Fig 1.

NLRP3 and caspase-1 activation, and IL-1β and IL-18 spontaneous secretion, by monocytes of patients with COVID-19. A, Above: ASC speck detection in monocytes from patients with severe COVID-19 by FACS. Representative dot plots of CD14⁺ monocytes from HDs (left), patients with COVID-19 (center), and HDs treated with LPS + ATP, used as a positive control. ASC specks are detected gating as ASC pulse width vs ASC pulse area. Percentages of ASC speck + cells are shown. Below: left, quantification of ASC speck⁺ CD14⁺ monocytes as identified by FACS; right, percentage of cells positive for active caspase-1 quantified by FACS, using FLICA-Caspase-1. B, Representative confocal images of NLRP3 and ASC speck formation in monocytes from HDs and patients with COVID-19. Images are a result of a 3-dimensional reconstruction of Z stack. Cells were stained with anti-ASC antibody (yellow), NLRP3 (green), and DAPI (blue). Scale bar is 5 μm. C,In vitro inflammatory cytokine secretion by monocytes as measured in culture supernatant after 18 hours with or without stimulation with the indicated stimuli. BF, Brightfield; DAPI, 4'-6-diamidino-2-phenylindole, dihydrochloride; FACS, fluorescence-activated cell sorting; FLICA, fluorochrome-labeled inhibitors of caspases; HD, healthy donor; IQR, interquartile range; MCC, MCC950 NLRP3 inhibitor. Data are expressed as median ± IQR. ∗P < .05, ∗∗P < .01 as assessed by Mann-Whitney t test.