Sphingosine Kinase 1 and Sphingosine 1-Phosphate Receptor 3 Are Functionally Upregulated on Astrocytes under Pro-Inflammatory Conditions

Background: Reactive astrocytes are implicated in the development and maintenance of neuroinflammation in the demyelinating disease multiple sclerosis (MS). The sphingosine kinase 1 (SphK1)/sphingosine1-phosphate (S1P) receptor signaling pathway is involved in modulation of the inflammatory response in many cell types, but the role of S1P receptor subtype 3 (S1P(3)) signaling and SphK1 in activated rat astrocytes has not been defined.Methodology/Principal Findings: Using immunohistochemistry we observed the upregulation of S1P(3) and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. Increased mRNA and protein expression of S1P(3) and SphK1, as measured by qPCR and Western blotting respectively, was observed after treatment of rat primary astrocyte cultures with the pro-inflammatory stimulus lipopolysaccharide (LPS). Activation of SphK by LPS stimulation was confirmed by SphK activity assay and was blocked by the use of the SphK inhibitor SKI (2-(p-hydroxyanilino)-4-(p-chlorphenyl) thiazole. Treatment of astrocytes with a selective S1P(3) agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK)-1/2), which was further elevated with a LPS pre-challenge, suggesting that S1P(3) upregulation can lead to increased functionality. Moreover, astrocyte migration in a scratch assay was induced by S1P and LPS and this LPS-induced migration was sensitive to inhibition of SphK1, and independent of cell proliferation. In addition, S1P induced secretion of the potentially neuroprotective chemokine CXCL1, which was increased when astrocytes were pre-challenged with LPS. A more prominent role of S1P(3) signaling compared to S1P(1) signaling was demonstrated by the use of selective S1P(3) or S1P(1) agonists.Conclusion/Significance: In summary, our data demonstrate that the SphK1/S1P(3) signaling axis is upregulated when astrocytes are activated by LPS. This signaling pathway appears to play a role in the establishment and maintenance of astrocyte activation. Upregulation of the pathway in MS may be detrimental, e. g. through enhancing astrogliosis, or beneficial through increased remyelination via CXCL1

Accelerated Axonal Loss Following Acute CNS Demyelination in Mice Lacking Protein Tyrosine Phosphatase Receptor Type Z

Protein tyrosine phosphatase receptor type Z (Ptprz) is widely expressed in the mammalian central nervous system and has been suggested to regulate oligodendrocyte survival and differentiation. We investigated the role of Ptprz in oligodendrocyte remyelination after acute, toxin-induced demyelination in Ptprz null mice. We found neither obvious impairment in the recruitment of oligodendrocyte precursor cells, astrocytes, or reactive microglia/macrophage to lesions nor a failure for oligodendrocyte precursor cells to differentiate and remyelinate axons at the lesions. However, we observed an unexpected increase in the number of dystrophic axons by 3 days after demyelination, followed by prominent Wallerian degeneration by 21 days in the Ptprz-deficient mice. Moreover, quantitative gait analysis revealed a deficit of locomotor behavior in the mutant mice, suggesting increased vulnerability to axonal injury. We propose that Ptprz is necessary to maintain central nervous system axonal integrity in a demyelinating environment and may be an important target of axonal protection in inflammatory demyelinating diseases, such as multiple sclerosis and periventricular leukomalacia. (Am J Pathol 2012, 181:1518-1523; http://dx.doi.org/10.1016/j.ajpath.2012.07.011)UK Multiple Sclerosis SocietyMultiple Sclerosis International FederationUniv Cambridge, Dept Vet Med, Cambridge CB3 0ES, EnglandUniv Cambridge, Wellcome Trust & MRC Cambridge Stem Cell Inst, Cambridge CB3 0ES, EnglandUniversidade Federal de São Paulo, Dept Biosci, Santos, BrazilMerck Serono Int, Geneva Res Ctr, Geneva, SwitzerlandUniversidade Federal de São Paulo, Dept Biosci, Santos, BrazilWeb of Scienc

Bridging the species divide: transgenic mice humanized for type-I interferon response.

We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These "HyBNAR" (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNβ, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is

Influence of smoking and smoking cessation on levels of urinary 11-dehydro thromboxane B2

Background: Thromboxane is a key clinical risk endpoint of smoking-induced inflammation which has been associated in the pathogenesis of cardiovascular disease. The goal of this review is to quantify the effect of smoking and smoking cessation on one of its urinary metabolites, 11-dehydrothromboxaneB2. Methods: PubMed and SCOPUS were searched to identify publications which report urinary 11-dehydrothromboxaneB2 levels in smokers and non-smokers, as well as articles reporting the effect of smoking cessation on urinary 11-dehydrothromboxaneB2 excretion. Results: We found ten studies assessing urinary 11-dehydrothroboxaneB2 levels in smokers and non-smokers. Four papers reported the amount of urinary 11-dehydrothromboxaneB2 excreted in 24 h while six reported the amount excreted adjusted for creatinine. The meta-analyses comparing the excretion of urinary 11-dehydrothromboxane in current smokers to non-smokers report increased levels in current smokers (mean difference = 0.31 μg/24-h [95%CI: 0.27–0.34] and 166.45 pg/mg creatinine [95%CI: 120.51–212.40]). There were not enough publications to perform meta-analyses on the effects of smoking cessation on urinary 11-dehydrothromboxaneB2 excretion. Conclusions: Urinary 11-dehydrothromboxaneB2 levels are increased in cigarette smokers, however, more data are needed to elucidate the effects of smoking cessation on urinary 11-dehydrothromboxaneB2 excretion. Keywords: Smoking, Inflammation, Thromboxane, Meta-analysi

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

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

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 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