Abstract
The skin has a dual function as a barrier and a sensory interface between the body and the environment. To protect against invading pathogens, the skin harbours specialized immune cells, including dermal dendritic cells (DDCs) and interleukin (IL)-17-producing γδ T (γδT17) cells, the aberrant activation of which by IL-23 can provoke psoriasis-like inflammation1,2,3,4. The skin is also innervated by a meshwork of peripheral nerves consisting of relatively sparse autonomic and abundant sensory fibres. Interactions between the autonomic nervous system and immune cells in lymphoid organs are known to contribute to systemic immunity, but how peripheral nerves regulate cutaneous immune responses remains unclear5,6. We exposed the skin of mice to imiquimod, which induces IL-23-dependent psoriasis-like inflammation7,8. Here we show that a subset of sensory neurons expressing the ion channels TRPV1 and Nav1.8 is essential to drive this inflammatory response. Imaging of intact skin revealed that a large fraction of DDCs, the principal source of IL-23, is in close contact with these nociceptors. Upon selective pharmacological or genetic ablation of nociceptors9,10,11, DDCs failed to produce IL-23 in imiquimod-exposed skin. Consequently, the local production of IL-23-dependent inflammatory cytokines by dermal γδT17 cells and the subsequent recruitment of inflammatory cells to the skin were markedly reduced. Intradermal injection of IL-23 bypassed the requirement for nociceptor communication with DDCs and restored the inflammatory response12. These findings indicate that TRPV1+Nav1.8+ nociceptors, by interacting with DDCs, regulate the IL-23/IL-17 pathway and control cutaneous immune responses.
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Change history
05 June 2014
An author was added to the author list and relevant contributions added to the Author Contributions section.
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Acknowledgements
We thank G. Cheng, M. Flynn and S. Omid for technical support; M. Perdue, L. Jones and E. Nigro for secretarial assistance; E. Gray for providing detailed protocols for flow cytometry of skin samples; J. Harris for discussion and providing detailed RNA isolation protocols of skin samples; R. T. Roderick for histopathology analysis; T. Liu and J. Ru-Rong for providing the RTX denervation protocol; M. Ghebremichael for statistical advice; F. Winau and J. L. Rodriguez-Fernandez for critical reading of the manuscript and members of the von Andrian laboratory for discussion and advice. This work was supported by National Institutes of Health (NIH) grants AI069259, AI078897, AI095261 and AI111595 (to U.H.v.A.), NIH 5F31AR063546-02 (to J.O.-M.) and the Human Frontiers Science Program, Charles A. King Trust and National Psoriasis Foundation (to L.R.-B.).
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Contributions
L.R.-B., J.O.-M., and U.H.v.A. designed the study. L.R.-B., J.O.-M., M.P., E.N., A.T. and D.A. performed and collected data from experiments, and L.R.-B., J.O.-M., A.T. and E.N. analysed data. J.N.W. provided reagents and gave conceptual advice. S.P. contributed to experiments in Extended Data Fig. 7d and provided technical support. J.O.-M., L.R.-B. and U.H.v.A. wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 6OHDA treatment ablates sympathetic nerve function and reduces ear swelling, but does not ameliorate the inflammatory response to IMQ treatment.
a, The experimental protocol was as follows. Mice were injected intraperitoneally with 6OHDA, resulting in a reversible chemical sympathectomy lasting for approximately 2 weeks. After a rest period of 3 days animals were challenged topically on the ear with IMQ. b, Representative section of splenic white pulp showing B cells (B220, white), T cells (CD3, red), and TH+ (green) nerve fibres in vehicle (ascorbic acid)-treated and sympathectomized (6OHDA) mice. c–i, Analysis of the inflammatory response in ears of vehicle (ascorbic acid)-treated and sympathectomized (6OHDA) mice after daily topical IMQ challenge: timecourse of change in ear thickness of IMQ-treated ear relative to the contralateral ear (n = 10; *P < 0.05; **P < 0.01) (c) and total number of infiltrating monocytes (d) and neutrophils (e), and the amount of IL-17A (f), IL-17F (g), IL-22 (h) and IL-23p40 (i) in protein extracts of IMQ exposed ears at day 3 (*P < 0.05; n = 5). NS, not significant.
Extended Data Figure 2 RTX treatment diminishes noxious heat sensation and decreases the expression of nociceptor markers on dorsal root ganglia.
a, Schematic protocol of nociceptor ablation and induction of psoriasiform skin inflammation. RTX was injected subcutaneously into the back in three escalating doses (30 μg kg−1, 70 μg kg−1 and 100 μg kg−1) on consecutive days and mice were allowed to rest for at least 4 weeks before IMQ treatment. b, Denervation was confirmed by immersing the tail of mice into a temperature-controlled water bath maintained at 52 °C and the latency to the first tail movement to avoid water was measured (n = 6). c, Total RNA was isolated from dorsal root ganglia (level C1–C7) of vehicle (DMSO)- and RTX-treated mice and the levels of Trpv1, Scn10a (Nav1.8), Tac1 (substance P), Mrgprd, Trpm8 and Trpa1 mRNA relative to Gapdh were determined (n = 3).
