Thymic stromal lymphopoietin controls hair growth

Abstract

Skin tissue regeneration after injury involves the production and integration of signals by stem cells residing in hair follicles (HFSCs). Much remains unknown about how specific wound-derived factors modulate stem cell contribution to hair growth. We demonstrate that thymic stromal lymphopoietin (TSLP) is produced in response to skin injury and during the anagen phase of the hair cycle. Intradermal injection of TSLP promoted wound-induced hair growth (WIHG), whereas neutralizing TSLP receptor (TSLPR) inhibited WIHG. Using flow cytometry and fluorescent immunostaining, we found that TSLP promoted proliferation of transit-amplifying cells. Lgr5^CreER-mediated deletion of Tslpr in HFSCs inhibited both wound-induced and exogenous TSLP-induced hair growth. Our data highlight a novel function for TSLP in regulation of hair follicle activity during homeostasis and wound healing.

Keywords: TSLP; hair follicle; hair follicle stem cell; human; immunology; mouse; regeneration; tissue stem cell.

Graphical abstract

Figure 1

TSLP is produced in the skin in response to injury (A) Schematic illustration representing locus for human TSLP. (B) qRT-PCR analysis of TSLP variants in human skin 24 h after wounding. Data presented are from 4 independent experiments using 4 different human donors in technical duplicates. (C) Immunostaining human skin of TSLP_total or long-form TSLP (lfTSLP; red) and KRT15 (green). Images presented are representative of 3 independent experiments using 3 different human donors. Scale bar: 100 μm. (D) qRT-PCR analysis of lfTSLP and sfTSLP of human skin 24 h after ex vivo wounding. Data are from technical duplicates or triplicates of 4 experiments using 4 different human donors. (E) qRT-PCR analysis of Tslp in RNA isolated from healing back skin wounds normalized to NW. Data are from 3 independent experiments, 4 pooled wounds per animal, n = 3 mice (NW, 4 DPW, 30 DPW) or n = 6 mice (1 DPW, 7 DPW) in technical triplicates. (F) ELISA of TSLP in whole-tissue lysates of skin wounds (2 independent experiments, n = 2 or 3 mice per group). (G) Immunostaining for TSLP (red) and ITGα6 (green) in non-wounded skin (top) or 7 DPW (bottom) skin from WT C57BL6 female mice. Scale bar: 20 μm. (H) Immunostaining for TSLP (red) and CD45 (green) 7 DPW. All scale bars: 100 μm. ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. Error bars represent ±SEM. FC, fold change; NW, non-wounded; W, wounded; DPW, days post-wounding. See also Figures S1 and S2.

Figure 2

TSLP is expressed throughout the hair cycle and accelerates onset of wound-induced hair growth (A) Tslp expression by qRT-PCR. Data represent 10–31 mice per group from 4 independent experiments. “Telogen” denotes telogen following a complete hair cycle. (B) TSLP concentration in skin tissue by ELISA. Data represent 9–24 mice per group from 4 independent experiments. (C) Immunostaining of skin samples collected from mice in anagen 8 days after depilation. Red, TSLP; green, KRT6a; blue, nuclei. Scale bars: 100 μm. (D) Experimental timeline indicating hair follicle stages and treatments of vehicle (0.01% BSA) or recombinant mouse TSLP (100 ng/wound) following 4 mm punch biopsy. (E) Quantification of skin area that entered anagen; represents data from 3 experiments using n = 8 mice per group. (F–H) Immunostaining for Ki67 (F), (G) Ki67 quantification, and (H) hair follicle length measurement from mouse skin 13 days after wound and treatment with vehicle or TSLP. Data are from 2 experiments using n = 3 mice per group; 11–30 images per group were analyzed. Scale bars: 50 μm. Error bars represent ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. See also Figure S3.

