Fig. 2.

Caspase-1 inhibition by VX-765 prevents inflammasome activation and pyroptosis in nigericin-exposed human microglia. Human microglia were exposed to nigericin (5 μM, 4 h) alone or following 4 h pretreatment with VX-765 (50 μM). (A and B) Control (A) and nigericin-exposed (B) microglia were immunolabeled for GSDMD (A, ii and B, ii) and IL-1β (A, iii and B, iii) and were labeled for F-actin (A, iv and B, iv). Nigericin-exposed microglia displayed the ring-of-fire phenotype characterized by a rounded morphology and the accumulation of GSDMD⁺/IL-1β⁺ pyroptotic bodies on the plasma membrane (arrows, B, i). (Scale bars, 23 μm.) (C) Cell size was quantified for 100–150 microglia per condition (control, CTRL, or nigericin-exposed, NIG). Data represent mean ± SEM and incorporate measurements from two biological donors. (D and E) IL-1β (D) and GSDMD (E) mean fluorescence intensity (MFI) was assessed. Data represent mean ± SEM and incorporate measurements from two biological donors. (F) To quantify the formation of the ring-of-fire phenotype, 100–150 microglia per condition were classified morphologically. Data represent the percentage of microglia demonstrating the phenotype and incorporate measurements from two biological donors (χ² test). (G and H) ELISA was used to assess IL-1β (G) and IL-18 (H) levels in supernatant from nigericin-exposed microglia. Data represent mean cytokine levels ± SEM (Student’s t test). (I) Loss of cell membrane integrity was assessed using an LDH activity assay (Student’s t test). LDH and ELISAs were performed with technical replicates of three to six wells per condition, and data were replicated in a minimum of three donor samples. *P < 0.05, **P < 0.01, ****P < 0.0001.