Research ArticleT CELLS

Regulatory T cells in skin are uniquely poised to suppress profibrotic immune responses

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Science Immunology  06 Sep 2019:
Vol. 4, Issue 39, eaaw2910
DOI: 10.1126/sciimmunol.aaw2910
  • Fig. 1 Bulk cell and single-cell analysis of Tregs in murine skin reveals a skewing toward TH2 differentiation.

    (A to C) FoxP3-GFP+CD25+ Tregs were sorted from SDLNs and skin of FoxP3GFP reporter mice for whole transcriptome RNAseq. (A) Volcano plot comparing expression profiles of skin versus SDLN Tregs. (B) Ingenuity Pathway Analysis of differentially expressed genes between skin and SDLN Tregs. (C) GSEA of Gata3-regulated genes in skin and SDLN Tregs. (D to G) FoxP3-GFP+CD25+ Tregs were sort-purified from skin of FoxP3GFP reporter mice for scRNAseq using the 10X Genomics platform. (D) t-SNE plot of skin Tregs. (E) Heatmap with genes enriched in each skin Treg cluster. (F) Feature plot of specific transcription factors and (G) genes regulated by Gata3. (H) Combined clustering t-SNE plot of skin and lung Tregs. (I) Feature plot of specific transcription factors and (J) genes regulated by Gata3 in combined t-SNE plot of skin and lung Tregs. (K) Representative contour plots and flow cytometric quantification of GATA3 expression in Tregs isolated from SDLNs, skin, and lungs of healthy WT mice. Cells are pregated on live CD45+CD3+CD4+ T cells. MFI, mean fluorescence intensity. Data are representative of three independent experiments, n = 3 to 5 mice per group, per experiment. Data are mean ± SEM. One-way ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

  • Fig. 2 Tregs preferentially regulate TH2 cytokines in skin.

    C57/B6 (WT) or FoxP3DTR+/+ (FoxP3DTR) mice (homozygous for the FoxP3DTR transgene) were treated with DT every 2 days, and skin and lung tissues were harvested on day 5. Cells from skin and lung from WT (black circles) and FoxP3DTR mice (red circles) were stimulated with PMA/ionomycin, and intracellular cytokine production was quantified by flow cytometry. (A) Representative flow cytometric plot for each cytokine tested and quantification shown below. (B) qRT-qPCR for cytokine genes using Qiagen RT2 Profiler Arrays on mRNA isolated from whole skin or lung of WT or FoxP3DTR mice 7 days after beginning treatment with DT with three replicates each. Log2-fold change in expression levels of cytokine genes with a statistical significance of P < 0.05 is shown.

  • Fig. 3 Tregs regulate fibroblast activation and profibrotic gene expression in skin.

    (A) αSMA-RFP mice (WT) or FoxP3DTR+/+/αSMA-RFP (FoxP3DTR) mice (homozygous for the FoxP3DTR transgene) were treated with DT every 2 days, and skin was harvested on day 5. (B) Representative αSMA staining of dorsal skin tissue sections on days 5 and 11 after DT treatment and quantification of αSMA staining intensity. (C) Representative histograms and flow cytometric quantification of αSMA expression on dermal fibroblasts (live CD45PDPN+PDGFRα+ cells) after Treg depletion on day 5. (D) qRT-qPCR for fibrosis-associated genes using Qiagen RT2 Profiler Array on mRNA isolated from whole skin of WT or FoxP3DTR mice on day 5 (n = 3). Fold change in expression levels of profibrotic, antifibrotic, and TGF-β pathway is shown. Unpaired t test, *P < 0.05 **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 Chronic Treg reduction results in dermal fibrosis.

    (A) αSMA-RFP mice (WT) or FoxP3DTR−/+/αSMA-RFP (FoxP3DTR−/+) mice (heterozygous for the FoxP3DTR transgene) were treated with DT every 3 days, and skin was harvested on day 28. (B) Representative histograms and flow cytometric quantification of αSMA expression on dermal fibroblasts on day 28. (C) Representative αSMA staining of dorsal skin tissue sections on day 28 after DT treatment and quantification of αSMA staining intensity. (D) Representative Masson’s trichrome staining of dorsal skin tissue sections and quantification of collagen density. (E) Representative H&E stain and quantification of dermal thickness indicated by red line on the image. (F) qRT-qPCR for fibrosis-associated genes on mRNA isolated from whole skin of WT or FoxP3DTR−/+ mice on day 28. Fold change in expression levels of profibrotic, antifibrotic, TGF-β pathway, and cytokine genes is shown. All data are representative of three independent experiments, n = 5 mice per group, per experiment. Data are mean ± SEM. Unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 100 μm.

  • Fig. 5 Chronic Treg reduction exacerbates bleomycin-induced skin fibrosis.

