Figure 4.
IFN-I signaling, caspase-11, and GSDMD are activated in neutrophils of mice with SCD. (A) Experimental scheme: control or mice with SCD were IV administered 10 µmol/kg oxy-Hb; venous blood was collected 3 hours later, and neutrophils were isolated from blood using a negative selection approach. Purity was confirmed (>98%) using flow cytometry, and neutrophils were used in messenger RNA (B-H) or western blot analysis (I-N). Refer to supplemental methods for details. (B) Heat map showing relative gene expression of the type I IFN-I pathway components, IFN-stimulated genes (ISGs) and NETs-related genes in SCD or control mice neutrophils. ISGs marked by grey squares are significantly altered between SCD and control mice. The data are presented as Log2-Fold change (relative expression) for 3 control and 3 mice with SCD. Each column reflects a single mouse. Significantly altered genes marked by asterisk in B. Log2-fold changes (relative expression) in panels (C) Ifna (IFN-α), (D) Ifnb1 (IFN-β), (E) Ifnar1 (IFN-α receptor 1 subunit), (F) Tyk2 (tyrosine kinase 2), (G) Casp11 (caspase-11), and (H) Gsdmd (GSDMD) genes were higher by several-fold in neutrophils of mice with SCD (n = 3) compared with control mice (n = 3) IV administered oxy-Hb. (I) Representative western blot micrograph showing presence of both uncleaved (45 kDa) and cleaved (20 and 25 kDa) caspase-11 in neutrophils of mice with SCD IV administered oxy-Hb. The expressions of both cleaved and uncleaved caspase-11 were below the detection limit in neutrophils of control mice IV administered oxy-Hb. (J-K) Densitometric analyses of western blot micrographs revealed significantly higher (J) uncleaved and (K) cleaved caspase-11 in neutrophils of SCD (n = 4) mice than control (n = 3) mice administered IV oxy-Hb. (L) Representative western blot micrograph showing presence of both uncleaved GSDMD (50 kDa) and cleaved GSDMD-NT (30 kDa) in neutrophils of mice with SCD IV administered oxy-Hb. The expressions of both GSDMD and GSDMD-NT were below the detection limit in neutrophils of control mice IV administered oxy-Hb. (M-N) Densitometric analyses of western blot micrographs revealed significantly higher (M) uncleaved GSDMD and (N) cleaved GSDMD-NT in neutrophils of SCD (n = 4) mice than control (n = 4) mice administered IV oxy-Hb. Data represent mean ± SE. *P < .05; **P < .01. Means compared using the Student t test. β-Tubulin (50 kDa) was the loading control protein. Uncropped images of western blot micrographs I and L shown in supplemental Figure 15A-B, respectively.

IFN-I signaling, caspase-11, and GSDMD are activated in neutrophils of mice with SCD. (A) Experimental scheme: control or mice with SCD were IV administered 10 µmol/kg oxy-Hb; venous blood was collected 3 hours later, and neutrophils were isolated from blood using a negative selection approach. Purity was confirmed (>98%) using flow cytometry, and neutrophils were used in messenger RNA (B-H) or western blot analysis (I-N). Refer to supplemental methods for details. (B) Heat map showing relative gene expression of the type I IFN-I pathway components, IFN-stimulated genes (ISGs) and NETs-related genes in SCD or control mice neutrophils. ISGs marked by grey squares are significantly altered between SCD and control mice. The data are presented as Log2-Fold change (relative expression) for 3 control and 3 mice with SCD. Each column reflects a single mouse. Significantly altered genes marked by asterisk in B. Log2-fold changes (relative expression) in panels (C) Ifna (IFN-α), (D) Ifnb1 (IFN-β), (E) Ifnar1 (IFN-α receptor 1 subunit), (F) Tyk2 (tyrosine kinase 2), (G) Casp11 (caspase-11), and (H) Gsdmd (GSDMD) genes were higher by several-fold in neutrophils of mice with SCD (n = 3) compared with control mice (n = 3) IV administered oxy-Hb. (I) Representative western blot micrograph showing presence of both uncleaved (45 kDa) and cleaved (20 and 25 kDa) caspase-11 in neutrophils of mice with SCD IV administered oxy-Hb. The expressions of both cleaved and uncleaved caspase-11 were below the detection limit in neutrophils of control mice IV administered oxy-Hb. (J-K) Densitometric analyses of western blot micrographs revealed significantly higher (J) uncleaved and (K) cleaved caspase-11 in neutrophils of SCD (n = 4) mice than control (n = 3) mice administered IV oxy-Hb. (L) Representative western blot micrograph showing presence of both uncleaved GSDMD (50 kDa) and cleaved GSDMD-NT (30 kDa) in neutrophils of mice with SCD IV administered oxy-Hb. The expressions of both GSDMD and GSDMD-NT were below the detection limit in neutrophils of control mice IV administered oxy-Hb. (M-N) Densitometric analyses of western blot micrographs revealed significantly higher (M) uncleaved GSDMD and (N) cleaved GSDMD-NT in neutrophils of SCD (n = 4) mice than control (n = 4) mice administered IV oxy-Hb. Data represent mean ± SE. *P < .05; **P < .01. Means compared using the Student t test. β-Tubulin (50 kDa) was the loading control protein. Uncropped images of western blot micrographs I and L shown in supplemental Figure 15A-B, respectively.

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