Fig. 4

Spermidine exhibits direct anti-inflammatory effects on microglia. a Hemispheres of wild type (WT) and APPPS1 mice were coronally sliced and treated with the indicated spermidine concentration, LPS (10 µg/ml) and ATP (5 mM) as depicted. The IL-1β and IL-6 concentration in the supernatant was determined by ELISA; n = 3–5, two-way ANOVA, Tukey’s post hoc test. b–f Neonatal microglia (neoMG) were either treated with LPS (1 µg/ml) and ATP (2 mM), with poly I:C (50 µg/ml) or with oligomeric Aβ (Aβo, 5 µM) and the indicated spermidine concentrations as depicted in the schemes. b–d Amount of cytokines in the cell supernatant was determined by electrochemiluminescence (MesoScale Discovery panel); n = 4–5. b IFN-γ, IL-10, IL-12, IL-2: Kruskal–Wallis, Dunn’s multiple comparison; IL-1β, IL-4, IL-5, IL-6, KC/GRO, TNF-α: one-way ANOVA, Dunnett’s post hoc test. c INF-γ, IL-2, IL-4: Kruskal–Wallis, Dunn’s multiple comparison; IL-10, IL-12, IL-1β, IL-5, IL-6, KC/GRO, TNF-α: one-way ANOVA, Dunnett´s post hoc test. d IL-10, IL-12, IL-4, KC/GRO: Kruskal–Wallis, Dunn’s multiple comparison; INF-γ, IL-1β, IL-2, IL-5, IL-6, TNF-α: one-way ANOVA, Dunnett’s post hoc test. e The gene expression of Tnf-α and Il-6 was assessed by RT-qPCR after treatment of neonatal microglia as depicted in b. Their expression was normalized to Actin and displayed as fold change compared to non-treated control cells; n = 4. Il-6: one-way ANOVA, Dunnett’s post hoc test; Tnf-α: Kruskal–Wallis, Dunn’s multiple comparison. f Levels of phosphorylated NF-κB (pNF-κB) and NF-κB were determined by western blot in neonatal microglia treated as depicted in b. Representative images are shown and protein levels are displayed as fold changes compared to non-treated controls normalized to ACTIN; n = 7. NF-κB: Kruskal–Wallis, Dunn’s multiple comparison; pNF-κB: one-way ANOVA, Dunnett’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001