Commensal–dendritic-cell interaction specifies a unique protective skin immune signature

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Abstract

The skin represents the primary interface between the host and the environment. This organ is also home to trillions of microorganisms that play an important role in tissue homeostasis and local immunity1,2,3,4. Skin microbial communities are highly diverse and can be remodelled over time or in response to environmental challenges5,6,7. How, in the context of this complexity, individual commensal microorganisms may differentially modulate skin immunity and the consequences of these responses for tissue physiology remains unclear. Here we show that defined commensals dominantly affect skin immunity and identify the cellular mediators involved in this specification. In particular, colonization with Staphylococcus epidermidis induces IL-17A+ CD8+ T cells that home to the epidermis, enhance innate barrier immunity and limit pathogen invasion. Commensal-specific T-cell responses result from the coordinated action of skin-resident dendritic cell subsets and are not associated with inflammation, revealing that tissue-resident cells are poised to sense and respond to alterations in microbial communities. This interaction may represent an evolutionary means by which the skin immune system uses fluctuating commensal signals to calibrate barrier immunity and provide heterologous protection against invasive pathogens. These findings reveal that the skin immune landscape is a highly dynamic environment that can be rapidly and specifically remodelled by encounters with defined commensals, findings that have profound implications for our understanding of tissue-specific immunity and pathologies.

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Figure 1: Remodelling of skin immunity by commensal colonization.
Figure 2: Distinct commensal species impose specific immune signatures in the skin.
Figure 3: Distinct dendritic cell subsets cooperate to mediate host–commensal interaction in the skin.
Figure 4: Commensal-driven CD8+ T cell response is specific for S. epidermidis antigen.

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Primary accessions

Sequence Read Archive

Data deposits

454 sequencing data are deposited in the Sequence Read Archive under accession number SRP039428.

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Acknowledgements

This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases (NIAID) and by the Human Frontier Science Program (C.W.). We thank the NIAID animal facility staff, in particular A. Gozalo (isolation of S. xylosus); D. Trageser-Cesler and C. Acevedo (NIAID gnotobiotic facility); K. Holmes, C. Eigsti and E. Stregevsky (NIAID sorting facility); K. Frank and F. Stock (MALDI-TOF analysis); B. Malissen (Langerin–GFP reporter mice); H. C. Morse (Irf8−/− mice); D. Kaplan (Langerin-DTA mice); R. Bosselut (B2m−/− mice); S. B. Hopping (collection of human skin tissue samples); J. Oh, K. Loré, and the Brenchley laboratory (technical advice and reagents); and K. Beacht and L. Martins dos Santos for technical assistance. We also thank the Belkaid laboratory for critical reading of the manuscript.

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Contributions

S.N., N.B., and Y.B. designed the studies. S.N. and N.B. performed the experiments and analysed the data. J.L.L. assisted with in vitro co-culture studies and S.-J.H. with innate cell analysis and imaging. O.J.H. and C.W. provided technical assistance. S.C. and C.D. provided technical advice and performed 454 pyrosequencing. S.C. and M.Q. analysed 454 pyrosequencing data. S.H. assisted in processing of human and non-human primate skin tissue samples. A.L.B. performed NanoString data analysis. J.M.B. and H.H.K. provided technical advice and skin tissue samples from non-human primates and human patients, respectively. R.T., K.M.M. and M.M. assisted with design of dendritic cell depletion strategies. J.A.S. helped to design sequencing studies and provided guidance on bacterial isolates. S.N., N.B. and Y.B. wrote the manuscript.

Corresponding author

Correspondence to Yasmine Belkaid.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Assessment of Foxp3+ regulatory T cells and cytokine production by effector T cells after S. epidermidis topical application and/or intradermal inoculation.

