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Glucocorticoid signaling and regulatory T cells cooperate to maintain the hair-follicle stem-cell niche

Nature Immunology volume 23, pages 1086–1097 (2022)Cite this article

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Abstract

Maintenance of tissue homeostasis is dependent on the communication between stem cells and supporting cells in the same niche. Regulatory T cells (Treg cells) are emerging as a critical component of the stem-cell niche for supporting their differentiation. How Treg cells sense dynamic signals in this microenvironment and communicate with stem cells is mostly unknown. In the present study, by using hair follicles (HFs) to study Treg cell–stem cell crosstalk, we show an unrecognized function of the steroid hormone glucocorticoid in instructing skin-resident Treg cells to facilitate HF stem-cell (HFSC) activation and HF regeneration. Ablation of the glucocorticoid receptor (GR) in Treg cells blocks hair regeneration without affecting immune homeostasis. Mechanistically, GR and Foxp3 cooperate in Treg cells to induce transforming growth factor β3 (TGF-β3), which activates Smad2/3 in HFSCs and facilitates HFSC proliferation. The present study identifies crosstalk between Treg cells and HFSCs mediated by the GR–TGF-β3 axis, highlighting a possible means of manipulating Treg cells to support tissue regeneration.

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Fig. 1: Impaired hair regeneration in GR cKO mice.
Fig. 2: GR signaling is required for Treg cell-facilitated HFSC activation and proliferation.
Fig. 3: GR signaling induces TGF-β3 expression by Treg cells.
Fig. 4: GR and Foxp3 cooperate to regulate TGF-β3 expression in Treg cells.
Fig. 5: Treg cell GR deficiency drives defective activation of TGF-β3:pSmad2/3 signaling in HFSCs.
Fig. 6: TGF-β3 produced by Treg cells promotes HFSC proliferation and hair regeneration.

Data availability

Sequence data (RNA-seq and ChIP–seq) have been deposited in the Gene Expression Omnibus under the accession no. GSE183808. Source data are provided with this paper.

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Acknowledgements

We thank T. Hua and C. Gordon for mouse colony management, C. O’Connor for assistance in flow cytometry, U. Manor for assistance in confocal microscopy, N. Hah for assistance in RNA-seq and ChIP–seq experiments, X. Li (Tufts) for CRISPRi vectors, M. Kurita, J. C. I. Belmonte, N. He and R. M. Evans for helpful discussion, and S. P. Bapat (University of California, San Francisco), L. F. Lu (UCSD), C. Wu (National Cancer Institute (NCI)) and T. Mann for reviewing the manuscript. Z.L. was supported by a NOMIS fellowship. J.Y. and M.N.S were supported by the National Institutes of Health (grant nos.: NCI CCSG P30-014195, NIA P01-AG073084, NIA-NMG RF1-AG064049 and NIA P30-AG068635) and the Leona M. and Harry B. Helmsley Charitable Trust. Y.Z. was supported by the NOMIS Foundation, the Crohn’s and Colitis Foundation, the Leona M. and Harry B. Helmsley Charitable Trust and the National Institutes of Health (grant nos. R01-AI107027, R01-AI1511123, R21-AI154919 and S10-OD023689). This work was also supported by the NCI-funded Salk Institute Cancer Center Core Facilities (grant no. P30-CA014195).

Author information

Authors and Affiliations

  1. NOMIS Center for Immunobiology and Microbial Pathogenesis, Salk Institute for Biological Studies, La Jolla, CA, USA

    Zhi Liu, Xianting Hu, Yuqiong Liang & Ye Zheng

  2. Department of Otolaryngology Head and Neck Surgery, Eye and ENT Hospital, Fudan University, Shanghai, China

    Xianting Hu & Huabin Li

  3. Razavi Newman Integrative Genomics and Bioinformatics Core, Salk Institute for Biological Studies, La Jolla, CA, USA

    Jingting Yu & Maxim N. Shokhirev

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Contributions

Z.L. and Y.Z. conceived the project. Z.L. provided the methodology. Z.L., X.H. and Y.L. carried out the investigations. H.L. and Y.Z. provided the resources. Z.L., J.Y. and M.N.S. carried out the formal analysis. Z.L. and J.Y. curated the data. Y.Z. supervised the project. Y.Z. acquired the funding. Z.L. and Y.Z. wrote the original draft of the paper. Z.L. and Y.Z. wrote, edited and reviewed the paper.

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Correspondence to Ye Zheng.