Extended Data Figure 3 Gating strategy for myeloid cells and T-cell subsets from digested ear skin.
a, The ear skin of mice challenged for 3 days with IMQ was digested as described in Methods and, after doublet exclusion and gating on defined FSC-A, SSC-A parameters, infiltrating myeloid cells were gated as CD45+ I-Ab (Class-II)−, CD11b+ CD11c−, and then subdivided into inflammatory monocytes and neutrophils based on Ly-6C and Ly-6G staining. b, The ear skin of naive mice was digested as described in Methods and, after doublet exclusion and gating on defined FSC-A, SSC-A parameters, cutaneous T cells were gated on CD45+, Thy1+, and then divided into subsets based on staining for δ-TCR and β-TCR.
Extended Data Figure 4 RTX treatment reduces the immune cell infiltrate upon IMQ treatment in the skin but does not affect reservoirs of inflammatory monocytes and neutrophils at steady state.
a, The ear skin of vehicle (DMSO) or sensory denervated (RTX) mice was treated with topical IMQ cream daily and the total numbers of CD45+ cells were determined on day 3 as explained in Methods (n = 10). b, Representative histological sections of untreated and IMQ-treated ears at day 3 stained by haematoxylin and eosin (×20) (n = 5 per condition). c, d, Total inflammatory monocytes (CD45+, CD11b+, Ly-6Chigh) and neutrophils (CD45+, CD11b+, Ly-6Ghigh) were determined by flow cytometry (n = 5–10 mice per time point). Two-way ANOVA was run to compare total numbers of inflammatory monocytes and neutrophils between DMSO and RTX conditions over days 3–6 (****P < 0.0001). One-way ANOVA was run to compare total inflammatory monocytes and neutrophil numbers over days 3–6 within DMSO or RTX conditions (**P < 0.003). e, Bone marrow was isolated from wild-type and RTX mice from one femur and the frequency of inflammatory monocytes (CD45+, CD11b+, Ly-6Chigh, Ly-6G−) and neutrophils (CD45+, CD11b+, Ly-6Cmid, Ly-6G+) relative to CD45+ cells was determined by flow cytometry (n = 5). f, Spleens from wild-type and RTX mice were processed for flow cytometry and the frequency of inflammatory monocytes (CD45+, CD11b+, Ly-6Chigh, Ly-6G−) and neutrophils (CD45+, CD11b+, Ly-6Cmid, Ly-6G+) relative to CD45+ cells was determined (n = 5). NS, not significant.
Extended Data Figure 5 Leukocyte rolling fractions in skin venules of control and RTX-treated mice analysed by intravital microscopy.
Combined results are shown for 26 venules from 5 control mice and for 20 venules from 4 RTX-treated mice. Data are expressed as mean ± s.e.m. of four experiments. NS, not significant.
Extended Data Figure 6 Dermal γδ T cells represent a major source of IL-17F and IL-22 in skin during IMQ challenge and already express IL-23R at steady state.
a, Wild-type mice were challenged with IMQ and the total numbers of IL17F+ dermal γδ T cells and αβ T cells at 3 days (n = 15) or 6 days (n = 10) were quantified. b, Representative flow plots (related to those depicted in Fig. 2g) of gating for IL-22+ cells within dermal γδ T cells after 6 days of IMQ treatment. c, Ears of DMSO- or RTX-treated mice were exposed for 6 days to IMQ and the frequency of IL-17F+ and IL-22+ cells within αβ T cells was determined (n = 5). d, Auricular lymph node (aLN) cells from IL-23RGFP/+ mice were analysed by flow cytometry for expression of IL-23R–GFP+ cells within the γδ T cells and αβ T cell compartment at steady state (representative FACS plot from eight mice analysed). e, The ear skin from IL-23RGFP/+ mice was digested and analysed by flow cytometry and the distribution of T-cells subsets within IL-23R–GFP+ and IL-23R–GFP− fractions of Thy1+ cells determined (representative FACS plot from eight mice analysed). NS, not significant.
Extended Data Figure 7 TRPV1+ nociceptors regulate IMQ- and DNFB- induced dermatitis and are upstream of IL-23.
a–c, After 3 days of IMQ challenge, ears were harvested and processed for total RNA isolation and Il12b (a), Il23a (b) and Il12a (c) mRNA levels were analysed by qPCR (n = 5). d, DNFB (0.5% in acetone) was applied topically to DMSO and RTX mice. Time course of change in ear thickness of IMQ-treated ear relative to the contralateral ear is represented (n = 10). Two-way ANOVA was run to compare ear swelling under DMSO and RTX conditions over time (****P < 0.0001). e, Representative FACS plots from ears harvested after 24 h of DNFB application. f, IL-23RGFP/GFP mice were treated with RTX and then compared to wild-type and IL-23RGFP/GFP littermate controls during IMQ treatment. Ear thickness was calculated relative to the contralateral ear (n = 5). g, After two IL-23 injections into the ear skin of wild-type and IL-23RGFP/GFP mice, the frequency of IL-17F+ cells within dermal γδ T cells was determined by flow cytometry (n = 5). h, IL-23 was injected twice into the ear skin of vehicle- and RTX-treated mice and the total numbers of IL17A+ or IL-17F+ dermal γδ T cells per ear were determined by flow cytometry (n = 5). NS, not significant.
