Fig 2: SYK and caspase-8 activity are important for CARD9 regulation of NLRP3 activity in BMDMs, but not in BMDCs.(a–d) Effects of caspase-8 inhibition by Z-IETD-FMK on LPS-primed WT and Card9-/- LPS-primed BMDMs on cellular viability (a,c) and IL-1ß production (b,d) during S. Typhimurium infection (m.o.i. 10, 1 h) (a,b) or nigericin (10 µM, 1 h) stimulation (c,d). (e–h) Effects of SYK inhibition by R406 on WT and Card9-/- LPS-primed BMDMs on cellular viability (e,g) and IL-1ß secretion (f,h) in response to S. Typhimurium infection (m.o.i. 10, 1 h) (e,f) or nigericin stimulation (5 µM, 1 h) (g,h). (i,j) Stimulation of WT or Card9-/- BMDCs with nigericin (5 µM, 1 h). Cellular viability (i) and IL-1ß secretion (j). *P<0.05 (one-way analysis of variance with Tukey's multiple comparisons test. (a–j) Data from three independent experiments (mean and s.e.m.).

Fig 3: CARD9 selectively negatively regulates NLRP3-induced IL-1ß production.(a,b,e,f) Cellular viability and (c,d,g,h) IL-1ß secretion from unprimed WT and Card9-/- BMDMs infected with S. Typhimurium at m.o.i. 10 for 2 (a,c,e,g) and 6 (b,d,f,h) h in presence or absence of NLRP3 inhibitors glibenclamide (a–d) and MCC950 (e–h). (i,j) Cellular viability (i) and IL-1ß secretion (j) from LPS-primed BMDMs after nigericin stimulation (10 µM, 1 h). IL-1ß from LPS-primed cells without nigericin stimulation was below the level of detection. (k,l) Cellular viability (k) and IL-1ß secretion (l) from LPS-primed BMDMs after ATP stimulation (5 mM, 30 min). *P<0.05 in comparison with WT (one-way analysis of variance with Tukey's multiple comparisons test). (a–l) Data from three independent experiments (mean and s.e.m.).

Fig 4: CARD9 acts as a regulatory switch during infection.(a) Immunoblotting of CARD9 and ASC in cell lysates from unprimed WT BMDMs after infection with S. Typhimurium (m.o.i. 5) for 0, 30, 60 and 120 min. (b) Immunoblotting of CARD9 and ASC in cell lysates from unprimed BMDMs (WT, Card9-/- or Pycard-/-) after infection with S. Typhimurium (m.o.i. 5) for 0, 2 and 6 h. (c) Immunoblotting of CARD9 and ASC in cell lysates from uninfected, S. Typhimurium-infected (m.o.i. 5 for 2 h) or nigericin-stimulated (5 µM, 30 min) LPS-primed BMDMs (WT, Card9-/- or Pycard-/-). (a–c) Images are representative of three independent experiments.

Fig 5: CARD9 does not control IL-1ß produced via NLRC4.(a) IL-1ß production from LPS-primed BMDMs after transfection with ultrapure flagellin. (b–d) Cellular viability and (e–g) IL-1ß production from unprimed BMDMs after infection with S. Typhimurium WT or ?fliC?fljB?prgJ (deficient in NLRC4 activation) at an m.o.i. 10 for 2 (b,e), 6 (c,f) and 24 (d,g) h. *P<0.05 in comparison with WT (one-way analysis of variance with Tukey's multiple comparisons test). (a–g) Data from three independent experiments (mean and s.e.m.).