Research ArticleSKIN INFLAMMATION

RORα-expressing T regulatory cells restrain allergic skin inflammation

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Science Immunology  02 Mar 2018:
Vol. 3, Issue 21, eaao6923
DOI: 10.1126/sciimmunol.aao6923
  • Fig. 1 Skin-resident Tregs exhibit an activated signature and express the transcription factor RORα.

    (A) Representative flow cytometric analysis (left) and quantification (right) of FOXP3+ (CD3+CD4+YFP+) cells among CD4+ T cells in ear skin compared with dLNs from Foxp3eyfp-cre mice (n = 3 mice per group). (B) Scatterplot of log2 (RPKM + 1) values of genes expressed in skin Tregs (x axis) compared with LN Tregs (y axis) determined by NGS transcriptomic analysis. Genes that differ by more than twofold are shown in dark gray. Select genes are identified. (C) Representative flow cytometric analysis (left) and quantification (right) of CD44-, ICOS-, and ST2-expressing skin and dLNs Tregs and the mean fluorescence intensity (MFI) of these markers (n = 3 mice per group). (D) Rora expression levels in sorted Tregs from skin and dLNs from Foxp3eyfp-cre mice (n = 3 mice per group). (E) Rora expression levels in sorted Tregs (CD4+CD25+CD127lo) from blood and skin of healthy donors (n = 2). (F) Representative flow cytometric analysis (left) and quantification (right) of Rora+(YFP+)-expressing Tregs in skin and dLN of Roracre/cre Rosayfp/yfp mice (n = 2 mice per group). Columns and bars represent means and SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 RORα deficiency in Tregs results in exaggerated skin inflammation in response to topical application of MC903.

    (A to G) Quantification of ear thickness at day 7 (A); representative H&E-stained sections (B); quantification of dermal thickness (C); representative FACS analysis (left) and quantification of the percentages (middle) and numbers (right) of CD45+ cells (D), eosinophils (E), mast cells, neutrophils, and basophils (F), and CD4+FOXP3+ Tregs, CD4+FOXP3 Teffs, and ILCs (G) in vehicle or MC903-treated ears of Foxp3eyfp-creRorafl/fl mice and Foxp3eyfp-cre controls. n = 3 to 8 mice per group. Columns and bars represent means and SEM. *P < 0.05, ***P < 0.001.

  • Fig. 3 Increased expression of eotaxins and IL-5 in MC903-treated skin of Foxp3eyfp-creRorafl/fl mice.

    (A to E) Relative Ccl11 and Ccl24 mRNA expression (A); IL-5 levels (B); relative Il5 expression in sorted LinCD90+ ILCs (C); representative FACS analysis and quantitation of the percentages of CD4+IL-5+, CD4+IL-13+, and CD4+IL-4+ Teffs (D); and relative Ccl8 mRNA expression in MC903-treated skin of Foxp3eyfp-creRorafl/fl mice and Foxp3eyfp-cre controls (E). n = 4 to 7 mice per group. Columns and bars represent means and SEM. *P < 0.05, ***P < 0.001. ns, not significant.

  • Fig. 4 RORα deficiency in Tregs alters the expression of genes involved in Treg cell migration and function and skews Tregs to IL-4–producing effectors.

    (A) Heat map showing relative expression of genes clustered by K-mean values in skin Tregs of Foxp3eyfp-cre and Foxp3eyfp-creRorafl/fl mice in the steady state and after MC903 treatment (n = 4 to 5 mice per group). (B) Heat map showing the relative expression of select chemotaxis, function, and inflammation genes in skin Tregs from Foxp3eyfp-creRorafl/fl mice and controls (n = 4 to 5 mice per group). (C and D) RNA-seq tracing of Ccr6 and Nt5e expression (left), representative FACS analysis (middle), and MFIs (right) of CCR6 and CD73 expression in skin Tregs of Foxp3eyfp-cre and Foxp3eyfp-creRorafl/fl mice (n = 4 to 5 mice per group). The numbers in the FACS panels represent the percentage of positive cells relative to fluorescence minus one (FMO) control. (E) Relative Il4 mRNA levels in Tregs from MC903-treated skin of Foxp3eyfp-creRorafl/fl mice and controls (n = 4 to 5 mice per group). (F) Representative FACS analysis of IL-4 expression in CD4+ cells and of FOXP3 versus CD90 expression in IL-4+CD4+ cells (left) and quantitation of the percentage of IL-4+CD4+FOXP3+ cells among IL-4+CD4+ cells in the skin of MC903-treated Foxp3eyfp-creRorafl/fl mice and controls.

  • Fig. 5 RORα expression in Tregs promotes expression of the TL1A receptor DR3 and restrains TL1A-driven allergic inflammation elicited by cutaneous application of MC903.

