FIG 4.

Blockade of neutrophil recruitment reduces both extracellular DNA levels and the extent of damaged C. albicans. At 4 dpf, mpo:Rac2-D57N and WT sibling zebrafish were injected in the swim bladder with 4 nl of Caf2-dTom (10⁷ CFU/ml) or PVP. The fish were immediately screened, and only fish with 15 to 30 yeast cells in the swim bladder were selected. Fish were injected at 20 hpi with 2 nl of Sytox green (0.05 mM) and imaged by epifluorescence microscopy 4 h later. Each image was analyzed with ImageJ software as described in Materials and Methods for determination of the amount of C. albicans (number of pixels in the red channel) and extracellular DNA (number of pixels in the green channel). The number of punctae on filaments was also scored. Both the number of pixels (extracellular DNA) and the number of punctae (C. albicans damage) were normalized to the amount of C. albicans (number of pixels) in each image resulting in the ET/C. albicans ratio indicated in panel C or the ratio of the number of punctae/10⁵ pixels of C. albicans indicated in panel D. (A and B) Representative image of 2D staining for extracellular DNA and C. albicans damage. Images were processed by overlaying the projection of the sum of slices in both the red and green channels. (B) Zoomed image of the box in panel A, with white arrows pointing to punctae (green) on C. albicans filaments (red). Bars, 20 μm (A) and 5 μm (B). (C) Loss of neutrophil recruitment in the mpo:Rac2-D57N line decreases the amount of extracellular DNA. The ratio of green pixels (extracellular DNA) to total red pixels (C. albicans) was calculated using ImageJ software, and the average ratio per fish is shown for mpo:Rac2-D57N and WT siblings. Data from three independent experiments were pooled (n = 27 and 30 for mpo:Rac2-D57N and WT fish, respectively). Medians, boxes, and whiskers showing the minimum-maximum are represented with the results of the Mann-Whitney test (P = 0.0295). (D) Loss of neutrophil recruitment in the mpo:Rac2-D57N line decreases C. albicans damage. The number of punctae on each image was normalized to the amount of C. albicans, and the average ratio per fish for fish with WT Rac2 and Rac2-D57N neutrophils is shown. Data from three independent experiments were pooled (n = 27 and 30 for mpo:Rac2-D57N and WT fish, respectively). Medians, boxes, and whiskers showing the minimum-maximum are represented with the results of the Mann-Whitney test (P = 0.0201). *, P < 0.05.