions.

<p>Immunofluorescence co-localization studies confirmed that S1P₃ is predominately expressed by reactive astrocytes (A). SphK1 is also expressed on reactive astrocytes (B), but the major cell type expressing SphK1 is macrophage in lesions and perivascular cuffs (C–D). Scale bars are 10 µm.</p

LPS-induced astrocyte migration is SphK1-dependent, but proliferation-independent.

<p>Astrocyte were treated with increasing concentrations of S1P (10 nM, 100 nM, 1000 nM) (A) or LPS (1 ng/ml, 10 ng/ml, 100 ng/ml) (B), and cell proliferation was measured using a [³H]- thymidine uptake assay. Data are representative of three independent experiments ± SEM. One-Way ANOVA followed by Dunnett's post-test: (A) *p<0.05 <u>vs</u>. control, ***p<0.001 <u>vs</u>. control. Astrocytes were pre-treated or not with SKI (10 µg/ml, 1 h) and then stimulated with LPS (100 ng/ml) (C) or stimulated with S1P (500 nM) and LPS (100 ng/ml), respectively, in the presence or absence of 10 µM antimitotic treatment (D). The graphs show the SKI-mediated inhibition of LPS-induced migration (C) and the influence of antimitiotic treatment on S1P- or LPS-induced migration after 48 h incubation (D). The surface of area covered by GFAP immunoreactivity is plotted. Each scratch was evaluated with an average of four photographs, and each treatment group represented five to six replicates. Data are representative of three independent experiments ± SEM. One-Way ANOVA followed by Dunnett's post-test: (C) *p<0.05, **p<0.01; (D) *p<0.05 **p<0.01 <u>vs</u>. respective control.</p

S1P₃ and SphK1 mRNAs and protein are upregulated in rat primary astrocytes by LPS stimulation.

<p>Primary astrocytes were incubated in culture medium for 5 or 24 h with LPS (100 ng/ml). The mRNA levels of S1P₁ (A), S1P₃ (B), SphK1 (C) and SphK2 (D) were assessed after 5 h and 24 h incubation. Quantitative PCR results are shown as the percentage expression of HKG (GAPDH) and represent mean ± SEM of three independent experiments. LPS mediated sustained upregulation of S1P₃ (E–F) and SphK1 (G–H) as shown by Western blots of plasma membrane fractions. Primary rat astrocytes were incubated with LPS (100 ng/ml) in serum-free medium containing 0.25% BSA for 12 and 48 h. Representative immunoblots are shown. Graphs represent the mean ± SEM of three independent experiments and are reported as protein expression normalized to actin, expressed as fold change over basal level. One-Way ANOVA followed by Bonferroni's multiple comparison test: *p<0.05, **p<0.01 <u>vs</u>. respective control.</p

S1P₃ and SphK1 are upregulated in MS lesions.

<p>(A, B): Immunohistochemical peroxidise staining shows that S1P₃ receptor and SphK1 enzyme expressions are increased in a chronic-active MS lesion which was located in parietal subventricular white matter. S1P₃ expression is strong on reactive astrocytes in this lesion border (C), in the lesion (D) and on perivascular cells (G), but is weak in normal control brain white matter (H). SphK1 expression is increased in reactive astrocytes (E) and in macrophages in this MS lesion (F). A particularly high expression of SphK1 is seen in perivascular inflammatory cells (I), whereas its expression is very low in normal control brain white matter (J). This Figure shows representative stainings. Scale bars are 200 µm for A and B, and 20 µm for C–J. The sections were counterstained with haematoxylin.</p

Transgene expression on B16F1-HyBNAR mouse melanoma cells and its binding to human IFN-I.

<p>(A) FACS analysis of B16F1-HyBNAR cells using antibodies that specifically bind to the human variants of the extracellular domains of IFNAR1 and IFNAR2. Specificity of binding is demonstrated by the staining of non-transfected mouse B16F1 control cells. (B) YNS, an engineered tight-binding variant of IFNα2 was radiolabelled and incubated with mouse B16F1, B16F1-HyBNAR and human WISH cells. Tightly binding radiolabelled YNS could not be displaced by co-incubation with 100× molar excess of lower affinity Hu-IFNα2a, although high affinity human IFNβ and YNS completely displaced the hot ligand. Importantly, 100× molar excess of mouse IFNβ did not displace binding of hot YNS in this system.</p

Time and injection regiment affects response to IFN-Is.

<p>(A). Response of different injection regiments to 0.2 ug (weight adjusted per 20 g mouse weight) of human IFN<b>β</b>. Injections were by intraperitoneal (IP), intra venous (IV) or by sub-cutaneous (SC) route. (B). Mice were injected IP with the indicated increasing doses of YNSα8 and luminosity was measured in a time-course from live animal measurements. MX2-LUC mice doubly transgenic for HyBNAR (solid lines) or without presence of HyBNAR (dotted lines) were both measured. The relative luminosity values given are averaged from three animals (A) or two animals (B) per injection group and were measured from a defined region of maximal signal (found in the liver region) for each mouse as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084259#pone.0084259.s002" target="_blank">Figure S2</a>. In both experiments, baseline luminosity measurements were determined from the mice taken immediately prior to IFN induction.</p

Interspecies Relatedness for a Selection of Cell Surface Receptors.

<p>Interspecies protein sequences derived for IFNAR1, IFNAR2, the epidermal growth factor receptor (EGFR) and the Insulin Receptor (INSR) were each compared by respective pairwise BLASTP alignment. Percent amino acid identity scores are given. Pre-calculated percentage identity scores were extracted from the Homologene Server (NCBI, Release #67). Exceptionally, rat IFNAR2 protein sequence (accession #XP_001073550.1) was not available in the Homologene dataset, and was thus curated manually.</p