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The commensal skin microbiota triggers type I IFN–dependent innate repair responses in injured skin

Nature Immunology volume 21, pages 1034–1045 (2020)Cite this article

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Abstract

Skin wounds heal by coordinated induction of inflammation and tissue repair, but the initiating events are poorly defined. Here we uncover a fundamental role of commensal skin microbiota in this process and show that it is mediated by the recruitment and the activation of type I interferon (IFN)-producing plasmacytoid DC (pDC). Commensal bacteria colonizing skin wounds trigger activation of neutrophils to express the chemokine CXCL10, which recruits pDC and acts as an antimicrobial protein to kill exposed microbiota, leading to the formation of CXCL10–bacterial DNA complexes. These complexes and not complexes with host-derived DNA activate pDC to produce type I IFNs, which accelerate wound closure by triggering skin inflammation and early T cell–independent wound repair responses, mediated by macrophages and fibroblasts that produce major growth factors required for healing. These findings identify a key function of commensal microbiota in driving a central innate wound healing response of the skin.

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Fig. 1: Neutrophils produce CXCL10 that recruits pDC into injured skin.
Fig. 2: Human skin blisters recapitulate findings of the murine skin injury model.
Fig. 3: The skin microbiota triggers CXCL10 expression in neutrophils via TLR2.
Fig. 4: CXCL10 activates TLR9 in pDC by forming complexes with DNA.
Fig. 5: CXCL10 promotes the ability of pDC to sense the skin microbiota.
Fig. 6: CXCL10 generates bacterial DNA complexes by killing the skin microbiota.
Fig. 7: Skin microbiota promotes inflammation and closure of skin wounds.
Fig. 8: Type I IFNs induce the expression of growth factors in dermal macrophages and skin fibroblasts.

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All data supporting the findings of this study are available within the article and its supplementary information and from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank M. Malissen from INSERM U1104 laboratory in CIML, Marseille, France for providing the Genista mice; B. Humbel and J. Daraspe from the Electron Microscopy Facility of the University of Lausanne for the electron microscopy; H. Maby-El Hajjami, L. Cagnon, S.A. Maillard and F. Stuber for recruiting healthy volunteers and for establishing and running the human skin blister study; and I. Surbeck and A. Joncic for technical assistance. This work was funded by the Swiss National Science Foundation to M.G. (grant no. 310030_163360), a grant from the Fondation Pierre Mercier to J.D.D., and an Alfred and Annemarie von Sick grant to D.E.S. and M.G.

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Author notes

  1. These authors contributed equally: Jeremy Di Domizio, Cyrine Belkhodja.

Authors and Affiliations

  1. Department of Dermatology, CHUV University Hospital, University of Lausanne, Lausanne, Switzerland

    Jeremy Di Domizio, Cyrine Belkhodja, Anissa Fries, Olivier Demaria, Curdin Conrad & Michel Gilliet

  2. Laboratory of Experimental and Molecular Immunology and Neurogenetics, UMR 7355 CNRS-University of Orleans, Orleans, France

    Pauline Chenuet & Bernhard Ryffel

  3. Department of Oncology, CHUV University Hospital, University of Lausanne, Lausanne, Switzerland

    Timothy Murray, Paula Marcos Mondéjar & Daniel E. Speiser

  4. Department of Dermatology, Medical Faculty, Heinrich-Heine-University, Duesseldorf, Germany

    Bernhard Homey

  5. ETH Zurich, Institute of Molecular Health Sciences, Zurich, Switzerland

    Sabine Werner

  6. Division of Immunology, Department of Pathology, Institute of Infectious Disease and Molecular Medicine, Health Sciences Faculty, University of Cape Town, and South Africa Medical Research Council, Cape Town, South Africa

    Bernhard Ryffel

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Contributions

J.D.D. and C.B. performed and analyzed most of the experiments and participated in their design. P.C., A.F. and B.R. helped establish and perform GF experiments. T.M., P.M.M. and D.E.S. designed and performed the skin blister induction experiments. O.D. and C.C. helped with some experiments and, along with S.W., contributed to discussions throughout the study. B.H. performed immunohistology for CXCL10. M.G. conceived and supervised the study, was involved in the design and evaluation of all experiments and wrote the manuscript along with J.D.D. and C.B., with comments from coauthors.

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Correspondence to Michel Gilliet.

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The authors declare no competing interests.

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Peer review information Zoltan Fehervari 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 CXCL10 is expressed by skin-infiltrating neutrophils.

Images of histological sections of injured skins harvested before (0h) or 6, 12, and 24 hours postinjury. Sections were stained by chromogenic immunohistochemistry using DAB with control isotypes, anti-CXCL10, or anti-Ly6G antibodies. Skin sections were then counterstained with hematoxylin. Images are representative of 5 different mice. Scale bars are shown and represent 50 μM.

Extended Data Fig. 2 Accumulation of non-hemolytic catalase-positive cocci in the dermis of tape-stripped skin.

a FISH staining of bacterial 16S rRNA of uninjured (top) and tape-stripped (bottom) murine skin. Reverse probes for 16S rRNA were used as controls. Scale bars are shown on images. b, Colorimetric Gram staining of uninjured and injured skin, showing the presence of cocci in the dermis of stripped skin but not in uninjured skin. a-c, images are representative of 5 different mice. c, Photographs of a blood-agar plate showing non-hemolytic bacterial colonies grown overnight from tape-stripped skin of three mice (top line). One colony per mouse was tested for catalase activity by adding 3% H2O2 . Absence of gas bubbles indicates catalase negativity (middle line). Gram staining of bacteria harvested from the injured skin analyzed by light microscopy (bottom line).

