Figure 2.

NETosis in response to Candida albicans hyphae is independent of Peptidylarginine deiminase 4 (PAD4). (A) The release of extracellular DNA from murine bone marrow neutrophils was detected by Sytox green after stimulation for 2.5 h with the yeast-locked strain Δhgc1 (C.a. yeast), preformed hyphae of the control strain Δhgc1 + HGC1 (C.a. hyphae), or ionomycin as indicated. The increase in fluorescence intensity from stimulated relative to unstimulated neutrophils is shown. Each bar represents the mean with standard error of the mean (SEM) of each group (n = 3) with data pooled from three independent experiments. (B) Bone marrow neutrophils were stimulated with C. albicans yeast cells or preformed hyphae of the highly virulent lab strain SC5413 and the low-virulent strain 101 as indicated. The release of extracellular DNA was detected by Sytox green as in (A). Data are the mean with SEM of each group (n = 3) with data pooled from three independent experiments. (C,D) The release of DNA from WT and PAD4^−/− neutrophils that were stimulated and stained with Sytox as described in (A) was visualized by immunofluorescence microscopy. Representative images for each condition are shown in (C). Scale bar = 20 µm. The frequency of Sytox⁺ neutrophils among all C. albicans hyphae-associated neutrophils is shown in (D). The bars are the mean with SEM of 15 images analyzed per condition. (E) Human peripheral blood neutrophils from healthy donors that were treated with the PAD inhibitor Cl-amidine or left untreated and from patients with acquired myeloperoxidase-deficiency were stimulated for 2.5 h with the yeast-locked strain Δhgc1 (C.a. yeast), preformed hyphae of the control strain Δhgc1 + HGC1 (C.a. hyphae), or phorbol 12-myristate 13-acetate. Sytox was detected as described in (A). Each bar represents the mean with SEM of three technical replicates of each group. Data are representative of one out of two independent experiments.