Figure 5.
DSF inhibits CC–induced neutrophil pyroptosis in human neutrophils. (A) Human neutrophils were primed with LPS for 2 hours, then stimulated with CC or nigericin (10 μM) for 3 hours under shaking conditions. Cell-free supernatants were collected for IL-1β ELISA. (B) Human neutrophils were primed with LPS for 2 hours and stimulated with or without CC (1 mg/mL) for 3 hours under shaking conditions. Cell-free supernatants and cell lysates from the 3 wells were combined for each condition and collected for immunoblot analysis of GSDMD and IL-1β. β-actin was used as a loading control. (C-D) Human neutrophils were primed with LPS for 2 hours. After pretreatment with DSF, VX-765, necrostatin-1s (Nec-1s), and necrosulfonamide (NSA), cells were stimulated with CC (1 mg/mL) for 3 hours under shaking conditions. Cell-free supernatants were collected for the IL-1β ELISA (C) and lactate dehydrogenase (LDH) assay (D). (E) Representative immunofluorescent and live images of NETs. Human neutrophils were incubated on CC-precoated slides for 4 hours, with or without DSF or dimethyl sulfoxide (DMSO) treatment. NETs were visualized by immunofluorescent images, stained with CitH3 (green), myeloperoxidase (MPO, red), and DAPI (blue), and live cell images were stained with SG (green). (F) Human neutrophils were primed with CXCL8. After pretreatment with DSF, diphenyleneiodonium chloride (DPI), or DMSO, cells were stimulated with CC (0.6 mg/mL) for 3 hours under shaking conditions. Cells were stained with SG for flow cytometric analysis. (G) Representative histogram of SG-positive neutrophils. (H) Heparinized whole blood samples from WT mice were perfused over the collagen-coated surface at 1000 s−1 in the presence or absence of CC using a flow chamber system. In the second-step flow chamber assay, RedDeep Tracker-fluorescently labeled WT and Gsdmd−/− bone marrow neutrophils were perfused over platelet-rich thrombi through the chamber at 500 s−1. (I) Platelets and neutrophils were stained with anti-CD41 (green), RedDeep (cyan), and CitH3 (red) antibodies, and visualized using immunofluorescence confocal microscopy. Nuclei were stained with DAPI (blue). (J-K) Quantification of the ratio between CD41 (platelet marker) and RedDeep-neutrophils (J) and CitH3 signals (K) (n = 4 per group). Scale bars: for panel E, 20 μm (immunofluorescent images) and 100 μm (live images), for panel I, 10 μm. The data represent mean ± standard error of the mean (SEM) for panels A,C-D,F or SD for panels J-K. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 using 1-way ANOVA with Dunnett multiple comparisons test for panels A,C-D,F), or 2-way ANOVA with Bonferroni multiple comparisons test for panels J-K. Data are representative of at least 2 independent experiments.

DSF inhibits CC–induced neutrophil pyroptosis in human neutrophils. (A) Human neutrophils were primed with LPS for 2 hours, then stimulated with CC or nigericin (10 μM) for 3 hours under shaking conditions. Cell-free supernatants were collected for IL-1β ELISA. (B) Human neutrophils were primed with LPS for 2 hours and stimulated with or without CC (1 mg/mL) for 3 hours under shaking conditions. Cell-free supernatants and cell lysates from the 3 wells were combined for each condition and collected for immunoblot analysis of GSDMD and IL-1β. β-actin was used as a loading control. (C-D) Human neutrophils were primed with LPS for 2 hours. After pretreatment with DSF, VX-765, necrostatin-1s (Nec-1s), and necrosulfonamide (NSA), cells were stimulated with CC (1 mg/mL) for 3 hours under shaking conditions. Cell-free supernatants were collected for the IL-1β ELISA (C) and lactate dehydrogenase (LDH) assay (D). (E) Representative immunofluorescent and live images of NETs. Human neutrophils were incubated on CC-precoated slides for 4 hours, with or without DSF or dimethyl sulfoxide (DMSO) treatment. NETs were visualized by immunofluorescent images, stained with CitH3 (green), myeloperoxidase (MPO, red), and DAPI (blue), and live cell images were stained with SG (green). (F) Human neutrophils were primed with CXCL8. After pretreatment with DSF, diphenyleneiodonium chloride (DPI), or DMSO, cells were stimulated with CC (0.6 mg/mL) for 3 hours under shaking conditions. Cells were stained with SG for flow cytometric analysis. (G) Representative histogram of SG-positive neutrophils. (H) Heparinized whole blood samples from WT mice were perfused over the collagen-coated surface at 1000 s−1 in the presence or absence of CC using a flow chamber system. In the second-step flow chamber assay, RedDeep Tracker-fluorescently labeled WT and Gsdmd−/− bone marrow neutrophils were perfused over platelet-rich thrombi through the chamber at 500 s−1. (I) Platelets and neutrophils were stained with anti-CD41 (green), RedDeep (cyan), and CitH3 (red) antibodies, and visualized using immunofluorescence confocal microscopy. Nuclei were stained with DAPI (blue). (J-K) Quantification of the ratio between CD41 (platelet marker) and RedDeep-neutrophils (J) and CitH3 signals (K) (n = 4 per group). Scale bars: for panel E, 20 μm (immunofluorescent images) and 100 μm (live images), for panel I, 10 μm. The data represent mean ± standard error of the mean (SEM) for panels A,C-D,F or SD for panels J-K. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 using 1-way ANOVA with Dunnett multiple comparisons test for panels A,C-D,F), or 2-way ANOVA with Bonferroni multiple comparisons test for panels J-K. Data are representative of at least 2 independent experiments.

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