In vivo imaging reveals a pioneer wave of monocyte recruitment into mouse skin wounds

Abstract

The cells of the mononuclear phagocyte system are essential for the correct healing of adult skin wounds, but their specific functions remain ill-defined. The absence of granulation tissue immediately after skin injury makes it challenging to study the role of mononuclear phagocytes at the initiation of this inflammatory stage. To study their recruitment and migratory behavior within the wound bed, we developed a new model for real-time in vivo imaging of the wound, using transgenic mice that express green and cyan fluorescent proteins and specifically target monocytes. Within hours after the scalp injury, monocytes invaded the wound bed. The complete abrogation of this infiltration in monocyte-deficient CCR2(-/-) mice argues for the involvement of classical monocytes in this process. Monocyte infiltration unexpectedly occurred as early as neutrophil recruitment did and resulted from active release from the bloodstream toward the matrix through microhemorrhages rather than transendothelial migration. Monocytes randomly scouted around the wound bed, progressively slowed down, and stopped. Our approach identified and characterized a rapid and earlier than expected wave of monocyte infiltration and provides a novel framework for investigating the role of these cells during early stages of wound healing.

Figure 1. MP wound infiltration was localized to the deep skin layer.

(a) Representative picture of skin wound excision. Pictures represent (b) front and (c) transversal two-photon laser scanning microscopy (TPLSM) 3D reconstruction of the wound site of a C57Bl6 mouse, 3 h after injury. The second harmonic generation (SHG) signal is in blue, and autofluorescent hairs in green. (d) Representative dot plot showing GFP and ECFP expression on distinct blood myeloid subsets from MacBlue×CX3CR1^gfp/+ mice. (e) Front and (f) transversal TPLSM 3D reconstruction of the wound site in MacBlue×CX3CR1^gfp/+ mouse, 4 h post-wounding. The SHG signal is in blue, the ECFP signal in cyan, and the GFP signal in green. Legends represent 1: Skull; 2: Deep skin layers; 3: Wound edge; 4: Skin. Inf Mo: Inflammatory monocytes; Np: neutrophils; Resident Mo: resident monocytes.

Figure 2. The skin wound induced a rapid and transient wave of monocyte recruitment.

(a) Representative TPLSM picture of the wound deep skin layer from MacBlue×CX3CR1^gfp/+ and MacBlue×CX3CR1^gfp/+ CCR2^−/− mice at several time points after wounding. The SHG signal is in blue, the ECFP signal in cyan, and the GFP signal in green. (b) Quantification of ECFP⁺ cell density from TPLSM pictures taken 15 to 360 min post-wounding in MacBlue×CX3CR1^gfp/+ and MacBlue×CX3CR1^gfp/+ CCR2^−/− mice. Each dot represents a mean number of ECFP cells calculated from 30 to 50 different fields (100×100 µm) from 3–8 mice. Time points were compared using one way ANOVA followed by Bonferroni adjustment. (c) Representative flow cytometry dot plots of Ly6C and Ly6G expression by ECFP⁺ blood myeloid cells (gated on CD11b⁺, NK1.1⁻) from MacBlue×CX3CR1^gfp/+ mice before and 4 hours after wounding. (d) Quantification of ECFP⁺ Ly6C^high inflammatory and Ly6C^low resident monocytes in the blood of MacBlue×CX3CR1^gfp/+ and MacBlue×CX3CR1^gfp/+ CCR2^−/− mice before and 4 hours after injury (n = 4–7 from 3 independent experiments). Inf Mo: Inflammatory monocytes; Resident Mo: Resident monocytes. *p<0.05; **: p<0.01; ***: p<0.001.

Figure 3. Monocytes infiltrated the wound bed concomitantly with neutrophils.

(a): Time lapse picture series representing concomitant invasion of the wound bed by monocytes (black arrows) and neutrophils (red arrows). The SHG signal is in blue, the ECFP signal in cyan, and the PE signal in red. Representative track paths are represented by white dashed lines. (b) Representative TPLSM pictures of the wound deep skin layer from MacBlue×CX3CR1^gfp/+ mice at several time points after wounding. Mice were injected intravenously with 10 µg of Ly6G-PE antibody before wounding. The SHG/ECFP signal is in blue, and the PE signal in red. (c) The graph shows the quantification of Ly6G⁺ and ECFP⁺ cell density from TPLSM pictures taken 15 to 360 min post-wounding in MacBlue×CX3CR1^gfp/+ mice. Each dot represents a mean of number of ECFP⁺ cells calculated from 30 to 50 different fields (100×100 µm) from 3 to 8 different mice. Time points were compared using one way ANOVA followed by Bonferroni adjustment. *p<0.05; **: p<0.01.

Figure 4. ECFP⁺ monocytes infiltrated the wound bed through areas of microhemorrhages.

(a) Front and (b) transversal TPLSM 3D reconstruction of the wound site in MacBlue×CX3CR1^gfp/+ mice 30 min post-wounding. 70 kDa fluorescein-dextran (green) was injected i.v. before the imaging session for vasculature staining. The SHG signal is in blue. Legends represent: 1: Skull, 2: Bone marrow vasculature, 3: Superficial vasculature, 4: Deep skin layer. (c) TPLSM pictures at different time point after wounding of MacBlue×CX3CR1^gfp/+ mice. Vasculature staining was performed with high molecular weight (2 MDa) rhodamine-dextran (red). The ECFP signal is in cyan. (d) Time lapse image series representing monocyte (arrows) egress from a microhemorrhage area toward the wound bed. Colored arrows depict 4 distinct infiltrating monocytes. (e) Representative ECFP⁺ cell morphology during egress from the blood.

Figure 5. Wound-infiltrating monocytes migrated actively and then stopped within the matrix.

(a) Representative TPLSM picture of monocyte migratory behavior 210 min after injury of MacBlue×CX3CR1^gfp/+ mice. Track pathways of ECFP⁺ cells (cyan) are indicated. Quantification of ECFP⁺-cell mean velocity (b) and arrest coefficient (c) at indicated time range after injury. (d) Graphs represent cell frequency distribution as a function of their mean velocity at indicated time range after injury. The percentages of cells with a mean velocity higher than 12 µm/min are indicated. (e) Quantification of track straightness of ECFP⁺ cells at the indicated time range after injury. (f) Representation of the ECFP⁺-cell mean displacement as a function of the square root of time at 210 min after injury (all quantifications represent a pool of 27 to 781 cells from 3 to 6 different videos performed on 2 to 5 different mice). *p<0.05; ***p<0.001.