Figure 1: Ly6C+ inflammatory macrophages contribute to WIH-A and WIHN. | Nature Communications

Figure 1: Ly6C+ inflammatory macrophages contribute to WIH-A and WIHN.

From: Macrophages induce AKT/β-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF

Figure 1

(a) Wounding the skin of 8-week-old mice induced a transition of HF from the telogen phase to the anagen phase around wound areas. n=56. (b) The longitudinal section of the wound at the 15th day post-wound (PWD-15) (upper panel) and of the dermal side of the skin showed the pigmented anagen HFs around the wound (lower panel). (c) Flow cytometry analysis of wound tissue single cell suspensions showed the extensive depletion of F4/80+/CD11b+ macrophages after clodronate liposome (Clo) treatment. Mice receiving clodronate, n=7; control mice, n=12. (d) WIH-A analysis in Clo treated (n=8) and the control mice (n=11). (e,f) Clo treatment resulted in complete elimination of WIHN (n=7) (e), and the number of neogenic HFs in the two groups of mice was quantified (f). (g) Immunofluorescence (IF) analysis of wound tissue showed the infiltration of both Ly6C+ inflammatory macrophages and CX3CR1+ resident macrophages at PWD-3. For all IF analyses, representative images from 8 to 16 tissue sections of wounds in 4–6 mice are shown. (h) IF image showed CX3CR1-YFP cells (green) in the dermis of the normal skin of CX3CR1CreER/+:R26iDTR/+ mice. Flow cytometry analysis indicated that TM and DT treatments depleted over 90% of CX3CR1-EYFP cells in the blood of CX3CR1CreER/+:R26iDTR/+ mice (n=9). (i) Flow cytometry analysis showed that the DT treatments depleted over 95% of Ly6C+ cells in the blood of LysM-Cre:R26iDTR/+ mice (n=9). (j) IF staining of Ly6C+ macrophages in the wound tissue (PWD-3) of LysM-Cre:R26iDTR/+ mice with or without DT treatment. (k,l) Treatment of LysM-Cre:R26iDTR/+ mice with DT resulted in the complete inhibition of WIH-A. n=8 for DT- mice; n=10 for DT+ mice. (m,n) Deletion of Ly6C+ macrophages resulted in no neogenic HF in the wound (n=7), but the deletion of CX3CR1+ macrophages showed no obvious effect on the number of neogenic HF (n=8 mice). W, wound; HA, hair follicle anagen analysis; HN, hair follicle neogenesis analysis; TM, tamoxifen; DT, diphtheria toxin; WT, wild type. Scale bars, 50 μm. Data are expressed as the mean±s.e.m. ***P<0.005, unpaired t-test, two-tailed.

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