a, Frequencies and absolute numbers of skin regulatory (CD45+ TCRβ+ CD4+ Foxp3+) T cells in unassociated mice (Ctrl, n = 4) and mice associated with S. epidermidis (n = 4) at day 14 post first topical application. b, Absolute numbers of effector T cells producing IL-17A after PMA/ionomycin stimulation in the skin (ear pinnae and flank), the lung or the small intestine lamina propria (gut) at day 14 post topical association (Ctrl, n = 4–5; S. epi., n = 4–5). c, d, Enumeration of colony-forming units and absolute numbers of effector T cells producing IFN-γ or IL-17A (PMA/ionomycin) from the skin 2 weeks post application with different doses (107, 108 or 109 c.f.u. per ml) of S. epidermidis (n = 4 per group). e, Frequencies and absolute numbers of neutrophils and monocytes in the skin of mice 14 days after the first topical application or intradermal inoculation with S. epidermidis (n = 4 per group). f, Assessment of cytokine production (mean ± s.e.m., n = 3 per time point) by leukocytes from the ear skin tissue 24 and 48 h after topical association with S. epidermidis. Unassociated mice were used as controls. No significant amounts of IL-4, IL-5, IL-17A, IL-18, IL-21 or IL-22 could be detected at the time of analysis. g, IFN-γ and IL-17A production by skin effector T cells in mice 7 days after S. epidermidis topical application or intradermal inoculation. h, Frequencies of IFN-γ and IL-17A-producing effector T cells in the skin of mice 7 and 14 days after the first topical application or intradermal inoculation of S. epidermidis (n = 4–5 mice per group). All results shown are representative of 2–3 experiments with similar results. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not statistically significant as calculated by Student’s t-test.

Extended Data Figure 2 Assessment of CD8+ T-cell responses in the skin of specific pathogen-free and germ-free mice after topical application with skin commensals.

a, Mice were left unassociated (Ctrl, n = 5) or topically associated with S. epidermidis human isolate (n = 5), S. xylosus (n = 3), S. epidermidis murine isolate (S.epi 42E03, n = 2), S. lentus (n = 2), R. nasimurium (n = 2), S. aureus (n = 5), C. pseudodiphtheriticum (n = 3) or P. acnes (n = 3). Quantification of colony-forming units from the ears after topical application is shown 2 weeks after first association. b, Frequencies and numbers of effector (CD45+ TCRβ+ CD4+ Foxp3) T cells producing IFN-γ or IL-17A after PMA/ionomycin stimulation in the skin of mice from a at day 14 post first topical application. Bar graphs represent the mean value from two mice. c, Frequencies of skin CD4+ and CD8β+ effector T cells in mice from a at day 14 post first topical application. d, Absolute numbers of IFN-γ- and IL-17A-producing CD8β+ effector T cells in the skin of unassociated (Ctrl) mice or mice associated with different doses (107, 108 or 109 c.f.u. per ml) of S. epidermidis (n = 4 per group). e, Absolute numbers of skin CD8β+ effector T cells in unassociated (Ctrl, n = 3) mice or mice associated with 1 ml (n = 5) or 5 ml (n = 5) of a suspension (109 c.f.u. per ml) of S. epidermidis. f, Flow cytometric assessment of the frequencies of CD4+ and CD8β+ effector T cells and absolute numbers of CD8β+ effector T cells in SPF (n = 3 per group) and germ-free (GF, n = 4 per group) mice 2 weeks after S. epidermidis topical application. g, Absolute numbers of IFN-γ- and IL-17A-producing CD8β+ effector T cells in the skin of unassociated (Ctrl) or S. epidermidis-associated C57BL/6 and BALB/c mice at 14 days post first topical application (n = 5 per group). For dg, all results shown are representative of 2–3 independent experiments with similar results. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not statistically significant as calculated with Student’s t-test. h, Quantification of colony-forming units from the ears of adult mice born from S. epidermidis-associated (S. epi+, n = 3) or unassociated (Ctrl, n = 3) breeder pairs. Flow plots and bar graphs (mean ± s.e.m.) illustrate the frequencies of CD4+ and CD8β+ effector T cells and absolute numbers of CD8β+ effector T cells, respectively. n.d., not detected; **P < 0.01 as calculated with Student’s t-test.

Extended Data Figure 3 CD8+ T cells accumulate preferentially in the epidermidis after topical application of S. epidermidis.

a, Proportion of effector (CD45+ TCRβ+ Foxp3) CD8β+ T cells in the epidermal and dermal compartments of the ear skin tissue 2 weeks after the first S. epidermidis topical application. b, Representative imaging volume projected along the x axis of ears from Langerin–GFP reporter mice at 14 days post first topical application with S. epidermidis. Scale bars, 30 μm. c, Numbers of CD3+ CD8β+ cells producing IFN-γ or IL-17A (after PMA/ionomycin stimulation) from normal nonhuman primate (NHP) skin (n = 8). d, Assessment of IL-17A production in the supernatant of CD8β+ T cells purified from the skin of mice topically associated with S. epidermidis and cultured overnight in presence of anti-CD3ε alone (Ctrl) or with IL-1α and IL-1β (+ IL-1). Bars represent the mean value ± s.e.m. (n = 3, **P < 0.01 as calculated with Student’s t-test). Results shown in a, c and d are representative of 2–3 experiments with similar results.