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Nature Immunology thanks Ming Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. N. Bernard was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Normal development and function of Treg cells in GR cKO mice.

a, b, Flow cytometric analysis and quantification of CD4+ T cells and CD8+ T cells (a), and CD25+Foxp3− Treg progenitors, CD25−Foxp3+ Treg progenitors, CD25+Foxp3+ Treg cells (b) in the thymus of 2-month-old WT and GR cKO mice (n = 5 mice per condition). NS, P > 0.05. c, d, Flow cytometric analysis and quantification of CD4+ T cells and CD8+ T cells (c) and Foxp3+ Treg cells (d) in the spleen of 6-month-old WT (n = 7) and GR cKO (n = 8) mice. NS, P > 0.05. e, f, g, Flow cytometric analysis and quantification of CD44highCD62Llow activated/memory CD4+ T cells (e), IFNγ or IL-17 producing CD4+ T cells and CD8+ T cells (f), IL-5 or IL-13 producing CD4+ T cells (g) in the spleen of 6-month-old WT (n = 7) and GR cKO (n = 8) mice. NS, P > 0.05. h, Suppression of proliferation of wild-type naive CD4+ T responder cells (Tresp) by WT and GR cKO Treg cells in an in vitro suppression assay (n = 3 biologically independent replicates per condition). NS, P > 0.05. i, Measurement of WT and GR cKO Treg cells suppressive function in a T cell transfer-induced colitis model by body weight loss (n = 7 mice per condition). NS, P > 0.05. Statistical analysis was performed using a two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. Representative data of two (a-i) independent experiments are shown.

Source data

Extended Data Fig. 2 Specific deletion of GR in Treg cells.

a, b, Quantification of Foxp3+ Treg cells (a), IFNγ or IL-17 producing in CD4+ T cells and CD8+ T cells (b) from the skin of WT (n = 6) and GR cKO mice (n = 5). NS, P > 0.05. c, d, Flow cytometric analysis and quantification of the expression of GR protein in Treg cells, CD4+ Teff cells, CD8+ T cells, dermal γδT cells, DETC from the skin of WT and GR cKO mice (n = 8 mice per group). FMO: Fluorescence minus one control. ****P < 0.0001; NS, P > 0.05. Statistical analysis was performed using a two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. Representative data of two (a-d) independent experiments are shown.

Source data

Extended Data Fig. 3 Transcriptomic analysis of HFSCs isolated from WT and GR cKO mice.

a, Immunofluorescence staining of Ki67 in HFs from WT and GR cKO mice 3 days post-depilation (n = 3 mice per condition). Arrows indicate hair germ location. Scale bars, 50 μm. b, EdU was intraperitoneally injected on day 2 post-depilation. Immunofluorescence staining of EdU in HFs from WT and GR cKO mice 3 days post-depilation (n = 3 mice per condition). Scale bars, 50 μm. c, RT-qPCR comparison of genes related HFSC proliferation between HFSCs from WT and GR cKO mice 4 days post-depilation (n = 5 mice per condition). **P = 0.0035 (Cdk1); *P = 0.047 (Cdk4); ****P < 0.0001 (Cdca3); ***P = 0.0005 (E2f8); ****P < 0.0001 (Bub1b); ***P = 0.0008 (Ccnd1). d, Heatmap of genes related to HFSC differentiation between HFSCs isolated from WT and GR cKO mice 4 days after depilation. e, Gene ontology analysis of genes down-regulated in HFSCs from WT relative to GR cKO mice. f, GSEA plot for the “Hallmarks – Abnormal hair growth” signature and heatmap of related gene expression in HFSCs from WT and GR cKO mice. Statistical analysis was performed using a two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. Representative data of three (a-b) or two (c) independent experiments are shown.

Source data

Extended Data Fig. 4 Gating strategy for flow cytometric analysis of skin lymphoid and myeloid cells.

a, Flow cytometric gating strategy to identify skin lymphoid cell lineages, including DETC, dermal γδT cells, CD4+ Teff cells, Treg cells, CD8+ T cells, and the production of proinflammatory cytokines IL-17 and IFNγ by these cells. b, Flow cytometric gating strategy to identify skin myeloid cell lineages, including neutrophils, eosinophils, macrophages and dendritic cells.

Extended Data Fig. 5 Normal immune homeostasis in the skin of GR cKO mice.

WT and GR cKO mice (n = 8 mice per condition) were depilated to induce hair regeneration. 5 days post-depilation, skin immune cell populations were analyzed by FACS. a, b, Representative flow cytometric plots of Treg cells and quantification of Treg cell number, Foxp3+ Treg cells ratio and Foxp3 protein level in the skin of WT and GR cKO mice (n = 8 mice per condition). NS, P > 0.05. c, Quantification of proinflammatory cytokine IFNγ and IL-17 production in DETCs and dermal γδ T cells, CD4+ Teff cells, and CD8+ T cells (n = 8 mice per condition). NS, P > 0.05. d, Quantification of cell numbers of DETCs, dermal γδ T cells, CD4+ Teff cells, and CD8+ T cells, neutrophils, eosinophils, macrophages, and dendritic cells in the skin (n = 8 mice per condition). NS, P > 0.05. e, Representative H&E staining of skin on day 5 post-depilation. Scale bars, 100 μm. Statistical analysis was performed using a two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. Representative data of two (a-d) independent experiments are shown.