Extended Data Fig. 3 Topical Neosporin ointment treatment efficiently reduces commensal bacteria in murine skin.

a, Photographs of a blood-agar plate showing bacterial colonies grown overnight from skin samples harvested from five different vehicle-treated and Neosporin-treated mice (left). The number of colony forming units (CFU) in each group was then calculated (right). Data represent the mean + s.e.m. n = 5. *P<0.01, **P<0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test). b, Quantification of bacterial 16S rDNA by qPCR in the skin of vehicle-treated, Neosporin-treated, or Neosporin-treated Staphylococcus epidermidis-recolonized SPF-housed mice, and untreated or Staphylococcus epidermidis-recolonized germ-free (GF) mice.

Extended Data Fig. 4 Only CXCL10 skin injection induces Ifna, but all CXCL9, CXCL10, and CXCL11 kill bacteria, bind DNA, and induce type I IFN production in pDC.

a, Expression of Ifna2 in the skin of mice 6 hours after intradermal injection of CXCL9, CXCL10, CXCL11, chemerin, or control saline. Data represent the mean + s.e.m. n = 4. *P<0.01 (one-way ANOVA followed by Tukey’s multiple comparisons test). b, Percentages of viable SytoGreen+ SytoxOrange- bacteria (Staphylococcus epidermidis) incubated for 24 hours with increasing concentrations of CXCL9, CXCL10, CXCL11, or Chemerin assessed by flow cytometry. Data represent the mean + sd of triplicates. c, Fluorimetric quantification of DNA staining by PicoGreen® dye after mixing of bacterial DNA with CXCL9, CXCL10, CXCL11, or Chemerin. A.U., arbitrary units. Data represent the mean + sd of triplicates. * P<0.05 (one-way ANOVA followed by Dunnett’s multiple comparisons test). d, Amounts of IFN-α produced by human blood-isolated pDC stimulated for 24 hours with purified DNA from bacteria in the presence or not of CXCL9, CXCL10, CXCL11, or Chemerin. Data represent the mean + sd. n=3. **** P<0.0001 (two-way ANOVA followed by Sidak’s multiple comparisons test).

Extended Data Fig. 5 Intradermal injection leads to bacterial translocation from the epidermis into the dermis.

a, Colorimetric Gram staining of murine skin 1 hour after intradermal injection of 50 ml of CXCL10-containing solution with a 30G needle showing bacterial cocci translocated into the dermis. b, FISH staining of bacterial 16S rRNA gene of the same intradermally injected murine skin than in (a). Reverse probes for 16S rRNA gene were used as controls. a-b, images are representative of 5 different mice. Scale bars are shown on images.

Extended Data Fig. 6 Expression of inflammatory and repair response genes in tape-stripped vs full-thickness skin injury.

Expression of Il1b, Ifna2, Cxcl10, Il6, Il12a, Il22, Il17a, Il23a, Il10, Tnf, Fgf2, Fgf7, Tgfb1, Vegfa, Pdgf, Ctgf, Egf, Il24, and Ifng genes in the uninjured or injured skin (tape-stripping or full thickness injury) at 12, 24, and 48 hours after skin injury assessed by RT-qPCR. Data are from five different mice per group and presented as a heatmap of fold changes. Data are representative of four independent experiments.

Extended Data Fig. 7 Neosporin treatment delays the wound closure rate, which is restored by re-colonizing mice with S.epidermidis or intradermal injection of IFN-α.

a, Photographs of skin wounds in untreated (vehicle), Neosporin-treated, Staphylococcus epidermidis-recolonized Neosporin-treated, and IFNα-intradermally injected Neosporin treated mice at the time of wounding (0) and everyday up to 6 days post-injury. b, Wound closure kinetics shown as percentages of initial wound size in the different mice groups as in (a). Data are the mean±SD of three mice per group are representative of 2 independent experiments. Scale bars are shown on images.

Extended Data Fig. 8 Skin injury activates dermal cells to produce growth factors.

a, Confocal microscopy images of FFPE murine skin sections at 0 (before) and 48 hours after injury (bottom) stained with DAPI (blue), and antibodies against vimentin (green), and FGF2 (red) or with isotype control (red). Images shown are representative of three different mice from two independent experiments. Scale bars shown on images represent 50 μm. b, Confocal microscopy images of frozen murine skin sections at 0 (before) and 48 hours after injury (bottom) stained with DAPI (blue), and antibodies against CD206 (green), and TGFb (red) or with isotype control (red). Images shown are representative of three different mice from two independent experiments. Scale bars shown on images represent 50 μm.

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Di Domizio, J., Belkhodja, C., Chenuet, P. et al. The commensal skin microbiota triggers type I IFN–dependent innate repair responses in injured skin. Nat Immunol 21, 1034–1045 (2020). https://doi.org/10.1038/s41590-020-0721-6

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