Abstract
The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11blo migratory DC2s—a DC2 population unique to the dermis—required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11bhi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4+GATA3+ helper T cells (TH), whereas antifungal IL-17+RORγt+ TH cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization.
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Data availability
The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files or are available from the authors upon reasonable requests. Sequencing data generated for this study including bulk and scRNA-seq data presented in Figs. 1, 2 and Extended Data Fig. 2: raw RNA-seq data as FASTQ files have been deposited in NCBI SRA under bioproject PRJNA668222. IL-13 KO mice are available from G.L.G., MIMR upon request and pending approval of a Material Transfer Agreement. Source data are provided with this paper.
Code availability
All code and associated parameters used for the analysis of bulk and scRNA-seq data as well as the reanalysis of publicly available dataset in Fig. 8 are available at: https://doi.org/10.5281/zenodo.5534993.
Change history
13 April 2022
A Correction to this paper has been published: https://doi.org/10.1038/s41590-022-01203-4
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Acknowledgements
We wish to thank G. Ogg, Oxford University, for sharing raw data of the scRNA-seq analysis of human skin blisters; P.M. Brunner and V. Vorstandlechner, Medical University of Vienna, for their advice on single-cell QC filtering and data analysis of scRNA-seq data of human skin biopsies and blisters cells; Y. Wang, Google, for developing the Shiny browser for scRNA-seq data and providing debugging help; M. Brewerton, Auckland DHB; A. Livingstone and T. Mosmann, University of Rochester; A. MacDonald, University of Manchester; S. Nutt, Walter and Eliza Hall Institute; and all colleagues at the Malaghan Institute of Medical Research for discussion and suggestions. The MR1 tetramer technology was developed jointly by J. McCluskey, J. Rossjohn and D. Fairlie, and the material was produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne, Australia. We also gratefully acknowledge the flow cytometry support of the members of the Hugh Green Cytometry Centre and the expert animal husbandry of the Biomedical Research Unit at the Malaghan Institute. This work was funded by an Independent Research Organization grant from the Health Research Council of New Zealand (HRC) to the Malaghan Institute (18-1003), an HRC Project grant to F.R. (18-510), and the Marjorie Barclay Trust. K.L.H. was supported by a Malaghan Institute Postdoctoral Fellowship and the Intramural Research Program of the NIAID, NIH. M.R.H. was supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant no. 105644/Z/14/Z), and a Lister Institute of Preventative Medicine Prize. R.G.D. was supported by an EMBO long-term fellowship (ALTF 1209-2019).
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J.U.M., O.L. and F.R. conceived the study; J.U.M., O.L., K.L.H., J.S.C., R.G.D., J.Y., G.R.W., L.M.-E., K.A.W., E.J.H., S.-C.T., S.C.C., S.V.D. and L.M.C. designed, carried out and analyzed experiments; D.A.E. and S.I.O. carried out bioinformatics analyses, wrote code and visualized results; C.R.M., F.B., A.S., R.T., D.G.O., D.J. and G.L.G. provided essential reagents and/or expertise; O.L., A.S., M.R.H. and F.R. supervised and/or funded research; J.U.M., O.L. and F.R. wrote the original draft; J.U.M., O.L., K.L.H. and F.R. finalized the submission and revision with input from all authors. O.L. and F.R. prepared the final manuscript for publication.
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Extended data
Extended Data Fig. 1 Gating strategy for DC and DC2 populations in the skin, lung and small intestine of naive mice and their draining lymph nodes.
a, Gating strategy for migratory DC2 subsets in skin, lung and small intestine (SI) dLNs. Generic gating on live single CD3−B220−Ly6G− (Lin−) and Ly6C− cells was performed for all samples before gating on dLN-specific DC2 subsets. b, Gating strategy for DC2 subsets in skin, lung and SI. Generic gating on live single CD45+CD3−B220−Ly6G− (Lin−) and CD64−Ly6C− cells was performed for all samples before gating on tissue-specific DC2 subsets. c, Numbers of DC2s in the skin, lung and SI dLN of Irf4f/f and Irf4f/f CD11c-Cre mice. Bar graphs show mean ± SEM for 5-8 mice pooled from 2 independent experiments. Each symbol refers to one mouse. P values were determined using a two-tailed Student’s t-test. *P < 0.05; ***P < 0.001; only significant comparisons are indicated.
