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. 2018 Jul 9;9:1573. doi: 10.3389/fimmu.2018.01573

Figure 4.

Figure 4

Peptidylarginine deiminase 4 (PAD4)-deficiency results in increased clustering of neutrophils to Candida albicans hyphae but does not affect their antifungal activity. (A–C) Neutrophils were isolated from the bone marrow of naïve WT and PAD4−/− mice (A,B) or from peripheral blood of a healthy donor and treated or not with the PAD inhibitor Cl-amidine (C). Neutrophils were incubated with C. albicans hyphae and then stained with DAPI (blue). The number of neutrophils interacting with each C. albicans hypha was quantified after 30 min (A) or 2.5 h (B,C) of stimulation. Representative images are shown on the left (Scale bar = 20 µm), summary plots are shown on the right. Bars are the mean with standard error of the mean (SEM) of each group with n = 2–3. (D) The cell wall integrity of C. albicans hyphae was assessed by transmission electron microscopy after 2.5 h incubation with WT or PAD4−/− bone marrow neutrophils. Representative images of each group are shown in the upper row. Lower row: some hyphae exposed to neutrophils showed membrane retraction and extensive cytoplasm disintegration (Di) or vacuolation, nuclear alterations, and swelling of the cell wall (Dii). Scale bar = 1 µm. (E) The frequency of C. albicans hyphae that were affected by WT and PAD4−/− neutrophils as described in (Di,Dii) was quantified. Bars are the mean with SEM of fungal elements from 18 EM images analyzed per condition. (F) WT and PAD4−/− bone marrow neutrophils were incubated with GFP-expressing C. albicans yeast cells and the degree of Candida uptake was determined by flow cytometry. Bars are the mean with standard error of the mean (SEM) of each group with n = 2. (G) Reactive oxygen species (ROS) production by WT and PAD4−/− bone marrow neutrophils in response to C. albicans yeast or hyphae was detected by chemiluminescence using the cell-impermeable substrate lucigenin to detect extracellular ROS (left) or the cell-permeable substrate luminol to detect total ROS (right). (H) WT and PAD4−/− bone marrow neutrophils were incubated for 3 h with opsonized or unopsonized C. albicans hyphae. The percentage of C. albicans killing was assessed by alamar blue assay. Bars are the mean with SEM of each group with n = 4. Data are pooled from (A,B,H) or representative of (G) two independent experiments.