Fig. 6

Chronic NF-κB activation in OLs triggers non-cell-autonomous expansion and activation of microglia cells. a Cell number of isolated Cd11b⁺ microglia cells from IKK2-CA^{PLP−CreERT2} mice is significantly increased compared to Cd11b⁺ microglia cells derived from control mice at 3 and 20 wpi (3 wpi: Control: 0.37 ± 0.03 × 10⁶, IKK2-CA^{PLP−CreERT2}: 1.3 ± 0.24 × 10⁶ n = 20–23, 20 wpi: Control: 0.5 ± 0.05 × 10⁶, IKK2-CA^{PLP−CreERT2}: 0.85 ± 0.1 × 10⁶, n = 10–23, two brains with identical genotype were pooled and set to n = 1). b Principal component analysis determines distinct differential gene expression between control and IKK2-CA^{PLP−CreERT2} Cd11b⁺ microglia at 3 and 20 wpi. c IKK2-CA^{PLP−CreERT2} Cd11b⁺ microglia are activated towards an M1 pro-inflammatory phenotype. RNA Sequencing analysis reveals upregulation of M1-like and downregulation of M2-like marker genes in IKK2-CA^{PLP−CreERT2} Cd11b⁺ microglia compared to control Cd11b⁺ microglia as depicted in the heatmap. All deregulated genes shown reached significance at 3 wpi, while only Lgals3, IL1β and Cd14 reached significance at 20 wpi. d and e Seahorse analysis of Cd11b⁺ microglia from IKK2-CA^{PLP−CreERT2} mice revealed decreased oxidative phosphorylation (D, OCR normalized to control; IKK2-CA^{PLP−Cre−ERT2}: 55.2 ± 4.9%, n = 3–4) and increased glycolysis (E, ECAR normalized to control; IKK2-CA^PLPCreERT2: 128.2 ± 3.1%, n = 3–4) 3 wpi. fEx-vivo analysis of primary Cd11b⁺ microglia revealed distinct morphological differences between control and IKK2-CA^{PLP−CreERT2} Cd11b⁺ microglia. IKK2-CA^{PLP−CreERT2} Cd11b⁺ microglia show a rather bushy, activated phenotype. Data are shown as mean ± SEM. Grey bars, control; red bars, IKK2-CA.^{PLP−CreERT2}. Statistical analysis: Two-way ANOVA multiple comparison test followed by Bonferroni´s post hoc test, Two-tailed Mann–Whitney-U Test, (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 NS, non-significant (p > 0.05))