FIGURE

Fig. S3

ID
ZDB-FIG-121212-11
Publication
Pase et al., 2012 - Neutrophil-Delivered Myeloperoxidase Dampens the Hydrogen Peroxide Burst after Tissue Wounding in Zebrafish
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Fig. S3

Zonal Variations in Tissue [H2O2] Related to the Path of an Individual Neutrophil Migrating towards the Wound Zone, Related to Figure 4, and a Model of the H2O2/Myeloperoxidase Interaction after Wounding
(A) Still images over 1 h showing path of a single lyz:DsRED2-marked neutrophil (upper row) and associated HyPer ratiometic data reflecting the [H2O2] data for the wound zone. Note the asymmetrical regional reduction on the dorsal (upper) half of the fin where the DsRED2-marked neutrophil is located, most evident by the loss of extent of yellow signal on the heat map. Stills are from Movie S5. Scale bar, 100 μm.
(B) (i) Merged transmission and red fluorescence image at 98 min after injury, overlayed with the migratory paths of lyz:DsRED2 neutrophils (4 green paths, only one of which is relevant to regions 1 and 2) and lyz:DsRED2-negative macrophages (purple, blue and cyan paths, all relevant to region 3). Regions 1-4 are: (1) and (2), regions where the single DsRED2-marked neutrophil passes in sequence; (3) region where three DsRED2-negative macrophages pass; (4) control region, distal to wound. Scale bar, 100 μm. (ii) HyPer ratiometic data reflecting the temporal [H2O2] data for the 4 regions of the injured zebrafish larvae detailed in (i); data are median HyPer ratio for each of the regions shown. Note that the median [H2O2] decreases sequentially in region 1 then 2 as the neutrophil passes through them, but remains elevated in region 3 where only macrophages pass. The [H2O2] remains at background in the control region (4).
(C) Representative HyPer heat maps depicting [H2O2] across the tail fin of a WT embryo 30 and 60 min after embedding only (i.e. without wounding by tail transection) showing resolution of small focal H2O2 bursts at minor nick injuries that were sustained during embedding (white arrowheads). Adjacent overlaid transmission (Trans) and DsRED images document that this resolution occurred without the arrival of any lyz:DsRED2-marked neutrophils. Scale bar, 200 m.
(D) Table showing the extent of overlap of leukocyte marker transgene expression in compound transgenic zebrafish embryos at 2-3 dpf. * = actual genotype Tg(mpeg1:GAL4)x(10xUAS:Kaede) na = not applicable ¶ = Wound margin (WM) corresponds to cell within 100 μm of the wound edge 2 h after amputation.
(E) Model of the H2O2/myeloperoxidase interaction in the wound zone. Schematic diagrams at three timepoints before (i), just after (ii) and 60-90 min after (iii) wounding, showing for each time point the stage in the post-wounding H2O2 burst (upper panels), a whole tail overview (middle panels), and a detail of a neutrophil (lower panels). Prior to wounding (i), intra-neutrophil and surrounding tissue [H2O2] is low. Neutrophils carry the Src-family kinase Lyn capable of H2O2 sensing (green), and myeloperoxidase (Mpx, blue) packaged in primary granules.
Immediately after wounding (ii), a tissue-scale [H2O2] gradient (red, low-high [H2O2] as indicated in scale) is generated by Dual oxidase (Duox, yellow). H2O2 is sensed by Lyn in neutrophils, initiating their migration. Initially, within migrating neutrophils the sum of their own endogenous H2O2 production (possibly generated by NADPH oxidase 2 (Nox2) and superoxide dismutase (SOD), and/or as a by-product of mitochondrial respiration) plus the diffusion of tissue-sourced H2O2 results in the net outcome of higher intracellular [H2O2] than surrounds.
The decline from peak tissue gradient H2O2 levels (iii) coincides with neutrophil arrival and is dependent on their delivery of enzymatically-active Mpx which interacts intracellularly with H2O2. Some migrating neutrophils approaching the wound develop and sustain an intracellular [H2O2] that is lower than their surrounds – they are persistent net consumers of endogenous and tissue-sourced H2O2. These neutrophils function as intracellular H2O2 sumps. At the same time, some other wound-proximate neutrophils display higher internal [H2O2] than their surrounds. However, the net effect of Mpx-dependent intracellular enzymatic HH2O2 consumption is dampening of the overall wound-stimulated tissue H2O2 burst.

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