Neutrophil recruitment and maturation are impaired in Gsdmd^−/− mice with focal TMA. (A) Representative gating of flow cytometric analysis for the quantification of neutrophils (CD45⁺ CD11b⁺ Ly6G⁺) and monocytes (CD45⁺ CD11b⁺ Ly6C⁺) in blood and kidney from WT and knockout (Gsdmd^−/−) mice with focal TMA (24 hours). (B-C) Percentage of neutrophils among CD45+ cells (B) and their absolute number (C) in the blood from healthy mice (n = 6) and focal TMA mice (WT: n = 8, Gsdmd^−/−: n = 8). (D-E) Percentage of neutrophils among CD45⁺ cells (D) and their absolute number (E) in sham (n = 4) and TMA kidneys from WT (n = 12) and Gsdmd^−/− (n = 11) mice. (F) Representative gating of flow cytometric analysis for the quantification of mature (CD45⁺ CD11b⁺ Ly6G⁺ CD101⁺) and immature (CD45⁺ CD11b⁺ Ly6G⁺ CD101⁻) neutrophils in the bone marrow, spleen, and blood from WT and Gsdmd^−/− mice with focal TMA. (G-I) Percentage of mature neutrophils among CD45⁺ CD11b⁺ Ly6G⁺ neutrophils in the bone marrow (G), spleen (H), and blood (I) from healthy mice (n = 9) and focal TMA mice (WT: n = 4, Gsdmd^−/−: n = 3). (J-L) Percentage of mature and immature neutrophils among CD45⁺ cells in the bone marrow (J), spleen (K), and blood (L) from healthy mice (n = 9) and focal TMA mice (WT: n = 4, Gsdmd^−/−: n = 3). (M) Bone marrow cells isolated from WT and Gsdmd^−/− healthy mice were incubated for 24 hours with or without granulocyte colony–stimulating factor (G-CSF; 100 ng/mL) or tumor necrosis factor alpha (TNFα; 20 ng/mL). The expression levels of CXCR2 in mature neutrophils (Ly6G⁺ CD101⁺) were quantified as mean fluorescence intensity (MFI) using flow cytometry. Data are representative of 3 independent experiments. (N) Expression levels of β₂ integrin MAC-1 in bone marrow neutrophils from healthy mice (n = 5) and focal TMA mice (WT: n = 8, Gsdmd^−/−: n = 7), shown as MFI quantified by flow cytometry. The data represent mean ± SD. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 using 1-way ANOVA with Tukey post hoc test for panels B-E,G-I,N or 2-way ANOVA with Bonferroni multiple comparisons test for panels J-M.