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Direct control of regulatory T cells by keratinocytes

Nature Immunology volume 18, pages 334–343 (2017)Cite this article

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Abstract

Environmental challenges to epithelial cells trigger gene expression changes that elicit context-appropriate immune responses. We found that the chromatin remodeler Mi-2β controls epidermal homeostasis by regulating the genes involved in keratinocyte and immune-cell activation to maintain an inactive state. Mi-2β depletion resulted in rapid deployment of both a pro-inflammatory and an immunosuppressive response in the skin. A key target of Mi-2β in keratinocytes is the pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP). Loss of TSLP receptor (TSLPR) signaling specifically in regulatory T (Treg) cells prevented their activation and permitted rapid progression from a skin pro-inflammatory response to a lethal systemic condition. Thus, in addition to their well-characterized role in pro-inflammatory responses, keratinocytes also directly support immune-suppressive responses that are critical for re-establishing organismal homeostasis.

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Figure 1: Loss of Mi-2β in the epidermis causes rapid keratinocyte activation.
Figure 2: Mi-2β actively represses genes involved in keratinocyte activation and immune cell regulation.
Figure 3: TSLPR's protective role in skin pro-inflammatory responses.
Figure 4: Activation of skin-associated Treg cells is dependent on TSLPR signaling.
Figure 5: TSLPR signaling in Treg cells supports immunosuppression and organismal homeostasis.
Figure 6: Transcriptional effects of TSLPR signaling in skin effector Treg cells.

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Acknowledgements

We thank P. Chambon (Institute of Genetics and Cellular and Molecular Biology) and V.K. Kuchroo (Brigham and Women's Hospital) for the Krt14-Cre-ERT2 mice and Foxp3-IRES-EGFP mice, respectively. We thank M.E. Bigby for consultation on lymphocyte isolation from the skin and L.M. Francisco and A.H. Sharpe for consultation on Treg cell and DC analysis. We thank E. Wu and B. Czyzewski for mouse husbandry, A. Cho, M. Ahl, J.E. King and J. Brandollni for bone marrow transplantation, and T. Minegishi for R platform support. We also thank J.M. Park, H. Cantor, H.J. Kim, F. Gounari and K. Khashayarsha for critical comments on the manuscript. This research was supported by NIH R21 AR055813, RO1 AR064390 and R01AR069132 to K.G., NIH RO1 AI068731 and PO1 HL098067 to S.F.Z., and NIH R01 AR055256 to B.A.M. K.G. is an MGH scholar supported by J. de Gunzburg. J.H. was supported by a Shiseido grant. High-throughput RNA sequencing was performed at the Bauer Center for Genomic research Harvard University, Cambridge.

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Authors and Affiliations

  1. Cuteneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA

    Mariko Kashiwagi, Junichi Hosoi, Bruce A Morgan & Katia Georgopoulos

  2. Immunology Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA

    Jen-Feng Lai & Steven F Ziegler

  3. Department of Cell Biology, State University of New York Downstate Medical Center, Brooklyn, New York, USA

    Janice Brissette

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Contributions

M.K. designed and performed experiments and analyzed experimental data. J.H. designed and executed cytokine studies on primary cultured keratinocytes and advised on DC and DETC studies. J.-F.L. provided experimental materials and advised on immunological studies. J.B. advised on keratinocyte transcriptional studies. S.F.Z. provided experimental materials and guidance throughout the project. K.G and B.A.M supervised research and analyzed data. M.K., K.G. and B.A.M wrote the manuscript.

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Correspondence to Mariko Kashiwagi or Katia Georgopoulos.

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Integrated supplementary information

Supplementary Figure 1 Pro-inflammatory responses in the Mi-2β deficient skin

(a) Clinical pictures of mice with Mi-2β depleted epidermis showing flaky skin in the shaved dorsal area and around the eyes. (b) Flow cytometric analysis of single cell suspension from wild-type and Mi2Δ ear skin stained for CD45, CD4 and CD3ɛ. The clinical pictures shown in (a) was seen with more than ten Mi2D mice. Data shown in (b) were representative of four independent experiments performed on a total of WT N=6 and Mi2Δ N=6 mice.

Supplementary Figure 2 The role of Mi-2β in the regulation of keratinocyte-specific gene networks

(a) Flow cytometric analysis of single cell suspension stained for ITGA6, CD45 and CD34 in wild-type and Mi2Δ epidermis. mRNAs were isolated from FACS-sorted ITGA6+CD45–CD34– basal keratinocytes and subjected to RNA-sequencing. (b) Expression of genes relevant to keratinocytes activation and migration are shown as normalized exon mapping reads (mean+s.e.m.). (c) A model is shown of Mi-2β-based negative regulation of gene networks in keratinocytes in steady state epidermis and how this effect is reversed upon Mi-2β depletion. Data shown in (a-b) were generated from two independent experimental groups with pooled samples from WT N=6 and Mi2Δ N=4 mice.

Supplementary Figure 3 Activation of Mi2Δ keratinocytes and DETCs is independent of TSLPR signaling

(a) Representative images of hematoxylin and eosin staining of dorsal skin from Mi2Δ, TMKO, RMKO and littermate controls were shown at day 7 and day 9 after induction of Mi-2β deletion in the skin. At day 9, skin hyperplasia was seen in Mi-2β mutant skin regardless of the presence of lymphocytes (Mi2Δ and RMKO), but was milder in RMKO mice. Scale bar, 50μm. WT and Mi2Δ sections shown for day 9 are also used in Fig. 1d. (b) DETCs (CD3+) and LCs (CD207+) in epidermal sheets from the ear were detected by immunofluorecence. The rounded morphology of DETCs, indicative of their activation, was seen in the Mi2Δ epidermis regardless of TSLPR signaling. Scale bar, 20μm. Data shown were representative of three independent experiments with more than five mice per group.