    αSMA-RFP: FoxP3DTR+/− mice (heterozygous for the FoxP3DTR transgene) were treated with either bleomycin only or with bleomycin and DT for 14 days. αSMA-RFP mice treated with DT were used as baseline controls. (A) Representative αSMA staining of dorsal skin on day 14 and histologic quantification of αSMA staining intensity from control (αSMA-RFP + DT, black circles), αSMA-RFP: FoxP3DTR+/− treated with bleomycin (B) alone (gray circles), and αSMA-RFP: FoxP3DTR+/− treated with bleomycin and DT (red circles). (B) Representative Masson’s trichrome staining of dorsal skin on day 14 and histologic quantification of collagen density. (C) Representative H&E stain on day 14 and quantification of dermal thickness. (D) Representative histograms and flow cytometric quantification of αSMA expression on dermal fibroblasts after Treg reduction. Data are representative of three independent experiments, n = 3 to 5 per group, per experiment. Data are mean ± SEM. One-way ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 100 μm.

  • Fig. 6 Treg expression of Gata3 plays a major role in controlling dermal fibrosis.

    FoxP3CreERT2 or FoxP3CreERT2/Gata3fl/fl mice were treated with bleomycin (B) and tamoxifen (T) for 14 days. FoxP3CreERT2/Gata3fl/fl treated with PBS (no bleomycin or tamoxifen) were used as baseline controls. (A) Representative contour plots and flow cytometric quantification of GATA3 expression in skin CD4+ T cells on day 14. (B) Flow cytometric quantification of Tregs (live CD45+CD4+FoxP3+ cells) isolated from skin, SDLNs, and lungs on day 14. (C) Flow cytometric quantification of percent of cytokine (IL-17, IL-13, IL-4, and IFN-γ)–producing Teffs (live CD45+CD4+FoxP3) after PMA/ionomycin stimulation for 4 hours from skin. (D) Fold change in the percentage of CD4+ TH subsets from skin and lung of bleomycin- and tamoxifen-treated FoxP3CreERT2/Gata3fl/fl mice versus FoxP3CreERT2 mice. (E) Percentage of neutrophils (CD45+MHCIILy6G+) and inflammatory macrophages (CD45+MHCII+CD11b+F4/80+Ly6C+). (F) Representative αSMA tissue staining of dorsal skin and histologic quantification of αSMA staining intensity on day 14. (G) Representative Masson’s trichrome staining and histologic quantification of collagen density. (H) Representative H&E staining and quantification of dermal thickness. Data are representative of two independent experiments, n = 4 to 7 per group, per experiment. Data are mean ± SEM. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 100 μm.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/39/eaaw2910/DC1

    Fig. S1. scRNAseq of lung Tregs and comparison with skin Tregs.

    Fig. S2. TH2-associated chemokine and cytokine receptors are preferentially enriched in skin Tregs compared with lung Tregs.

    Fig. S3. TH2 cells are enriched in skin.

    Fig. S4. Quantification of cell subsets in the FoxP3DTR+/+ mice after DT administration.

    Fig. S5. Gating strategy for dermal fibroblasts.

    Fig. S6. Tregs are reduced in skin after DT administration in FoxP3DTR+/− mice.

    Fig. S7. Immune phenotyping of skin Tregs after Gata3 deletion.

    Fig. S8. No significant differences in neutrophils and inflammatory macrophages after Gata3 deletion in Tregs.

    Fig. S9. No evidence of lung fibrosis after subcutaneous bleomycin injection and Gata3 deletion in Tregs.

    Fig. S10. Blockade of TH2 cytokines partially rescues fibrosis observed in mice with inducible deletion of Gata3 in Tregs.

    Table S1. Raw data Excel file.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. scRNAseq of lung Tregs and comparison with skin Tregs.
    • Fig. S2. TH2-associated chemokine and cytokine receptors are preferentially enriched in skin Tregs compared with lung Tregs.
    • Fig. S3. TH2 cells are enriched in skin.
    • Fig. S4. Quantification of cell subsets in the FoxP3DTR+/+ mice after DT administration.
    • Fig. S5. Gating strategy for dermal fibroblasts.
    • Fig. S6. Tregs are reduced in skin after DT administration in FoxP3DTR+/− mice.
    • Fig. S7. Immune phenotyping of skin Tregs after Gata3 deletion.
    • Fig. S8. No significant differences in neutrophils and inflammatory macrophages after Gata3 deletion in Tregs.
    • Fig. S9. No evidence of lung fibrosis after subcutaneous bleomycin injection and Gata3 deletion in Tregs.
    • Fig. S10. Blockade of TH2 cytokines partially rescues fibrosis observed in mice with inducible deletion of Gata3 in Tregs.

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