Figure 3.

Neutrophil function in D3KO mice. (A) Hematopoietic bone marrow populations in D3KO mice (n = 6) and WT littermates (n = 7) quantified using flow cytometry. Percentage of total cells is shown for early hematopoietic blast cells, neutrophil precursors (PMN prec), monocyte precursors (mono prec), monocytes (mono), and neutrophils (PMN). See Supplemental Fig. 1 ^{(620KB, pdf)} for gating. Neutrophils were quantified in venous blood derived from the cava vein of D3KO (n = 6 male, n = 5 female) and WT (n = 7 male, n = 3 female) mice. (B) Ex vivo survival of neutrophils incubated at 37°C. Samples were stained for Annexin V and propidium iodide (PI). Percentage of healthy cells (Annexin V⁻/PI⁻) and apoptotic cells (Annexin V⁺) are shown (n = 3 to 4 mice per genotype per time point). (C) Neutrophils (D3KO, n = 7; WT, n = 5) were incubated with zymosan fluorescently labeled with pHrodo green (MOI = 5) for 1 hour at 37°C or on ice. pHrodo+ cells (see Supplemental Fig. 2 ^{(620KB, pdf)} for gating) were considered phagocytosing neutrophils. MFI, median fluorescence intensity. (D) D3KO and WT neutrophils were incubated with PMA. Fluorescence, indicating H₂O₂ release, was measured. The effect of D3KO on NADPH oxidase activity was present only in cells from female mice (D3KO, n = 5; WT, n = 3) and was not observed in cells from male mice (D3KO, n = 5; WT, n = 4). Area under the curve (AUC) was analyzed using the unpaired two-tailed Student t test (*P < 0.05). RFU, relative fluorescent unit. (A–D) All data represent mean ± standard error of the mean. The n values indicate number of animals used. Assays were performed in triplicate. Experiments were repeated independently two to four times. P values for two-way analysis of variance are indicated. Post hoc analysis (Bonferroni) P values: *P < 0.05, **P < 0.01, ***P < 0.001. PMN, polymorphonuclear leukocytes.