Supplementary Figure 4 Activation of Mi2Δ skin DCs is independent of TSLPR signaling

(a-b) Flow cytometric analysis of MHCclassIIhi dendritic populations in sDLNs from wild-type, TSLPRΔ, Mi2Δ, TMKO (a) and RagΔ and RMKO (b) mice. Three major populations were revealed by staining for CD207 (Langerin), EpCAM, and CD11c. The activation and maturation state of DCs was measured by expression of CD40, and CD86 in MHCclassIIhiEpCAMhiCD207+ LCs (red) and MHCclassIIhiEpCAMloCD207– DCs (blue). In Mi2Δ skin both LCs and other DCs were increased and showed an activated phenotype, regardless of the Rag or TSLPR mutations. Data shown were representative of three independent experiments with more than five mice per group.

Supplementary Figure 5 Activation of Mi2Δ skin Treg cells is dependent for TSLPR signaling

(a) Absolute cell numbers of CD4+ and CD8+ T cells in sDLNs are shown. *P < 0.05, **P < 0.01, ***P < 0.0001 (two-tailed unpaired t-test). (b) A full version of the Treg cell and Teff cell analysis shown in Fig. 4b is presented here. Expression of CD25, CD44, TNFR2, CTLA4, KLRG1, CD103 and the Ki67 antigen was analyzed in CD4+Foxp3+ Treg cells and CD44 and CD69 was analyzed in CD4+Foxp3- Teff cells from wild-type, TSLPRΔ, Mi2Δ, and TMKO sDLNs. Percent of cells in the gate or MFI for these markers are shown. (c) In vitro Treg cell immunosuppressive assay. CD4+CD62L+Foxp3-GFP- cells (Teff) were co-cultured with CD4+Foxp3-GFP+ cells (Treg) from Mi2Δ sDLN at indicated ratios (Teff: Treg) and stimulated with anti-CD3ɛ in the presence of irradiated-APCs for 3 days. The effect on Teff proliferation was assessed by the change in PI+ S phase cells. Treg cells from sDLNs or spleen of wild-type mice were used as a control. (d) CD4+Foxp3- Teff cells were stimulated in vitro and tested for intracellular expression of pro-inflammatory cytokines. CD4+Foxp3-GFP– T cells from wild-type, Mi2Δ and TMKO sDLN were stimulated with anti-CD3ɛ and anti-CD28 for 16 h. Cells were re-stimulated with PMA and ionomycin for 4 h and stained for intracellular IL-2, TNF and IFNγ. (–); without PMA or ionomycin, (+); with PMA and ionomycin. Data shown in (a) were generated from five independent experiments with a total of WT N=5, TSLPRΔ N=5, Mi2Δ N=10, and TMKO N=10 mice (mean+/– s.e.m). Data shown in (b) were representative of four independent experiments with WT N=5, TSLPRΔ N=5, Mi2Δ N=4, TMKO, N=4 mice, in (c-d) were representative of two experiments with pooled samples from WT N=14 and Mi2Δ N=5 mice.

Supplementary Figure 6 Direct induction of Treg cell activation by TSLPR signaling

(a-b) Analysis of CD4+Foxp3+CD25+ Treg cells in the skin and sDLNs of chimeric wild-type or Mi2D mice is shown. TSLPRΔ (donor) and wild-type (recipient) Treg cells were distinguished by Foxp3-GFP expression in the recipient population. (c) Ratios of TSLPRΔ to wild-type Treg cells in skin and sDLN are shown. *P < 0.01 (two-tailed unpaired t-test). (d) Disease development and staging for 4-OHT treated wild-type and Mi2Δ chimeras is shown. Data were generated from three independent experiments with WT N=5 and Mi2Δ N=5 chimeric mice.

Supplementary Figure 7 Treg cell immunosuppression is directly dependent on TSLPR signaling

Flow cytometric analysis of CD4+ T cells from skin and sDLNs, for Foxp3 and CD25 expression is shown in (a), and for Foxp3, CTLA4 and CD103 expression is shown in (b). Both a reduction in activation and number of TSLPRΔ Treg cells was seen. Data were representative of four independent experiments with Rag: TSLPR+ Treg N=5, Rag:TSLPRD Treg N=5, RMKO:TSLPR+ Treg N=4, and RMKO:TSLPRD Treg N=4 chimeric mice.

Supplementary Figure 8 IL-2R signaling is not required for Treg cell activation in Mi2Δ skin

(a) Schema of anti-CD25 treatment and induction of Mi-2β deletion with 4-OHT with time points of analysis. (b-c) Flow cytometric analysis of single cell suspension staining for CD3ɛ, CD4, CD25 and Foxp3 in WT and Mi2Δ skin with or without anti-CD25 treatment. Data shown are representative of three independent experiments, two at day 8 and one at day 14 after Mi-2β deletion with WT:PBS N=3, WT:anti-CD25 N=4, Mi2Δ:PBS N=2 and Mi2Δ:anti-CD25 N=4 mice.

Supplementary Figure 9 Direct control of regulatory T cells by keratinocytes

A model on how a pro-inflammatory signal overrides the repressive activity of the Mi-2β/NuRD complex in keratinocytes to induce TSLP expression and activate TSLPR signaling in skin-associated Treg cells. TSLPR signaling in Treg cells leads to the rapid induction of soluble and membrane bound immunosuppressive factors that are critical for an early Treg cell immunosuppressive function that prevents development of a systemic inflammatory response with dire for the organism consequences.

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Kashiwagi, M., Hosoi, J., Lai, JF. et al. Direct control of regulatory T cells by keratinocytes. Nat Immunol 18, 334–343 (2017). https://doi.org/10.1038/ni.3661

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