Figure 1.
GSDMD is expressed in neutrophils during their maturation and is activated upon focal crystalline TMA. (A) Illustration of the study design presented in the published data set. Cells were sorted from the kidney, blood, and spleen of C57BL/6J mice before (D0) and on days 1 (D1) and 3 (D3) after unilateral ischemia-reperfusion injury (uIRI). (B) Uniform manifold approximation and projection (UMAP) plot of 80 829 cells after quality control. Clusters 3 and 15 were identified as neutrophils based on the representative neutrophil gene expression. (C) UMAP plot of 7467 neutrophils categorized into 8 clusters (G0-4 and G5a-c) representing different maturation stages. Clusters G0, G1, G2, G3, and G4 corresponded to granulocyte-monocyte progenitor, proneutrophil, preneutrophil, immature neutrophil, and mature neutrophil in bone marrow. The names, proneutrophil, preneutrophil, immature neutrophil, and mature neutrophil, were adopted from the previous report.20 Clusters G5a-c represent the most mature neutrophils present in peripheral blood. (D) Dot plot representing the expression profile of Gsdmd in each cluster. The dot color indicates the average gene expression level in each cluster, whereas the dot size represents the percentage of cells in each cluster. (E) UMAP plot of Gsdmd expression in neutrophils from different organs. (F) Representative MACSima images of CC-induced TMA and contralateral sham kidneys from WT mice. High-magnification views 1, 2, and 4 of the TMA kidney represents intravascular thrombotic occlusion with platelets and NETs, whereas high-magnification view 3 represents neutrophil accumulation in the peri-infarct region. Gray: DAPI (4’,6-diamidino-2-phenylindole), red: Ly6G, yellow: CitH3, purple: smooth muscle actin (SMA), blue: cleaved caspase-3, cyan: Ly6C (in the sham kidney and low magnification of TMA kidney) or CD41 (in high-magnification views 1-4 of TMA kidney). (G) Representative immunofluorescent images of thrombotic occlusion of the kidney artery. TER-119–positive erythrocytes (red) and CD41-positive platelets (green) within αSMA-positive arteries (cyan) in TMA and sham kidneys of WT mice. The nuclei were visualized using DAPI (blue). (H) Representative immunohistochemical images of GSDMD staining of TMA and sham kidneys from WT and Gsdmd−/− mice. (I) Immunoblot analysis of GSDMD (pro and cleaved p30 [N-terminal fragment]) and caspase-1 (pro and cleaved p20) in WT kidneys of 2 sets of healthy control (HC) and focal TMA mice. β-actin was used as a loading control. (J) Immunoblot analysis of GSDMD in immune cells isolated from TMA and healthy kidneys of WT mice. Immune cells from 4 kidneys per group were pooled and analyzed. β-actin was used as a loading control. (K) Flow cytometric quantification of caspase1 activation within neutrophils (CD45+ CD11b+ Ly6G+) in TMA and sham kidneys from WT mice. (L) Representative histogram of caspase1 activation in neutrophils from the kidneys. Scale bars: for panel F, 1000 μm in the sham kidney and low magnification of the TMA kidney or 50 μm in high-magnification views 1 to 4 of the TMA kidney. For panels G-H at 20 μm. The data represent mean ± standard deviation (SD). ∗∗∗P < .001 using unpaired Student t test.

GSDMD is expressed in neutrophils during their maturation and is activated upon focal crystalline TMA. (A) Illustration of the study design presented in the published data set. Cells were sorted from the kidney, blood, and spleen of C57BL/6J mice before (D0) and on days 1 (D1) and 3 (D3) after unilateral ischemia-reperfusion injury (uIRI). (B) Uniform manifold approximation and projection (UMAP) plot of 80 829 cells after quality control. Clusters 3 and 15 were identified as neutrophils based on the representative neutrophil gene expression. (C) UMAP plot of 7467 neutrophils categorized into 8 clusters (G0-4 and G5a-c) representing different maturation stages. Clusters G0, G1, G2, G3, and G4 corresponded to granulocyte-monocyte progenitor, proneutrophil, preneutrophil, immature neutrophil, and mature neutrophil in bone marrow. The names, proneutrophil, preneutrophil, immature neutrophil, and mature neutrophil, were adopted from the previous report.20 Clusters G5a-c represent the most mature neutrophils present in peripheral blood. (D) Dot plot representing the expression profile of Gsdmd in each cluster. The dot color indicates the average gene expression level in each cluster, whereas the dot size represents the percentage of cells in each cluster. (E) UMAP plot of Gsdmd expression in neutrophils from different organs. (F) Representative MACSima images of CC-induced TMA and contralateral sham kidneys from WT mice. High-magnification views 1, 2, and 4 of the TMA kidney represents intravascular thrombotic occlusion with platelets and NETs, whereas high-magnification view 3 represents neutrophil accumulation in the peri-infarct region. Gray: DAPI (4’,6-diamidino-2-phenylindole), red: Ly6G, yellow: CitH3, purple: smooth muscle actin (SMA), blue: cleaved caspase-3, cyan: Ly6C (in the sham kidney and low magnification of TMA kidney) or CD41 (in high-magnification views 1-4 of TMA kidney). (G) Representative immunofluorescent images of thrombotic occlusion of the kidney artery. TER-119–positive erythrocytes (red) and CD41-positive platelets (green) within αSMA-positive arteries (cyan) in TMA and sham kidneys of WT mice. The nuclei were visualized using DAPI (blue). (H) Representative immunohistochemical images of GSDMD staining of TMA and sham kidneys from WT and Gsdmd−/− mice. (I) Immunoblot analysis of GSDMD (pro and cleaved p30 [N-terminal fragment]) and caspase-1 (pro and cleaved p20) in WT kidneys of 2 sets of healthy control (HC) and focal TMA mice. β-actin was used as a loading control. (J) Immunoblot analysis of GSDMD in immune cells isolated from TMA and healthy kidneys of WT mice. Immune cells from 4 kidneys per group were pooled and analyzed. β-actin was used as a loading control. (K) Flow cytometric quantification of caspase1 activation within neutrophils (CD45+ CD11b+ Ly6G+) in TMA and sham kidneys from WT mice. (L) Representative histogram of caspase1 activation in neutrophils from the kidneys. Scale bars: for panel F, 1000 μm in the sham kidney and low magnification of the TMA kidney or 50 μm in high-magnification views 1 to 4 of the TMA kidney. For panels G-H at 20 μm. The data represent mean ± standard deviation (SD). ∗∗∗P < .001 using unpaired Student t test.

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