Fig. 5.

Spermidine regulates neuroinflammation beyond transcription by interfering with inflammasome assembly. Neonatal microglia (neoMG) were treated with LPS (1 µg/ml) and spermidine at indicated concentrations for 1.45 h and ATP (2 mM) as depicted in the scheme (a). b IL-1β concentration in the cell supernatant was determined by ELISA; n = 4–8; Kruskal–Wallis, Dunn´s multiple comparison. c IL-18 concentration in the cell supernatant was determined by ELISA; n = 3; Kruskal–Wallis, Dunn’s multiple comparison. d Pro-IL-1β protein levels were determined by western blot and normalized to ACTIN. Representative images are shown and values are displayed as fold changes compared to LPS/ATP-treated cells; n = 8–9; Kruskal–Wallis, Dunn’s multiple comparison. e Cellular Pro-CASP1 and cleaved CASP1 p20 levels in the supernatant were determined by western blot (* non-specific band). Pro-CASP1 was normalized to ACTIN (n = 4–8) and CASP1 p20 was normalized on whole protein content determined by Ponceau S staining (n = 3). Values are displayed as fold changes compared to LPS/ATP-treated cells; Pro-CASP1: Kruskal–Wallis, Dunn´s multiple comparison; cleaved CASP1: one-way ANOVA, Dunnett’s post hoc test. f Neonatal microglia were stimulated as shown in a and MCC950 was added 15 min before addition of ATP. Cells were stained for ASC to visualize inflammasomes and with DAPI for nuclear staining. The percentage of ASC specks in respect to the number of total cells (DAPI positive cells) was determined (left). The IL-1β concentration in the cell supernatant was assessed by ELISA (right); n = 3; one-way ANOVA, Dunnett’s post hoc test. g Neonatal WT and Casp1^−/− microglia were stimulated as shown in a but with 4 mM ATP to increase the number of inflammasomes. Cells were stained for ASC (red) to visualize inflammasomes and with DAPI (blue) for nuclear staining as shown in the representative images (scale bar = 100 µm). Arrowheads highlight ASC specks within microglia. The percentage of ASC specks in respect to the number of total cells (DAPI positive cells) was determined (left). The IL-1β concentration in the cell supernatant was assessed by ELISA (right); WT: n = 8–16; Casp1^−/−: n = 3. Kruskal–Wallis, Dunn’s multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001