Extended Data Figure 8 Selective depletion of migratory and skin-resident myeloid cell subsets in ear skin and gating strategy used for sorting to isolate RNA from MHC-II+ cells in skin.
a, Wild-type mice were treated with anti-Gr-1 (clone RB6-8C5 to deplete neutrophils and inflammatory monocytes) or matched isotype control, challenged with IMQ for 3 days and skin was digested to quantify the total numbers of inflammatory monocytes and neutrophils per ear. Shown are representative plots pre-gated on CD45+ cells and quantification of cell numbers from n = 3 mice. b, DTX treatment resulted in depletion of both subsets of DDCs as well as Langerhans cells (LCs) but not macrophages. Cells were gated as shown in Extended Data Fig. 8c and normalized to levels in wild-type mice based on the frequency within the CD45+ population from n = 4 mice. c, Ear skin from naive mice was digested and analysed by flow cytometry for the indicated subsets. Shown is a representative plot pre-gated on CD45+ Class II+ cells from which further subsets were divided based on CD11b and CD11c expression and then F4/80 and CD103 as indicated. d, Total RNA from sorted cells was isolated and qPCR for Il12a relative to Gapdh was performed from naive and IMQ-treated ears after 6 h from n = 20 pooled mice.
Extended Data Figure 9 DDCs are found in close apposition to Nav1.8+ nociceptors and characterization of RTX-treated and Nav1.8-DTA mice.
a, Representative confocal micrographs of CD11c–YFP mice stained for β3-tubulin, Lyve-1 (lymphatics) and CD31 (blood and lymphatic endothelial cells). b, Three-dimensional quantification of DDC proximity to peripheral nerves in naive and 6 h post-IMQ treatment ears binned into contact (<0 µm), proximal (0–7 µm) and distal (>7 µm) fractions as explained in Methods (n of dendritic cells = 200). c, Total RNA from dorsal root ganglia (C1–C4) of littermate control and Nav1.8-DTA mice was isolated and levels of mRNA for Trpv1 (TRPV1), Scn10a (Nav1.8), Tac1 (substance P) and Trpa1 (TRPA1) were determined relative to Gapdh. This demonstrates the efficacy of the Nav1.8-DTA system and combined with the original reference characterizing the pain phenotype of these mice illustrates that a subset of peptidergic TRPV1+ nerve fibres is spared. d, Representative confocal micrograph of whole-mount ear skin of vehicle- and RTX-treated mice showing preserved nerve scaffold. e, Representative confocal micrographs of whole-mount ear skin of control and Nav1.8-DTA mice showing preserved nerve scaffold. Although dorsal root ganglia showed a loss of the hallmark ion channels of these nerve subsets (Extended Data Figs 1c, 9c), surprisingly we still observed that RTX mice and Nav1.8-DTA mice maintain a meshwork of nerves in the skin.
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CD11c-YFP+ dermal dendritic cells (DDC) show diverse interactions with NaV1.8+ nociceptor
NaV1.8-Cre-TdTomato mice in which nociceptors express fluorescent TdTomato protein (red) were reconstituted with CD11c-YFP bone marrow to visualize interactions of DDC (green) with cutaneous nerves in intact ears of anesthetized mice. Videos are displayed as maximum intensity projections of time-lapse recordings of 3D image stacks generated on a multi-photon intravital microscope (Prairie Technologies) using a 20X water immersion objective (Nikon). Scale bar = 40 µm. Playback is accelerated 300x over real-time. (MOV 20834 kb)
MHC-II-GFP+ dendritic cells migrate along GFAP+ Schwann cells in mouse skin
Glial fibrillary acidic protein (GFAP)-Cre-TdTomato mice in which Schwann cells express the fluorescent TdTomato protein (red) were reconstituted with MHC-II-GFP bone marrow to visualize MHC-IIbright dermal dendritic cell interactions (green) with cutaneous nerves in intact ears of anesthetized mice. Videos are displayed as maximum intensity projections of time-lapse recordings of 3D image stacks generated on a multi-photon intravital microscope (Prairie Technologies) using a 20X water immersion objective (Nikon). Scale bar = 10 µm. Playback is accelerated 300x over real-time. (MOV 2345 kb)
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Riol-Blanco, L., Ordovas-Montanes, J., Perro, M. et al. Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation. Nature 510, 157–161 (2014). https://doi.org/10.1038/nature13199
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DOI: https://doi.org/10.1038/nature13199
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