Fig 2: S1P3 and SphK1 are upregulated in MS lesions.(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.

Fig 3: General stress effects and S1P pathways analyses in the colon of WT and Sphk2 mice after sub-chronic stress exposure. Percentage of weight gain after the stress protocol (a), plasma corticosterone levels (b), relative spleen weight (c), colon S1P levels (d), Sphk1 mRNA levels (e), Sphk2 mRNA levels (f), SGPL1 mRNA levels (g), SGPP1 mRNA levels (h), SGPP2 mRNA levels (i), S1PR1 mRNA levels (j), S1PR2 mRNA levels (k), S1PR3 protein expression (l). Data are means ± SEM of 5–8 mice per group. The densitometric data of the respective bands of interest are normalized by ß-actin (lower band). Blots were cropped (black lines) to improve the clarity and conciseness of the presentation. Two-way ANOVA considering stress (S) and Sphk2-/- as independent variables followed by Tukey's post hoc for (a–l); *S p < 0.05, **S p < 0.01, ***S p < 0.001; #Sphk2-/- p < 0.05; @ (interaction) p < 0.05; +p < 0.05, ++p < 0.01 vs Control WT; &p < 0.05 vs Control Sphk2-/-. Two-tailed Student t-test for f; %%p < 0.01. Sphk2: sphingosine kinase 2; WT: wild-type; Sphk2-/-: Sphk2 knockout; S1P: sphingosine-1-phosphate; Sphk1: sphingosine kinase 1; SGPL1: sphingosine-1-phosphate lyase 1; SGPP1: sphingosine-1-phosphate phosphatase 1; SGPP2 sphingosine-1-phosphate phosphatase 2; S1PR1: sphingosine-1-phosphate receptor 1; S1PR2: sphingosine-1-phosphate receptor 2; S1PR3: sphingosine-1-phosphate receptor 3.

Fig 4: S1P3 and SphK1 are expressed by reactive astrocytes and macrophages in MS lesions.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.

Fig 5: S1P3 contributes to the S1P-induced CXCL1 release by primary astrocytes.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.