Figure 6. CXCL5 neutralization restores skin barrier function and HFSC differentiation in the absence of Treg cells.

FoxP3^DTR mice were treated as in Figure 1a. Mice were co-administered α-CXCL5 mab or isotype control with DT on days 0,1 and 3 and harvested on day 4.

(a) Representative flow cytometry plots of Ly6G⁺ CD11b⁺ neutrophils in skin. Plots are pregated on live CD45⁺ cells.

(b) Percent and absolute number of Ly6G⁺ CD11b⁺ neutrophils in skin.

(c) Transepidermal water loss (TEWL) 4 days after epidermal injury in Treg cell-depleted mice treated with either α-CXCL5 or isotype control antibody compared to injured Treg cell-sufficient cntrl mice.

(d) Lgr5-tdTom-FoxP3^DTR mice were treated as in Figure 2a and co-administered α-CXCL5 or isotype control mAb with DT on days 0,1 and 3 of recovery. Mice were harvested on day 4. Representative IF images of Lgr5-derived cell (tdTomato⁺) localization in the epidermis compared to Treg cell-sufficient Lgr5-tdTom-FoxP3^DTR mice. (e) Quantification of Lgr5-derived cell localization in the epidermis of the indicated strains and conditions 4 days after skin injury. tdTomato⁺/ HF and tdTomato⁺/hpf are cell numbers normalized per hair follicle and per high power field respectively

(f) Representative plots and

(g) quantification of CD34⁺ HFSCs in the epidermis of Cntrl and FoxP3^DTR mice treated with α-CXCL5 or isotype control. For all relevant panels, error bars are +/−SEM. *p < 0.05. Results of panels a-f are representative of 3–5 independent experiments (n=2–4 mice per group). Results of g are pooled data from 3 independent experiments. Scale bar in d is 100µm; Red- tdTomato⁺ Lgr5 stem cell progeny; Blue – DAPI.