Figure 2

Activation of LTB4 Biosynthesis Is Favored in Clustering Neutrophils

(A) Schematic of LTB4 biosynthesis. cPLA2 (calcium-dependent phospholipase A2) and 5-LO are recruited to the nuclear membrane and produce arachidonic acid (AA) and LTA4, respectively. LTA4 is metabolized into LTB4 by LTA4 hydrolase.

(B) Constructs for transgenic expression of a fluorescent fusion of 5-LO with tRFP in neutrophils (below). Schematic of neutrophil with 5-LO nuclear translocation is shown.

(C and D) Spinning-disk confocal projections of neutrophils in 3-dpf double-transgenic Tg(lyz:GCamp6F)xTg(lyz:tRFP-5LO) zebrafish larvae after two-photon LW in the ventral fin-CHT boundary (C) or mechanical wound (MW) in the ventral fin (D). Blue dotted lines indicate the wound area occupied by clustering neutrophils. Zoomed images of examples of neutrophils with 5-LO translocation are shown. Time in relation to translocation is indicated in minutes in the first example. Examples are from three (LW) or two (MW) different larvae. Scale bars represent 50 μm and 5 μm, respectively.

(E and F) Quantification of mean distance from the wound center (x; left) and normalized GCamp6F fluorescence intensity (right) for 5-LO-translocating cells versus non-translocating cells in laser wounds (E) and mechanical fin wounds (F). GCamp6F fluorescence intensity was normalized to the most fluorescent cell in the corresponding frame.

(E) n = 41 cells (for translocating cells, each dot is a cell; for non-translocating cells, each dot represents the mean of all non-translocating cells in the same field of view; left) and n = 31 cells from 8 larvae in 5 different experiments (right).

(F) n = 17 (left) and n = 16 (right) cells from 5 larvae in 3 different experiments. Colored dots represent examples shown in individual images in (D).

Wilcoxon matched-pairs signed rank test. Error bar represents 95% confidence intervals of medians. ^∗∗∗p < 0.0002. See also Figure S2 and Video S2.