Figure 4.
Figure 4. Topical ruxolitinib ameliorates skin GVHD, protects Lgr5+ HFSCs against skin GVHD, and promotes hair regeneration and wound healing. B6 or B6-Lgr5EGFP-cre/ER mice were transplanted as in Figure 1. (A-J) Recipients were treated daily with Vaseline (Ctrl) or ruxolitinib (Ruxo) ointment on their shaved back skin from day +1 after SCT. (A-E) Numbers of T cells (A), IFN-γ+ T cells (B), IL-17-eYFP+ T cells (C) and MFI of CXCR3 on T cells (D), and expression levels of Cxcl9, Cxcl10, and Cxcl11 (E) measured by Q-PCR on day +14. Data from 3 independent experiments (n = 6-10 per group). (F) H&E staining of skin samples on day +28 after SCT. Original magnification ×10. Scale bar, 100 μm. (G) Immunofluorescent staining of the skin samples on day +14 after SCT with EGFP (green), CK15 (red), and DAPI (blue). Original magnification ×40. Scale bar, 50 μm. (H) Numbers of EGFP+ cells per 50 follicles from 2 independent experiments (n = 6-7 per group). (I) Macroscopic images of hair regeneration in the treated skin on day +35 after SCT. The treated areas are surrounded by dashed lines. (J) Mice were treated with each ointment from day +1 to day +21 after SCT. Full-thickness wounds were made on the back skin of recipient mice on day +21. Wound areas of syngeneic (Syn; blue), allogeneic control (Allo; red), allogeneic plus BMV (green), and allogeneic plus ruxolitinib (Ruxo; orange) from 2 independent experiments. (K-O) Ruxolitinib ointment was stopped on day +15 (Stopped) or continued thereafter (Continued). The representative images of H&E staining (K; scale bar, 100 μm), pathology skin GVHD scores (L) and the numbers of Lgr5+ HFSCs in the treated skin (M) on day +21 after allogeneic SCT from 2 independent experiments (n = 5-6 per group) were shown. Representative images of Masson trichrome staining (N; scale bar, 100 μm) and dermal thickness of treated skin (O; n = 6-7 per group) on day +35 from 2 independent experiments. The Mann-Whitney U test or 1-way ANOVA followed by Tukey posttest was used to compare the data (*P < .05; **P < .01; ***P < .005). Data represent the mean ± SEM.

Topical ruxolitinib ameliorates skin GVHD, protects Lgr5+HFSCs against skin GVHD, and promotes hair regeneration and wound healing. B6 or B6-Lgr5EGFP-cre/ER mice were transplanted as in Figure 1. (A-J) Recipients were treated daily with Vaseline (Ctrl) or ruxolitinib (Ruxo) ointment on their shaved back skin from day +1 after SCT. (A-E) Numbers of T cells (A), IFN-γ+ T cells (B), IL-17-eYFP+ T cells (C) and MFI of CXCR3 on T cells (D), and expression levels of Cxcl9, Cxcl10, and Cxcl11 (E) measured by Q-PCR on day +14. Data from 3 independent experiments (n = 6-10 per group). (F) H&E staining of skin samples on day +28 after SCT. Original magnification ×10. Scale bar, 100 μm. (G) Immunofluorescent staining of the skin samples on day +14 after SCT with EGFP (green), CK15 (red), and DAPI (blue). Original magnification ×40. Scale bar, 50 μm. (H) Numbers of EGFP+ cells per 50 follicles from 2 independent experiments (n = 6-7 per group). (I) Macroscopic images of hair regeneration in the treated skin on day +35 after SCT. The treated areas are surrounded by dashed lines. (J) Mice were treated with each ointment from day +1 to day +21 after SCT. Full-thickness wounds were made on the back skin of recipient mice on day +21. Wound areas of syngeneic (Syn; blue), allogeneic control (Allo; red), allogeneic plus BMV (green), and allogeneic plus ruxolitinib (Ruxo; orange) from 2 independent experiments. (K-O) Ruxolitinib ointment was stopped on day +15 (Stopped) or continued thereafter (Continued). The representative images of H&E staining (K; scale bar, 100 μm), pathology skin GVHD scores (L) and the numbers of Lgr5+ HFSCs in the treated skin (M) on day +21 after allogeneic SCT from 2 independent experiments (n = 5-6 per group) were shown. Representative images of Masson trichrome staining (N; scale bar, 100 μm) and dermal thickness of treated skin (O; n = 6-7 per group) on day +35 from 2 independent experiments. The Mann-Whitney U test or 1-way ANOVA followed by Tukey posttest was used to compare the data (*P < .05; **P < .01; ***P < .005). Data represent the mean ± SEM.

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