Figure 2.
Figure 2. Macrophages regulate neutrophil resolution at wounds. (A) Real-time imaging of neutrophil behavior near wounds when neutrophils arrive first at the wound (a) or after macrophages (b) in 3-dpf Tg(mpx:DsRed) × Tg(mpeg1:Dendra2). Yellow lines indicate neutrophil tracks toward wounds, red line are tracks at wounds, and blue lines are tracks away from wounds. Dotted lines indicate the fin margin. The inset represents an enlargement of the dashed square. The asterisks identify the location of the same neutrophil at different time points. (B) Quantification of the duration that neutrophils stay at the wound in condition a and b. (C and D) Quantification of the directionality (C) and velocity (D) of neutrophils near the wound (red track) in condition a and b. In B–D, (n) = number of neutrophil tracks at the wound (red track) analyses for each condition. (E) Real-time imaging over 2 hpw in control or irf8 morphant (irf8 MO) Tg(mpx:DsRed) × Tg(mpeg1:Dendra2) larvae. Blue lines correspond to neutrophils that stay at wounds, and yellow lines correspond to neutrophils that resolve. (F) Number of neutrophils at wound 2 hpw in control and irf8 MO larvae. (G) Percentage of neutrophils that resolved from the wounded zone during the 2 hpw in control and irf8 MO larvae. In F and G, (n) = number of larvae. (H) Real-time imaging and neutrophil tracking in control or irf8 MO Tg(mpx:Dendra) larvae from 0–3.5 hpw. Yellow lines indicate neutrophils migrating toward the wound, red line shows migration at the wound, and blue lines shows migration away from the wound. (I) Quantification of duration that neutrophils stay at the wound in control and irf8 MO larvae. (J and K) Quantification of the directionality (J) and velocity (K) of neutrophils toward the wound, at the wound, and away from the wound in control and irf8 MO larvae. In I–K, (n) = number of neutrophil tracks analyzed for each condition. Ctrl, control. Horizontal lines indicate means. Error bars indicate SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, two-tailed unpaired t test. Triangles indicate needle wound location. Bars: (A) 50 µm; (E and H) 100 µm.

Macrophages regulate neutrophil resolution at wounds. (A) Real-time imaging of neutrophil behavior near wounds when neutrophils arrive first at the wound (a) or after macrophages (b) in 3-dpf Tg(mpx:DsRed) × Tg(mpeg1:Dendra2). Yellow lines indicate neutrophil tracks toward wounds, red line are tracks at wounds, and blue lines are tracks away from wounds. Dotted lines indicate the fin margin. The inset represents an enlargement of the dashed square. The asterisks identify the location of the same neutrophil at different time points. (B) Quantification of the duration that neutrophils stay at the wound in condition a and b. (C and D) Quantification of the directionality (C) and velocity (D) of neutrophils near the wound (red track) in condition a and b. In B–D, (n) = number of neutrophil tracks at the wound (red track) analyses for each condition. (E) Real-time imaging over 2 hpw in control or irf8 morphant (irf8 MO) Tg(mpx:DsRed) × Tg(mpeg1:Dendra2) larvae. Blue lines correspond to neutrophils that stay at wounds, and yellow lines correspond to neutrophils that resolve. (F) Number of neutrophils at wound 2 hpw in control and irf8 MO larvae. (G) Percentage of neutrophils that resolved from the wounded zone during the 2 hpw in control and irf8 MO larvae. In F and G, (n) = number of larvae. (H) Real-time imaging and neutrophil tracking in control or irf8 MO Tg(mpx:Dendra) larvae from 0–3.5 hpw. Yellow lines indicate neutrophils migrating toward the wound, red line shows migration at the wound, and blue lines shows migration away from the wound. (I) Quantification of duration that neutrophils stay at the wound in control and irf8 MO larvae. (J and K) Quantification of the directionality (J) and velocity (K) of neutrophils toward the wound, at the wound, and away from the wound in control and irf8 MO larvae. In I–K, (n) = number of neutrophil tracks analyzed for each condition. Ctrl, control. Horizontal lines indicate means. Error bars indicate SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, two-tailed unpaired t test. Triangles indicate needle wound location. Bars: (A) 50 µm; (E and H) 100 µm.

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