Source data

Extended Data Fig. 6 Transcriptomic analysis of WT and GR cKO skin-resident Treg cells.

a, b, Comparison of Treg cells signature genes (a) and genes associated with Treg cell function in the skin (b) between WT (n = 3) and GR cKO (n = 5) skin-resident Treg cells one day post-depilation. NS, P > 0.05. c, RT-qPCR analysis of the expression of Jag1 in skin Treg cells from WT (n = 3) and GR cKO (n = 4) mice one day post-depilation. **P = 0.0046. d, e, Flow cytometric analysis and quantification of the Jagged-1 protein in skin Treg cells from WT (n = 5) and GR cKO (n = 4) mice. NS, P > 0.05. f, Comparison of the Expression of Notch target genes in HFSCs between WT and GR cKO mice (n = 3 mice per condition). NS, P > 0.05. g, Summary of acquired data from NATMI, showing changes of all the differential pair weight of Tgfb3-Tgfbr and Jag1-Notch between WT and GR cKO mice. Statistical analysis was performed using a two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. Representative data of two (c-e) independent experiments are shown.

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Extended Data Fig. 7 Induction of TGF-β3 expression by dexamethasone in Treg cells is dependent on the Tgfb3 enhancers.

a, RT-qPCR analysis of the expression of Tgfb1 and Il7r in WT (n = 3) and GR cKO (n = 4) skin-resident Treg cells one day post-depilation. NS, P > 0.05; **P = 0.0015. b, Schematic for CRISPR knockout of GR/Foxp3-bound peaks by CRISPR-Cas9. c, RT-qPCR analysis of the expression of Tgfb3 and Ttll5 after CRISPR knockout of indicated GR- and Foxp3- bound sites in Treg cells (n = 3 biologically independent replicates per condition). For Tgfb3 (up): ***P = 0.0003 (Tgfb3 pro); **P = 0.0018 (Tgfb3 intron); **P = 0.0094 (Ttll5 Peak1); **P = 0.0071 (Ttll5 Peak2); **P = 0.0094 (Ttll5 peak3). For Ttll5 (bottom): NS, P > 0.05; *P = 0.0272 (Tgfb3 intron); *P = 0.0205 (Ttll5 Peak3). Statistical analysis was performed using a two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. Representative data of two (a, c) independent experiments are shown.

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Extended Data Fig. 8 HFSCs from GR cKO mice show an enrichment of BMP/pSmad1/5 signaling.

a, Immunofluorescence staining and quantification of pSmad1/5 and pSmad2/3 in HFs from WT and GR cKO mice before hair depilation (n = 10 HFs from 3 mice per condition). Scale bars, 50 μm. NS, P > 0.05. b, Immunofluorescence staining and quantification of pSmad1/5 and pSmad2/3 in HFs from WT and GR cKO mice 3 days post-depilation (n = 10 HFs from 3 mice per condition). Scale bars, 50 mm. ****P < 0.0001. c, GSEA plot for the BMP-responsive genes in HFSCs (Genander et al, 2014) from WT to GR cKO mice isolated 4 days post-depilation. d, RT-qPCR analysis of the expression of genes encoding ligands for Wnt and BMP pathways in the skin from WT and GR cKO mice one day post-depilation (n = 5 mice per condition). NS, P > 0.05. Statistical analysis was performed using a two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. Representative data of three (a, b) or two (d) independent experiments are shown.

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Extended Data Fig. 9 Intradermal injection of TGF-β3 in GR cKO mice results in activation of pSmad2/3 signaling and hair follicle regeneration.

a, Surface view of the BSA or TGF-β3 injected area in WT mice on day 20 post-depilation. Circle in the dashed line indicated injection sites. b, Representative H&E staining and quantification of HF length at the BSA or TGF-β3 injected area of the skin sections from GR cKO mice (n = 25 HFs from 4 mice per condition). The whole images were reconstructed from two adjacent images using stitching plugins from Image J. The area between two dashed lines was the injection site. Scale bars, 500 μm. ****P < 0.0001 c, Immunofluorescence staining and quantification of pSmad1/5 and pSmad2/3 at the BSA or TGF-β3 injected area from GR cKO mice (n = 20 HFs from 5 mice per condition). Scale bars, 50 μm. ****P < 0.0001. Statistical analysis was performed using a two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. Representative data of three independent experiments are shown.

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Extended Data Fig. 10 Normal T cell development and distribution in TGF-β3 cKO mice.

a, b, Flow cytometric analysis and quantification of CD4+ T cells and CD8+ T cells (a), and Foxp3+ Treg cells (b) in the thymus of 2-month-old WT and TGF-β3 cKO mice (n = 5 mice per condition). NS, P > 0.05. c, d, Flow cytometric analysis and quantification of CD4+ T cells and CD8+ T cells (c), Foxp3+ Treg cells (d) in the spleen of 2-month-old WT and TGF-β3 cKO mice (n = 5 mice per condition). NS, P > 0.05. e, Flow cytometric analysis and quantification CD44highCD62Llow activated/memory T cells in the spleen of WT and TGF-β3 cKO mice (n = 5 mice per condition). Data are mean ± SEM. NS, P > 0.05. Statistical analysis was performed using a two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. Representative data of two (a-e) independent experiments are shown.

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Liu, Z., Hu, X., Liang, Y. et al. Glucocorticoid signaling and regulatory T cells cooperate to maintain the hair-follicle stem-cell niche. Nat Immunol 23, 1086–1097 (2022). https://doi.org/10.1038/s41590-022-01244-9

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