Extended Data Figure 4 Depletion strategies for the different subsets of skin dendritic cells.

a, Gating strategy for various dendritic cell subsets in the skin. Cells are first gated on live CD45+ CD11c+ MHCII+. Subsets of dendritic cells are then defined as follows: Langerhans cells (LC) are gated on CD11b+ CD207(Langerin)+ cells, CD103+ dendritic cells (CD103 DC) on CD11b CD207+ cells and CD11b+ dermal dendritic cells (CD11b DC) on CD11b+ CD207 cells. b, Comparative assessment by flow cytometry of Langerhans cell, CD103 DC and CD11b DC in the ear skin of unassociated mice (control) and mice first topically associated with S. epidermidis 2 weeks earlier. c, Absolute numbers of Langerhans cell, CD103 DC and CD11b DC 2 weeks after the first topical application of S. epidermidis in wild-type (WT, n = 3), Langerin–DTA (Lan–DTA, n = 3), Batf3−/− (n = 3) or Irf8−/− (n = 3) mice and in mice treated with anti-CSF1R (n = 3) or isotype control (rat IgG, n = 3) antibodies. d, Absolute numbers of CD11chiMHCII+ CD8+ DEC205+ dendritic cells in the spleen and the skin draining lymph node (dLN) of wild-type (n = 5) and Batf3−/− (n = 6) mice. e, Phenotypic analysis of CD45+ MHCII+ CD11c+ cells by flow cytometry and absolute numbers of effector (CD45+ TCRβ+ Foxp3) CD8β+ T cells and IL-17A- or IFN-γ-producing CD8β+ T cells in wild-type (n = 3) and Irf8−/− (n = 3) mice 2 weeks after the first topical application of S. epidermidis. f, Assessment of IL-1 production by leukocytes from the ear skin tissue of S. epidermidis-associated mice treated with anti-CSF1R (n = 4) or isotype control (rat IgG, n = 5) antibodies. g, Frequencies of total and IFN-γ- or IL-17A- producing CD8β+ effector T cells in S. epidermidis-associated Irf4 fl/fl × CD11ccre+ (n = 3) and littermate control (n = 3) mice. All data shown in this figure are representative of 2–3 experiments with similar results. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not statistically significant as calculated with Student’s t-test.

Extended Data Figure 5 Commensal-driven CD4+ and CD8+ T-cell responses in the skin tissue and the skin draining lymph nodes are specific for commensal antigens.

a, Frequencies of IFN-γ- or IL-17A-producing CD8β+ T cells in overnight co-cultures of splenic dendritic cells (SpDC) and CD8β+ T cells purified from the skin draining lymph node (dLN) of mice first topically associated with S. epidermidis 2 weeks earlier. b, Frequencies of IFN-γ- and IL-17A-producing CD8β+ T cells in overnight co-cultures of SpDC and CD8β+ T cells purified from the skin of mice 14 days after the first S. epidermidis application. Dendritic cells were purified from either wild-type (WT) or Abb−/− B2m−/− mice. c, d, Frequencies of IFN-γ- and IL-17A-producing CD4+ T cells in overnight co-cultures of SpDC and CD8β+ T cells purified from the skin ear tissue or the skin dLN of mice 14 days after the first S. epidermidis application. For a, b and d, Ctrl, naive SpDC; S. epi, SpDC + heat-killed S. epidermidis; Abb/B2m S. epi, Abb−/− B2m−/− SpDC + heat-killed S. epidermidis. e, Frequencies of IFN-γ- and IL-17A-producing CD4+ T cells in overnight co-cultures of SpDC and CD8β+ T cells purified from the skin ear tissue or the skin dLN of mice 14 days after the first S. xylosus application. Ctrl, naive SpDC; S. xylo, SpDC + heat-killed S. xylosus; Abb/B2m S. xylo, Abb−/− B2m−/− SpDC + heat-killed S. xylosus. All data shown in ad are representative of three independent experiments. Graph bars represent the mean ± standard deviation of triplicate cultures. **P < 0.01, ***P < 0.0001, ****P < 0.0001 as calculated with Student’s t-test. f, S100a8 and S100a9 gene expression in dorsal skin biopsies of mice associated with different doses (107, 108 or 109 c.f.u. per ml) of S. epidermidis 2 weeks after the first topical application (n = 4 per group). Data are expressed as fold increase over gene expression in unassociated control mice.

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Naik, S., Bouladoux, N., Linehan, J. et al. Commensal–dendritic-cell interaction specifies a unique protective skin immune signature. Nature 520, 104–108 (2015). https://doi.org/10.1038/nature14052

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