Figure 3

TSLP expands CD34⁺ITGα6^lo transit amplifying cells (TACs) (A) Experimental timeline for hair growth analysis following s.c. TSLP (250 ng). (B) Quantification of skin area that has entered anagen. Data are from n = 3 mice per group. (C) Representative photos of mouse back skin after s.c. TSLP treatment. (D) Experimental timeline for analysis of TSLP-driven cell proliferation. (E) Representative flow cytometry plots of CD34⁺ cells. (F) Quantification of CD34⁺ cells pre-gated on live, single cells. (G) Quantification of total, live, EdU⁺ cells. (H) IFlow cytometry plots representing ITGα6 expression pre-gated on CD34⁺ cells. (I) Quantification of CD34⁺ITGα6 subpopulations. (J) Flow cytometry histogram overlay of CD34⁺ITGα6^lo cells from TSLP- and vehicle-treated groups. (K) Quantification of EdU⁺CD34⁺ITGα6^lo. Graphs represent means of 2 experiments using 6–8 mice per group ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. See also Figure S4.

Figure 4

Murine epithelial cells express TSLPR (A) Representative flow cytometry plots pre-gated on live, single cells. (B) Quantification of TSLPR⁺ relative cell abundance. (C) Mean fluorescence intensity of surface expression of TSLPR on cells from hair follicle (HF) and skin from non-wounded tissue. Data presented represent averages ± SEM from 4 experiments using n = 7 mice (HF) and n = 20 mice (skin). (D) Schematic showing location of tissue section portrayed in (E). (E) Immunostaining of hair follicles neighboring 7-day-old wound bed. Red, TSLPR; blue, KRT6a. Scale bars: 50 μm. (F) Flow cytometry for TSLPR⁺ cells after wounding. Data are from 2 experiments using n = 2–4 mice per group in technical duplicates. (G) Immunostaining TSLPR (red) of 7-day-old wound beds. Images 1 and 2 (insets) were enlarged to show detail. Scale bars: 200 μm. (H) TSLPR immunostaining in non-wounded mouse back skin in telogen. Scale bars: 50 μm. Green, keratin 6a (KRT6a); blue, nuclei. See also Figure S5.

Figure 5

TSLPR expression by LGR5⁺ HFSCs is essential for WHIG (A–G) Quantification of TSLPR⁺ cells by flow cytometry from Lgr5^CreER.Tslpr^fl/fl mice and Lgr5^CreER.Tslpr^fl/+mice treated daily for 3 consecutive days with 4-hydroxytamoxifen (4OHT). (A) LGR5-^eGFP+ cells pre-gated on total live cells. (B) TSLPR⁺ cells pre-gated on LGR5^eGFP+ cells. (C) Flow plot and (D) quantification of TSLPR⁺LGR5⁺ cells, pre-gated on live, single cells. (E) Histogram for TSLPR expression from total LGR5^eGFP+ cells. (F) Flow cytometry plot and (G) quantification of TSLPR⁺CD45⁺ cells, pre-gated on live, single cells. Data are from 2 independent experiments using 5–7 mice per group. (H) Experimental timeline of quantification of skin that has entered anagen following wounding. (I) Representative photos of mouse back skin after 4OHT treatment of Lgr5^CreER.Tsfpr^fl/fl and Lgr5^CreER.Tslpr^fl/+ mice. (J) Quantification of anagen skin area of mice treated with or without TSLP. Graph represents averages ± SEM of n = 4–6 mice per group across 3 experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. See also Figure S6.

Figure 6

TSLP promotes accumulation of progenitor factor DDX6 (A and B) qRT-PCR analysis of DDX6, KLF4, and FLG of RNA isolated from primary human keratinocytes treated with sfTSLP or lfTSLP for (A) 16 h or (B) 24 h. (C) Immunostaining of DDX6 (red) in primary human keratinocytes stimulated with human TSLP (100 ng/mL) for 20 h. Scale bars: 20 μm. (D) Quantification of DDX6. Graph represents average ± SEM DDX6 intensity from n = 17–28 images/group; ^∗∗∗∗p < 0.0001. (E) Representative histogram of Ki67 staining of primary human keratinocytes stimulated with vehicle control, 3 nM lfTSLP, or sfTSLP for 40 h. Isotype IgG used for control. (F and G) Quantification of Ki67⁺ (F) cell abundance and (G) Ki67 MFI in primary human keratinocytes stimulated with 3 nM lfTSLP and sfTSLP for 24 h. Data represent averages ± SEM of 3 independent experiments using 2 or 3 different human donors in technical triplicates. ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. See also Table S1.