Fig. 4
Treatment with FTY720 plus IFN-β is associated with a protective glial transcriptional program in astrocytes. Primary murine neonatal astrocytes were treated with vehicle, FTY720 + vehicle, or the combination of FTY720 and IFN-β overnight and subsequently stimulated with IL-1β and TNF-α for 4 h. (A) Heatmap of 550 differentially expressed genes in at least two out of the groups (n = 3 biological replicates). (B) Heatmap of selected genes differentially expressed in the FTY720 plus IFN-β condition relative to FTY720 alone. (C) Ingenuity Pathway Analysis identifying predicted downstream effects in astrocytes treated with FTY720 plus IFN-β compared with FTY720 alone. (D) Fold change in mRNA expression of the indicated genes, shown as log2 (combined/FTY720). (E) Measurement of Socs1 in activated astrocytes (n = 5 biological replicates). A p-value of p < 0.05 was considered significant and determined by one-way ANOVA followed by Tukey´s multiple-comparisons test. (F) Activated primary murine astrocytes were pre- treated with a SOCS1 inhibitor (pJAK2(1001–1013); 20 µM) or a control peptide (20 µM) at the beginning of the pre-treatment phase and maintained throughout cytokine stimulation with IL-1β (10 ng/mL) and TNF-α (5 ng/mL) for 4 h. qPCR was performed for indicated genes (n = 3 biological replicates). (G) Activated primary murine astrocytes were treated with FTY720/IFN-β and a control peptide or pJak2(1001–1013). qPCR was performed for indicated genes (n = 3 biological replicates). (H) Astrocyte-conditioned medium (ACM) was generated by activating primary astrocytes with IL-1β (10 ng/mL) and TNF-α (5 ng/mL) for 24 h, followed by treatment with FTY720/IFN-β and either the control peptide or pJAK2(1001–1013) (20 µM). The medium was replaced the next day and collected after an additional 24 h. The harvested ACM was added to the lower chamber of a transwell system, and 200,000 isolated monocytes were seeded into the upper insert for a 3 h migration assay. (I) Number of migrated monocytes was counted after 3 h. P-values of p < 0.05 were considered significant and determined by Student´s t-test.
