Figure 1. Socs3^ΔLysM mice exhibit brain-targeted, atypical EAE with infiltration of hyperactivated neutrophils.

EAE was induced in both Socs3^fl/fl and Socs3^ΔLysM mice, and cerebellar tissue was collected at the peak of disease for further analysis. (A) Demyelination was assessed on day 14 and quantified by Black Gold staining (n = 4). Arrows indicate demyelinated regions. (B–J) Immune cells isolated from the cerebellum on days 13–14 using a Percoll gradient were subjected to surface staining. (B) Overlay of cerebellar-infiltrating neutrophils from Socs3^fl/fl and Socs3^ΔLysM mice stained for CD11b, CXCR4, CD62L, and CXCR2 (n = 7–8). (C) Relative expression of surface markers of cerebellar-infiltrating neutrophils (n = 7–8). (D) Total number of cerebellar-infiltrating neutrophils (n = 7–8). (E) Degranulation was measured by analyzing the percentage of CD63⁺ neutrophils on days 13–14 (n = 4). (F–I) Before surface staining, isolated neutrophils were incubated with CM-H2DCFDA (1 μM) at 37°C for 30 minutes. (F) Percentage of ROS-producing neutrophils (n = 7–8). (G and H) ROS production by neutrophils measured as the MFI of CM-H2DCFDA staining (n = 7–8). (I) ROS production by different immune cell types from the cerebellum on days 13–14. Neutrophils (CD45⁺CD11b⁺Ly6C^loLy6G⁺), Ly6C⁺ monocytic cells (Ly6C⁺ Mo) (CD45⁺CD11b⁺Ly6C⁺Ly6G^–), Ly6C^– monocytic cells (Ly6C^– Mo) (CD45⁺CD11b⁺Ly6C^–Ly6G^–), microglia (CD45^loCD11b⁺), and other leukocytes (CD45⁺CD11b^–). Plot represents 8 individual samples. (J) Superoxide was measured using DHE (n = 4). (K) RNA was isolated from whole cerebellum on day 13, and Hmox1 expression was analyzed by quantitative reverse transcription PCR (qRT-PCR) (n = 4). All error bars represent ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by 2-tailed Student’s t test. MFI, mean fluorescence intensity; DHE, dihydroethidium.