    (A) RNA-seq tracing of Tnfrsf25 expression in skin Tregs from untreated and MC903-treated skin of Foxp3eyfp-cre and Foxp3eyfp-creRorafl/fl mice. (B) Representative FACS analysis (left) and MFIs (right) of DR3 expression by skin Tregs of Foxp3eyfp-creRorafl/fl mice and Foxp3eyfp-cre controls (n = 3 mice per group). The numbers in the FACS panels represent the percentage of positive cells relative to FMO control. (C) Representative FACS analysis of DR3 expression by ILCs from the skin of Foxp3eyfp-creRorafl/fl mice and Foxp3eyfp-cre controls. Results are representative of three independent experiments. The numbers in the FACS panels represent the percentage of positive cells relative to FMO control. (D) TL1A levels in vehicle and MC903-treated ear skin of Foxp3eyfp-creRorafl/fl mice and Foxp3eyfp-cre controls (n = 4 mice per group). (E) Representative FACS analysis (left) and quantification (right) of CD11b+Siglec-F+ eosinophils in MC903-treated ears of Tnfrsf25−/− mice and WT controls. (F) Representative FACS analysis (left) and quantification (right) of CD11b+Siglec-F+ eosinophils and CD11b+Gr1high neutrophils in TL1A-injected skin of Foxp3eyfp-creRorafl/fl mice and Foxp3eyfp-cre controls (n = 3 mice per group). (G to J) Representative H&E-stained sections (G), quantification of dermal thickness (H), quantification of CD45+ cells (right) and CD11b+Siglec-F+ eosinophils (left) (I), and relative mRNA expression of Il5 (right) and Ccl8 (left) (J) in MC903-treated ears of Foxp3eyfp-creRorafl/fl mice injected with anti-TL1A antibody or isotype control (n = 4 mice per group). Columns and bars represent means and SEM. *P < 0.05, **P < 0.01

  • Fig. 6 RORα deficiency in Tregs results in exaggerated skin inflammation in response to EC sensitization.

    (A) Schematic of the experimental mouse model. (B to H) Representative H&E-stained sections (B); quantification of epidermal thickness (C); number of CD45+ cells (D), CD11b+Siglec-F+ eosinophils (E), mast cells, neutrophils, and basophils (left), and CD4+FOXP3 Teffs, CD4+FOXP3+ Tregs, and ILCs (right) (F); relative Il4 (right) and Il13 (left) mRNA expression (G); and numbers of IL-5+ CD4+ T cells and ILCs (H) in saline and OVA-sensitized skin of Foxp3eyfp-creRorafl/fl mice (also designated as cKO) and Foxp3eyfp-cre controls (also designated as WT). n = 3 to 7 mice per group. Columns and bars represent means and SEM. *P < 0.05, ***P < 0.001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/21/eaao6923/DC1

    Fig. S1. RORα-expressing skin Tregs are HELIOS+ natural Tregs that express high levels of ST2 and ICOS.

    Fig. S2. Multiple skin-resident cell types express RORα.

    Fig. S3. CD4+ T cells are the only cells that express eGFP in Foxp3egfp mice.

    Fig. S4. Treg-specific RORα deficiency does not affect Treg numbers in the skin, nor their ability to produce IL-10.

    Fig. S5. Skin TSLP and serum IgE levels in Foxp3eyfp-creRorafl/fl mice and Foxp3eyfp-cre controls.

    Fig. S6. Effect of lack of RORα in Tregs on MC903-driven blood eosinophilia and on IL-4, IL-13, and chemokines in MC903-treated skin.

    Fig. S7. Analysis of correlation of RNA-seq samples.

    Fig. S8. Increased number and motility of Tregs in MC903-treated ear skin.

    Table S1. Summary of RNA-seq experiments.

    Movie S1. Intravital two-photon imaging of the untreated ear dermis of a Foxp3egfp animal.

    Movie S2. Intravital two-photon imaging of the MC903-treated ear dermis of Foxp3egfp mice.

  • Supplementary Materials

    Supplementary Material for:

    RORα-expressing T regulatory cells restrain allergic skin inflammation

    Nidhi Malhotra,* Juan Manuel Leyva-Castillo,* Unmesh Jadhav, Olga Barreiro, Christy Kam, Nicholas K. O′Neill, Francoise Meylan, Pierre Chambon, Ulrich H. von Andrian, Richard M. Siegel, Eddie C. Wang, Ramesh Shivdasani, Raif S. Geha*

    *Corresponding author. Email: nidhi.malhotra{at}elstartherapeutics.com (N.M.); manuel.leyvacastillo{at}childrens.harvard.edu (J.M.L.-C.); raif.geha{at}childrens.harvard.edu (R.S.G.)

    Published 2 March 2018, Sci. Immunol. 3, eaao6923 (2018)
    DOI: 10.1126/sciimmunol.aao6923

    This PDF file includes:

    • Fig. S1. RORα-expressing skin Tregs are HELIOS+ natural Tregs that express high levels of ST2 and ICOS.
    • Fig. S2. Multiple skin-resident cell types express RORα.
    • Fig. S3. CD4+ T cells are the only cells that express eGFP in Foxp3egfp mice.
    • Fig. S4. Treg-specific RORα deficiency does not affect Treg numbers in the skin, nor their ability to produce IL-10.
    • Fig. S5. Skin TSLP and serum IgE levels in Foxp3eyfp-creRorafl/fl mice and Foxp3eyfp-cre controls.
    • Fig. S6. Effect of lack of RORα in Tregs on MC903-driven blood eosinophilia and on IL-4, IL-13, and chemokines in MC903-treated skin.
    • Fig. S7. Analysis of correlation of RNA-seq samples.
    • Fig. S8. Increased number and motility of Tregs in MC903-treated ear skin.
    • Table S1. Summary of RNA-seq experiments.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Intravital two-photon imaging of the untreated ear dermis of a Foxp3egfp animal.
    • Movie S2 (.mov format). Intravital two-photon imaging of the MC903-treated ear dermis of Foxp3egfp mice.
    • Data file (Microsoft Excel format)

    Files in this Data Supplement:

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