Extended Data Fig. 2 Phenotypic and transcriptomic characterization of myeloid and DC2 populations in C57BL/6 and STAT6 KO mice.
a, Gating strategy for DC2 precursors in the bone marrow and skin. b, Representative Opt-SNE visualization of concatenated live single CD45+CD3−CD19− myeloid cells from the skin of naive C57BL/6 (C57) and STAT6 KO mice; combined data from both strains are shown in the color image. Opt-SNE was performed on 16648 events (2081 events/mouse, 4 mice per genoype, one experiment) using 12 parameters (XCR1, CD206, CD11b, Ly6c, CD11c, Ly6G, SIRPα, CD326, CD26, CD64, BST2, MHC-II) with default OMIQ settings and a perplexity of 30 with 1000 iterations. Population frequencies are shown in the bar graph. c, Numbers of migratory DC2s in the skin, lung and small intestine (SI) dLNs of naive C57 and STAT6 KO mice. d, Number of migratory DCs in the skin dLN of naive C57 and STAT6 KO mice. e, Relative frequencies (top) and numbers (bottom) of CD11bhi and CD11blo DC2s in the auricular (au), inguinal (i), axillary (ax), brachial (b) and popliteal (p) skin dLNs of naive C57 and STAT6 KO mice. f, Principal component (PC) analyses of all expressed genes in DC2 subsets from the skin, lung and SI dLNs of naive C57 and STAT6 KO mice. Each symbol refers to a biological replicate. g, UpSet plot showing the numbers of unique and shared differentially expressed genes (DEGs, including up- and downregulated genes) in the indicated DC2 subsets from the skin, lung and SI dLNs of STAT6 KO vs. C57 mice. b-e, Bar graphs show mean ± SEM for n=9 (b) n=4-5 (c) n=9-10 (d) n=5-8 (e) mice pooled from 2 independent experiments. Each symbol refers to one mouse. P values were determined using a two-tailed unpaired Student’s t-test. *P < 0.05; ***P < 0.001; only significant comparisons are indicated.
Extended Data Fig. 3 IL-13 signalling is necessary for the development of CD11blo DC2s in skin.
a, Experimental set-up of mixed wild type and STAT6 KO BM chimeras. The phenotype of skin DC2s from each donor BM is shown in the contour plots, subsets are quantified in the bar graph. b, Schematic of the genomic Il13 locus in wild type and IL-13 KO mice illustrating the deletion of exons 2 and 3. c, Phenotype of skin DC2s in naive mice of the indicated strains. All KO strains were on a C57BL/6 background except for the IL-13Rα1 KO which were on a BALB/c background. d, Relative frequencies of skin DC2 subsets in naive mice of the indicated strains. e,f, Frequencies of CD11blo DC2s (e) and DC2 subsets (f) in DC2 populations from skin, lung and small intestine (SI) dLNs of C57 controls, and IL-13 KO mice treated with either PBS or IL-13 fusion protein (IL-13fp). In (e), C57 controls are representative of several repeats. a,d,e,f, Bar graphs shows mean ± SEM for n=8 (a) n=5-15 (d) n=3-6 (e,f) mice pooled from 2 (a,d,e IL-13 KO, f) or 1 (e, C57 lung and SI dLNs) independent experiments. Each symbol refers to one mouse. P values were determined using two-way ANOVA with Sidak’s (a, lower graphs in d, IL-13 KO in e,f) or Tukey’s (d, top graphs) correction *P < 0.05; ***P < 0.001. Only relevant significant comparisons are indicated.
Extended Data Fig. 4 Identification of lymphoid cell populations in murine skin.
a, Gating strategy to identify lymphoid cell populations in the ear skin of naive 4C13R reporter mice. To define cells of lymphoid origin, cells were pre-gated on single, live, CD45+, myeloid-lineage negative (CD11b−Ly6G−Ly6C−CD11c−) cells. Il13-DsRed+ cells were gated within each cell population, or in the total CD45+ population as shown in the lower right panel. b, Gating strategy to identify innate lymphoid cells (ILCs) in the skin of naive 4C13R reporter or C57 mice. The Lineage AF4700 gate includes antibodies specific for B220, Ly6G, Ly6C, NK1.1, CD11b, and FCεRIα. A similar gating strategy was used to identify ILCs in lung and small intestine (SI). c, Frequencies of GATA3+, RORyt+, T-bet+ ILCs in the skin, lung and SI of naive C57 mice. Bar graphs show mean ± SEM for 7-8 mice pooled from 2 independent experiments. Each symbol refers to one mouse.
