Fig. 4

Fig. 4 Myeloperoxidase and H₂O₂ Interact Directly within Neutrophils at the Wound Zone(A) HyPer₄₈₀ images showing neutrophil-specific HyPer expression in Tg(lyz:HyPer) embryos, with HyPer ratio heat maps reflecting the corresponding intraneutrophil [H₂O₂] levels at four time points after wounding. Stills from Movie S3. Corresponding graphs show single neutrophil mean HyPer ratios plotted against distance from wound for four individual embryos, with individual animal regression lines (individual animals color-coded). The negative gradient at 30–50 min after wounding contrasts with the uniform intraneutrophil [H₂O₂] levels prior and later, and indicates higher internal [H₂O₂] within wound-proximate neutrophils at these time points.(B–F) Demonstration of a direct enzymatic Mpx/H₂O₂ interaction within the wound zone by histochemical staining for Mpx activity. For each reaction condition (B–F), the three panels are (i) an overview, (ii) details of cells in the caudal hematopoietic tissue, and (iii) wound zone. Stained cells (white arrows) indicate Mpx-dependent catalysis of p-phenylenediamine and catechol. When H₂O₂ is supplied as a reagent, cells stain throughout the embryo (B), but without supplemented H₂O₂, cells stain only at the wound margin (C). (D)–(F) are negative controls: (D) and (E) drf controls show that staining is mpx-dependent regardless of the H₂O₂ source; (F) WT uninjured control showing that detecting staining of wound-proximate cells is injury-dependent. x/y n values are as follows: (embryos with e1 positive cell in region)/(embryos scored).(G–I) The relative [H₂O₂] within and surrounding wound zone neutrophils, demonstrated using HyPer-mRNA-loaded Tg(lyz:HyPer) embryos. (G) Groups of neutrophils 29 and 85 min after wounding displayed by their HyPer₄₈₀ signal, with corresponding HyPer ratio images portraying intra- and extracellular [H₂O₂]. At 29 min, all neutrophils have higher [H₂O₂] internally than externally, but at 85 min, a cluster of 3 neutrophils displays lower [H₂O₂] internally than externally. Stills from Movie S4 are shown. (H) Tracks of nine neutrophils plotted in space (x,y) and time (z), with tracks colored in categories to indicate when the neutrophil internal [H₂O₂] exceeds (green), equals (blue), or is less than (red) the external [H₂O₂]. Legend gives percentage of exterior HyPer ratio category cut-offs. The nine cells are three neutrophils (C1-3) selected from three wounded embryos (W1–W3). (I) The difference between internal and external [H₂O₂] in time for these same nine cells expressed as the difference between internal and external mean HyPer ratios. Six out of nine cells in (H, I) are net [H₂O₂] consumers, and in 5/6 cases this phenotype is accentuated as they approach the wound margin (one cell does not migrate). Scale bars in (A) and (G) represent 50 μm. Unlabelled perspective-shortened y axis marks (H, left upper corner) indicate 50 μm steps.