Fig. 6: LPS alone is sufficient to trigger GSDMD cleavage and rapid IL-1β secretion in monocytes from CAPS patients
a Intracellular ROS accumulation was quantified by H2DCF-DA staining in unstimulated monocytes (Mo) from two healthy donors (Ctrl Mo) and two CAPS patients. The data are expressed as relative fluorescence units (RFU), and confirmed previous data obtained on a larger number of CAPS and control monocytes33,34. b, c Kinetics of IL-1β secretion in LPS or LRZ stimulated monocytes from healthy donors (Ctrl Mo) (b) or patients (CAPS Mo) (c) quantified by ELISA. Average of 5 experiments ± SEM. d Accumulation of pro-IL-1β in monocytes from CAPS patients or healthy donors. A representative Western blot out of 3 performed is shown. e Confocal [upper panel, one single stack of Z-stack series is shown (Z = 6)] and TIRF (lower panel, penetration depth = 110 nm) microscopy analyses of monocytes from a representative CAPS patient (out of three examined) stimulated for 5 h with LPS. Scale bar, 5μm. f IL-1β secreted by LPS-stimulated monocytes from CAPS patients, incubated in the absence or presence of latrunculin B (Lat), punicalagin (Pun), Ac-YVAD or Z-LEVD as indicated, was quantified by ELISA. Data are expressed as percent of IL-1β secretion relative to untreated cells. N = 6, mean ± SEM. g Live cell microscopy images of monocytes from healthy donors (Ctrl Mo) or CAPS patients (CAPS Mo) exposed for 6 h to LPS or LRZ as indicated. CAPS monocytes stimulated with LRZ died in the first 3 h from stimulations and detached from the well (not shown). N = 3 h This representative Western Blot (out of 3 performed) compares GSDMD cleavage and total levels in cell monocytes from CAPS patients or healthy donors, after 5 h with or without LPS. Data information: In a, f Unpaired t-test was performed, and the significance compared with untreated cells is indicated (*P < 0.05, **P < 0.01)
