Figure 5.
Inflammatory milieu in SCD promotes caspase-4–dependent activation of neutrophil-GSDMD. (A) Experimental scheme: control or SCD human blood with or without preincubation (15 minutes) with 20 μM hemin or 20 μM hemin + 20 μM caspase-4 inhibitor (LEVD-CHO) or 20 μM hemin + 20 μM antioxidant NAC was used for neutrophil isolation. Neutrophil purity was confirmed (∼97%) using flow cytometry, and neutrophils were used in western blot analysis. Refer to supplemental methods for details. (B) Representative western blot micrograph shows both uncleaved GSDMD (53 kDa) and the cleaved GSDMD-NT (31 kDa) present in patient neutrophils with SCD but only uncleaved GSDMD present in control human neutrophils. (C-G) Densitometric analyses of western blot micrographs (representative example shown in panel B) revealed significantly higher (C) GSDMD in neutrophils isolated from untreated SCD than control human blood, (D) GSDMD in neutrophils isolated from hemin treated SCD than control human blood, (E) GSDMD-NT in neutrophils isolated from untreated SCD than control human blood, (F) GSDMD-NT in neutrophils isolated from hemin treated SCD than control human blood, and (G) GSDMD-NT in neutrophils isolated from hemin-treated than untreated blood of same patients with SCD. Data representative of 7 control and 6 SCD human subjects (C, D), 5 control and 6 SCD human subjects (E, F), and 6 SCD human subjects (G). (H) Representative western blot micrograph and (I-K) densitometric analyses show significantly higher levels of caspase-4 (45 kDa) in neutrophils isolated from (I) untreated SCD than control human blood, (J) hemin-treated SCD than control human blood, and (K) hemin-treated than hemin + NAC-treated blood of same patients with SCD. Data representative of 5 control and 6 SCD human subjects (I-J) and 6 SCD human subjects (K). (L) Representative western blot micrograph and (M-N) densitometric analyses show significantly higher GSDMD-NT (31 kDa) in neutrophils isolated from (M) hemin-treated than hemin + LEVD-CHO–treated blood of same patients with SCD and (N) hemin-treated than hemin + NAC–treated blood of same patients with SCD. Data in panels M-N representative of 6 SCD human subjects. Pre- and posttreatment data point of each SCD human subject connected by a straight line in panels G, K, M, and N. Each data point in panels C-F and I-J represents a separate human subject. Mean ± SE shown in panels C-F and I-J compared using Student t test. Data in panels G, K, and M-N compared using paired Student t test. *P < .05; **P < .01. glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 37 kDa) was used as the loading control. Uncropped images of western blot micrographs panels B, H, and L shown in supplemental Figures 18-20, respectively.

Inflammatory milieu in SCD promotes caspase-4–dependent activation of neutrophil-GSDMD. (A) Experimental scheme: control or SCD human blood with or without preincubation (15 minutes) with 20 μM hemin or 20 μM hemin + 20 μM caspase-4 inhibitor (LEVD-CHO) or 20 μM hemin + 20 μM antioxidant NAC was used for neutrophil isolation. Neutrophil purity was confirmed (∼97%) using flow cytometry, and neutrophils were used in western blot analysis. Refer to supplemental methods for details. (B) Representative western blot micrograph shows both uncleaved GSDMD (53 kDa) and the cleaved GSDMD-NT (31 kDa) present in patient neutrophils with SCD but only uncleaved GSDMD present in control human neutrophils. (C-G) Densitometric analyses of western blot micrographs (representative example shown in panel B) revealed significantly higher (C) GSDMD in neutrophils isolated from untreated SCD than control human blood, (D) GSDMD in neutrophils isolated from hemin treated SCD than control human blood, (E) GSDMD-NT in neutrophils isolated from untreated SCD than control human blood, (F) GSDMD-NT in neutrophils isolated from hemin treated SCD than control human blood, and (G) GSDMD-NT in neutrophils isolated from hemin-treated than untreated blood of same patients with SCD. Data representative of 7 control and 6 SCD human subjects (C, D), 5 control and 6 SCD human subjects (E, F), and 6 SCD human subjects (G). (H) Representative western blot micrograph and (I-K) densitometric analyses show significantly higher levels of caspase-4 (45 kDa) in neutrophils isolated from (I) untreated SCD than control human blood, (J) hemin-treated SCD than control human blood, and (K) hemin-treated than hemin + NAC-treated blood of same patients with SCD. Data representative of 5 control and 6 SCD human subjects (I-J) and 6 SCD human subjects (K). (L) Representative western blot micrograph and (M-N) densitometric analyses show significantly higher GSDMD-NT (31 kDa) in neutrophils isolated from (M) hemin-treated than hemin + LEVD-CHO–treated blood of same patients with SCD and (N) hemin-treated than hemin + NAC–treated blood of same patients with SCD. Data in panels M-N representative of 6 SCD human subjects. Pre- and posttreatment data point of each SCD human subject connected by a straight line in panels G, K, M, and N. Each data point in panels C-F and I-J represents a separate human subject. Mean ± SE shown in panels C-F and I-J compared using Student t test. Data in panels G, K, and M-N compared using paired Student t test. *P < .05; **P < .01. glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 37 kDa) was used as the loading control. Uncropped images of western blot micrographs panels B, H, and L shown in supplemental Figures 18-20, respectively.

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