Fig. 2 |. Monocytes of patients with COVID-19 have activated inflammasomes, caspase-1 and GSDMD.

Monocytes from HDs, non-COVID-19 patients or patients with COVID-19 at the time of presentation were analysed by imaging flow cytometry for ASC, GSDMD, caspase-1 activation (FLICA) and/or Zombie dye uptake. a–c, The percentage of monocytes with activated ASC (a) or caspase-1 (b) (n = 8 (HD), n = 5 (non-COVID-19), n = 10 (COVID-19)) or colocalized ASC/caspase-1 specks (c) (n = 8 (HD), n = 4 (non-COVID-19), n = 8 (COVID-19)) (c). Representative images (top) and quantification of all samples (bottom) are shown. d, The percentage of ASC-speck-containing monocytes with colocalized activated caspase-1, NLRP3, AIM2 or pyrin specks. n = 6. e, f, Representative images of ASC (e) or Zombie dye (f) and GSDMD co-stained monocytes. n = 4 independent experiments. g, Lysates of purified monocytes of HDs and patients with COVID-19, and of LPS- and nigericin-treated monocytes of HDs (+) probed with a monoclonal antibody that recognizes full length GSDMD (GSDMD-FL) and the C-terminal of GSDMD (GSDMD-CT) (top), β-actin (middle) and COX-IV (bottom). Representative of n = 4 independent experiments. h, i, Representative images of ASC co-staining with NLRP3 (left; n = 5 (HD), n = 4 (non-COVID-19), n = 6 (COVID-19)), AIM2 (middle; n = 4 (HD), n = 3 (non-COVID-19), n = 4 (COVID-19)) and pyrin (right; n = 4 (HD), n = 4 (non-COVID-19), n = 5 (COVID-19)) (h), and quantification of monocytes showing ASC specks colocalized with the indicated inflammasomes (i). j, Representative images of co-staining of ASC, NLRP3 and AIM2. n = 3 independent experiments. For a–c, e, f, h, j, scale bars, 7 μm. For a–d, i, data are mean ± s.e.m. Statistical analysis was performed using one-way ANOVA with Tukey multiple-comparisons test (a–d) and two-way ANOVA with Tukey multiple-comparisons test (i); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.