Fig. 5.

Astrocyte-specific Dll4 inactivation induces downregulation of astrocyte reactivity through the downregulation of the IL-6-STAT3 pathway. (A-C) NA were cultured until 70% confluence and transfected with a CONTROL siRNA (20 µM) versus a DLL4 siRNA (20 µM), starved for 12 h and treated with 1X PBS versus IL-1β 10ng/mL for 12 h. (A-B) IL-6 (n = 8) and (A, C) P-STAT3 (n = 6) expression were quantified by western blot (WB). β-ACTIN (ACTB) and STAT3 total were used as references. (D) NA were cultured on Lab-Tek^® until 70% of confluence. They were then treated as above. GFAP (in green) and IL-6 (in red) localizations were evaluated by immuno-fluorescent staining. Nuclei were stained with DAPI (in blue). (E) Spinal cord from control tissues or EAE lesions (18 days post induction) from Dll4^ACKOP mice, Dll4^ACKOC mice and littermate controls were harvested. Dll4^ACKOP, Dll4^ACKOC and control tissues were immuno-stained with anti-GFAP (in green) and anti-IL-6 (in red) antibodies. Nuclei were stained with DAPI (in blue). Dll4^ACKOC and control tissues are shown. (F-G) IL-6 positive areas were quantified (F) in reactive NA (n = 8) and (G) in spinal cord lesions (3 lesions per tissue) (Dll4^ACKOP mice n = 6, WT n = 5) (Dll4^ACKOC mice n = 7, WT n = 6)). (H-J) NA were cultured until 70% confluence. They were then starved for 12 h and treated with 1X PBS versus IL-1β 10ng/mL with IgG or an anti-IL-6 receptor antibody Tocilizumab (1 µg/mL) for 24 h. (H-I) P-STAT3 (n = 5–6) and (H, J) DLL4 were quantified by western blot (n = 5–6). STAT3 total and ACTB were used as references. Statistical significance was determined by using a Mann-Whitney U test or Kruskal Wallis test followed by the Dunn’s multiple comparison test