Fig. 4: NADPH oxidase function plays a critical role in swarm dynamics.
From: Neutrophil swarming delays the growth of clusters of pathogenic fungi
Human neutrophils were incubated with different inhibitors to interrogate the mechanisms necessary to restrict the growth of C. albicans during swarming. The inclusion of 300 µM Apocynin disrupted the ability of neutrophils to swarm to C. albicans, while the inclusion of 10 µM DPI resulted in rapid formation and then dissolution of swarms, as shown in quantification of the neutrophil accumulation area. N = 36 swarms across 3 donors for DPI, N = 48 across 3 donors for Apocynin (a). The area of fungal growth after swarming with ROS inhibitors at 16 hours shown (b). N = 288 swarms for PMN, 96 swarms for Trolox, 286 swarms for Apocynin and 219 spots for the no PMN group, across 3 independent donors. CGD neutrophils exhibit exaggerated swarming at early points in the assay as shown in a panel of representative images and in the quantification of neurophil swarming area (c–e). N = 16 swarms for one donor for control and N = 46 swarms across three donors for CGD. The area of fungal growth after swarming with control or CGD neutrophils after 16 hours is shown (f). Results are normalized to growth of the C. albicans alone control. N = 96 swarms for one donor for control and N = 288 swarms across three donors for CGD. CGD samples exhibit reduced NETs. The MFI of Sytox Green staining was quantified for swarms from a healthy donor and from CGD neutrophils (g). Average of 16 swarms for control and 16 swarms for each individual CGD sample is shown. The number of nuclei/µm2 was quantified and is shown (h). N = 16 swarms for one donor for control and N = 16 swarms for each individual CGD sample.*p ≤ 0.05 (p = 0.0489 in e), **p ≤ 0.01 (p = 0.0068 for control 60 vs CGD 60, p = 0.0011 for control 180 vs CGD 180, p = 0.0030 for control 240 vs CGD 240 in e), ***p ≤ 0.001 (p=0.0001 for control 120 vs CGD 120 in e), ****p <0.0001 (a, b, f, h). Kuskal-Wallis with Dunn’s post-test for a-b, e, h; Mann-Whitney for f (unpaired, two-tailed). Error bars represent mean +/− standard deviation. Scale bar is 100 µm.
