Figure 7.
Circadian redistribution of neutrophils protects the surrounding tissue. (a) Representative imaging of skin wounds (at days 0 and 1; D0 and D1) visualized by SHG acquired through multiphoton imaging control mice at ZT5 (WT), neutropenic mice at ZT5 (iDTR), control mice injured at ZT13, and control mice injured at ZT5 and treated with ATI2341. Dotted lines show the rim of skin wounds, also shown at the bottom. (b) Quantification of the increase in wound size from D0 to D1 (24 h after injury) in the indicated mice; n = 3–4 mice per group, 2–5 wounds per mouse. (c) Whole-mount imaging of neutrophils (left) in and around skin wounds of WT (C57BL/6 or Ly6GTomato mice) injured at ZT5 and ZT13, and ATI2341-treated mice at ZT5, quantified in the bars at right; n = 3–6 mice per group (1–2 wounds per mouse). Solid white lines show the wound limits by the neutrophil rim, and dotted white lines show the distribution of neutrophil around the wounds. (d and e) Representative confocal images (left) and quantification (right) of Annexin V signal around skin wounds from mice injured at ZT5 or ZT13 (d; n = 6–8 mice per group, 3–5 wounds per mouse), or around skin wounds from vehicle- or ATI-treated mice (e; n = 6–7 mice per group, 2–4 wounds per mouse). (f and g) Representative images (f) and quantification (g) of neutrophil distribution in the infarcted myocardium of control and ATI2341-treated mice; n = 3 mice per group, two ischemic and four border regions quantified per mouse. The ischemic areas are identified by lack of vascular staining (VE-cadherin) and are delimited by white dotted lines. Neutrophils in and outside this area were identified by MPO staining. (h) Neutrophil numbers in the LV of infarcted hearts from sham (placebo surgery) and vehicle- and ATI2341-treated mice; n = 3–4 mice per group; 2–4 regions per mouse. (i) Summary cartoon showing neutrophil redistribution into wounds, driven by CXCR4 signaling, that occurs naturally at nighttime and can be induced by CXCR4 agonism, resulting in reduced collateral damage to the surrounding tissue. Data in a, b, and g are from single experiments. Data in c and d are pooled from three experiments. Data in e are pooled from four experiments. Data in h are representative of two experiments. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant, as determined by one-way ANOVA (c and h), unpaired t test (d and e), and two-way ANOVA (b and g).

Circadian redistribution of neutrophils protects the surrounding tissue. (a) Representative imaging of skin wounds (at days 0 and 1; D0 and D1) visualized by SHG acquired through multiphoton imaging control mice at ZT5 (WT), neutropenic mice at ZT5 (iDTR), control mice injured at ZT13, and control mice injured at ZT5 and treated with ATI2341. Dotted lines show the rim of skin wounds, also shown at the bottom. (b) Quantification of the increase in wound size from D0 to D1 (24 h after injury) in the indicated mice; n = 3–4 mice per group, 2–5 wounds per mouse. (c) Whole-mount imaging of neutrophils (left) in and around skin wounds of WT (C57BL/6 or Ly6GTomato mice) injured at ZT5 and ZT13, and ATI2341-treated mice at ZT5, quantified in the bars at right; n = 3–6 mice per group (1–2 wounds per mouse). Solid white lines show the wound limits by the neutrophil rim, and dotted white lines show the distribution of neutrophil around the wounds. (d and e) Representative confocal images (left) and quantification (right) of Annexin V signal around skin wounds from mice injured at ZT5 or ZT13 (d; n = 6–8 mice per group, 3–5 wounds per mouse), or around skin wounds from vehicle- or ATI-treated mice (e; n = 6–7 mice per group, 2–4 wounds per mouse). (f and g) Representative images (f) and quantification (g) of neutrophil distribution in the infarcted myocardium of control and ATI2341-treated mice; n = 3 mice per group, two ischemic and four border regions quantified per mouse. The ischemic areas are identified by lack of vascular staining (VE-cadherin) and are delimited by white dotted lines. Neutrophils in and outside this area were identified by MPO staining. (h) Neutrophil numbers in the LV of infarcted hearts from sham (placebo surgery) and vehicle- and ATI2341-treated mice; n = 3–4 mice per group; 2–4 regions per mouse. (i) Summary cartoon showing neutrophil redistribution into wounds, driven by CXCR4 signaling, that occurs naturally at nighttime and can be induced by CXCR4 agonism, resulting in reduced collateral damage to the surrounding tissue. Data in a, b, and g are from single experiments. Data in c and d are pooled from three experiments. Data in e are pooled from four experiments. Data in h are representative of two experiments. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant, as determined by one-way ANOVA (c and h), unpaired t test (d and e), and two-way ANOVA (b and g).

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