Figure 6. Neutrophils from septic... | American Society of Hematology

Figure 6.

$Neutrophils from septic patients who undergo NETosis express gasdermin D. (A) Gene set enrichment analysis identified “neutrophil degranulation,” “neutrophil-mediated immunity,” and “neutrophil activation involved in immune response gene sets” as the top 3 GO terms with the highest normalized enrichment score in nonsurvivor patients with septic shock at day 1 after ICU admission compared with healthy volunteers (adjusted P < .1). (B) Expression levels of Gsdmd in healthy volunteers and patients with septic shock (adjusted P < .1). (C) Heatmap representation of differentially expressed genes in surviving and nonsurviving patients with septic shock compared with healthy volunteers at days 1, 2, and 3 after ICU admission (adjusted P < .1). (D) Circulating amounts of MPO/DNA-NETs were quantified from the plasma of patients with sepsis and healthy volunteers. (E) Neutrophils from patients with sepsis and healthy volunteers were isolated and cultured for 4 hours at 37°C. Representative immunostaining images for DNA (DAPI, gray), myeloperoxidase (MPO, green), and the gasdermin D cleaved fraction (GSDMD, red) are shown. Scale bar, 50 µm at ×630 magnification. (F) GSDMD expression was quantified by Median Fluorescence Intensity (MFI) per field. (G) Magnified fluorescence microscopy images of panel E for DNA (DAPI, gray) and the gasdermin D cleaved fraction (GSDMD, red) are shown. Scale bar, 50 µm at ×630 magnification. Furthermore, to demonstrate the details of GSDMD in neutrophil membranes and its association with NETs (G, top), we performed a 4× digital zoom of the inset square (G, bottom). (H) Neutrophils from patients with sepsis and healthy volunteers were isolated, treated with disulfiram (30 µM) or vehicle, and cultured for 4 hours at 37°C. Representative fluorescence images of NETs stained for DNA (DAPI, gray), myeloperoxidase (MPO, green), and the gasdermin D cleaved fraction (GSDMD, red) are shown. Scale bar, 50 µm at ×630 magnification. (I) The concentrations of MPO/DNA-NETs in the neutrophil culture supernatant after 4 hours were determined using the picogreen test. The data are expressed as means ± SEM. *P < .05; Student t test (D), 1-way ANOVA, followed by Tukey’s test (I). The data are representative of ≥2 independent experiments.$

Neutrophils from septic patients who undergo NETosis express gasdermin D. (A) Gene set enrichment analysis identified “neutrophil degranulation,” “neutrophil-mediated immunity,” and “neutrophil activation involved in immune response gene sets” as the top 3 GO terms with the highest normalized enrichment score in nonsurvivor patients with septic shock at day 1 after ICU admission compared with healthy volunteers (adjusted P < .1). (B) Expression levels of Gsdmd in healthy volunteers and patients with septic shock (adjusted P < .1). (C) Heatmap representation of differentially expressed genes in surviving and nonsurviving patients with septic shock compared with healthy volunteers at days 1, 2, and 3 after ICU admission (adjusted P < .1). (D) Circulating amounts of MPO/DNA-NETs were quantified from the plasma of patients with sepsis and healthy volunteers. (E) Neutrophils from patients with sepsis and healthy volunteers were isolated and cultured for 4 hours at 37°C. Representative immunostaining images for DNA (DAPI, gray), myeloperoxidase (MPO, green), and the gasdermin D cleaved fraction (GSDMD, red) are shown. Scale bar, 50 µm at ×630 magnification. (F) GSDMD expression was quantified by Median Fluorescence Intensity (MFI) per field. (G) Magnified fluorescence microscopy images of panel E for DNA (DAPI, gray) and the gasdermin D cleaved fraction (GSDMD, red) are shown. Scale bar, 50 µm at ×630 magnification. Furthermore, to demonstrate the details of GSDMD in neutrophil membranes and its association with NETs (G, top), we performed a 4× digital zoom of the inset square (G, bottom). (H) Neutrophils from patients with sepsis and healthy volunteers were isolated, treated with disulfiram (30 µM) or vehicle, and cultured for 4 hours at 37°C. Representative fluorescence images of NETs stained for DNA (DAPI, gray), myeloperoxidase (MPO, green), and the gasdermin D cleaved fraction (GSDMD, red) are shown. Scale bar, 50 µm at ×630 magnification. (I) The concentrations of MPO/DNA-NETs in the neutrophil culture supernatant after 4 hours were determined using the picogreen test. The data are expressed as means ± SEM. *P < .05; Student t test (D), 1-way ANOVA, followed by Tukey’s test (I). The data are representative of ≥2 independent experiments.

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