Fig 2: Inhibition of Syk protects Aβ42‐induced inflammasome activation in LPS‐primed microglia. LPS‐primed microglia were pretreated with Syk inhibitor Bay 61‐3606 for 1 h followed by treatment with Aβ42 for 24 h. (a–e) Representative image (a) and quantification (b–e) of Western blot for the pSyk, Syk, pASC, ASC, pro‐IL‐1β, IL‐18, and β‐actin. (f–h) mRNA expressions of NLRP3 (f), IL‐1β (g), and IL‐18 (h) were analyzed using real‐time PCR. 18S rRNA was used for normalization. (i) Representative immunofluorescence image of primary microglia with anti‐caspase‐1 (green). DAPI represents the nuclear signal (blue). Scale bar: 20 μm. (j) Quantification of immunofluorescence of caspase‐1. (k) Immunofluorescence staining for ASC (red) speck. Scale bar: 10 μm. (l) Percentages of microglia containing ASC foci. (m) Immunoblot analysis of indicated proteins in cell lysates. (n–o) Quantification of pAMPK (n) and pAKT (o) normalized by total AMPK and AKT, respectively. (p) Relative phagocytic activity of microglia measured by bead uptake assay. All data are presented as mean ± SEM from at least four independent experiments and analyzed by one‐way ANOVA and Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig 3: SYK activity increases the number of caspase-8 specks in response to NLRP3 stimulation in Card9−/− BMDMs.(a–c) WT, Card9−/− or Pycard−/− LPS-primed BMDMs were incubated with nigericin (5 μM, 30 min) in the presence of caspase-1 and caspase-8 FLICA substrates, with or without the SYK inhibitor R406. (a) Pattern of caspase-1 and caspase-8 specks observed by immunofluorescence labelling of the protein. Percentage of cells containing (b) caspase-8 or (c) caspase-1 specks. *P<0.05 (one-way analysis of variance with Tukey's multiple comparisons test). (a) Image is representative of three independent experiments. (b,c) Data from three independent experiments (mean and s.e.m.). Scale bar, 10 μm.

Fig 4: CARD9 and SYK regulates NLRP3 activation upstream to speck formation.(a) CARD9 and ASC were immuno-labelled in WT, Card9−/− and Pycard−/− LPS-primed macrophages after nigericin stimulation (5 μM, 30 min). Green arrows indicate CARD9 aggregates. Red arrows indicate ASC specks. (b) Co-immunoprecipitation of ASC, CARD9 and SYK from cell lysates of uninfected, S. Typhimurium-infected (m.o.i. 10, 30 min) or nigericin-stimulated (10 μM, 30 min) LPS-primed BMDMs. (c) Immunoblot analysis of LPS-primed WT and Card9−/− uninfected, S. Typhimurium-infected (m.o.i. 10, 2 h) or nigericin-stimulated (10 μM, 30 min) BMDMs. (a–c) Images are representative of three independent experiments. Scale bar, 10 μm.

Fig 5: CARD9 modulates pro-IL-1β expression.(a–f) Quantitative PCR analysis of WT and Card9−/− BMDMs infected with S. Typhimurium (m.o.i. 5) for 2 h, compared with uninfected controls. (a) Pro-IL-1β, (b) NLRP3, (c) TNF-α, (d) RANTES, (e) caspase-8 and (f) SYK. (g) Expression of pro-IL-1β, SYK, pro-caspase-8, pro-caspase-1, CARD9, ASC, β-actin in cell lysates, and caspase-1 p10, caspase-8 p18 and IL-1β in culture supernatants from WT, Card9−/− and Pycard−/− BMDMs after 2 or 6 h of infection with S. Typhimurium (m.o.i. 5) and relative densitometry (Card9−/−/WT) in culture supernatants (h) or cell lysates (Card9−/−/WT, actin-normalized) (i). *P<0.05 in comparison with uninfected control, unless stated in the graph (one-way analysis of variance with Tukey's multiple comparisons test). (a–f,h,i) Data from three independent experiments (mean and s.e.m.). (g) Image is representative of three independent experiments.