Extended Data Fig. 5 IL-13 signalling drives the development of CD11blo DC2 in vitro in a dose and time dependent manner.
a, Gating strategy to identify Sirpα+ DC2 in FLT3L bone marrow DC (BMDC) cultures from C57BL/6 (C57) donors. Cultures were untreated or supplemented with 10 ng/ml rIL-13 for the last 72h of culture as indicated. b, Phenotype of Sirpα+ DC2 subsets in FLT3L BMDCs cultures from C57 donors. Cultures were treated with the indicated concentrations of rIL-13 for the last 72h. Frequencies of CD11blo DC2s are shown in the bar graph. c, Phenotype of Sirpα+ DC2 subsets in FLT3L BMDC cultures from C57 donors. Cultures were supplemented with 10 ng/ml rIL-13 at the indicated times before harvest. Frequencies of CD11blo DC2s are shown in the bar graph. d, IL-4Rα and IL-13Rα1 expression on DC2 subsets from the skin dLNs of male Klf4f/f→C57 and Klf4f/f Vav1-iCre→C57 chimeric mice. FMO: fluorescence-minus-one. Histograms show concatenated data from 3 mice. Median fluorescence intensities (MFI) are reported in the bar graphs. e, Representative images of FLT3L BMDC cultures from male C57, IL-4Rα KO and STAT6 KO donors that were set up as controls for the experiments in Figure 5e. Cultures were either unstimulated or treated with 100 ng/ml rIL-13 for 30 minutes before staining. Scale bars correspond to 50 µm. Frequencies of pSTAT6+ cells in the Sirpα+ population are quantified in the bar graph. An average of 2200 Sirpα+ cells/sample were assessed. Each symbol refers to one culture from one of two repeat experiments. b-d, Bar graph shows mean ± SEM for n=4 (b,c) cultures or n=10-11 (d) mice pooled from 2 (b,c) or 3 (d) separate experiments. Each symbol refers to one independently treated culture or one mouse. P values were determined using two-way ANOVA with Sidak’s correction (b,c) or one-way ANOVA with Tukey’s correction (d). **P < 0.01; ***P < 0.001; only significant comparisons are indicated.
Extended Data Fig. 6 IL-13 signalling in DC2s is required for optimal IL-4+ TH responses in skin-draining lymph node.
a, Experimental set-up of mixed bone marrow (BM) chimeras to compare the antigen-presenting function of C57 and STAT6 KO DCs. b, Phenotype of DC2 populations in the skin dLN of naive C57 and STAT6 KO chimeras. c, Relative frequencies of DC2 subsets from each donor BM in the skin dLN of naive C57 or STAT6 KO mixed BM chimeras. d, Gating strategy to identify cytokine-expressing CD45.1/CD45.2+CD4+ T cells in the skin dLN of Nb-immunized C57 or STAT6 KO mixed BM chimeras. Gating for IL-4+CD4+ T cells is shown; other cytokines were gated in a similar manner. e, Phenotype of DC2 subsets in the skin dLN of naive Il4raf/− and Il4raf/− zDC-Cre mice. f, Gating strategy to identify cytokine-expressing CD4+ T cells in the skin dLN of Nb-immunized Il4raf/− and Il4raf/− zDC-Cre mice. Gating for IL-4+ CD4+ T cells is shown; other cytokines were gated in a similar manner. g, Frequencies of CD44hiCD4+ T cells in the skin dLN of Il4raf/− and Il4raf/− zDC-Cre mice 5 days after intradermal immunization with Mycobacterium smegmatis (Ms), Nippostrongylus brasiliensis L3 larvae (Nb), Candida albicans (Ca) or PBS as a control. h, Gating strategy for AF488+ cells in the skin dLN of Il4raf/− and Il4raf/− zDC-Cre mice 48 hours after intradermal injection of AF488-labeled Nb (Nb-AF488). i, Numbers of AF488+ cells in the skin dLN of Il4raf/− and Il4raf/− zDC-Cre mice 48 hours after intradermal injection of Nb-AF488. j, Numbers of total DCs and monocytes in the skin dLN of Il4raf/− and Il4raf/− zDC-Cre mice 48 hours after intradermal injection of Nb-AF488 or PBS. c,g,i,j, Bar graphs show mean ± SEM for n=7-8 (c) n=6-8 (g) n=7 (i) n=3-9 (j) mice pooled from 2 independent experiments. P values were determined using two-way ANOVA with Sidak’s correction. **P < 0.01; ***P < 0.001; only significant comparisons are indicated. Each symbol refers to one mouse.
Extended Data Fig. 7 Enrichment of IL-4/IL-13 signature genes in DC2s from healthy human skin.
a, Heatmap showing the expression of GSEA core-enrichment genes of the IL-4 and IL-13 Reactome pathway in cDCs and monocytes from the blood and skin of healthy donors from a published scRNA-seq dataset (Chen et al, 2020). b, UMAP plot showing scRNA-seq subclusters of cells from skin biopsies and suction blisters of healthy donors from the published dataset GSE153760. EC: Endothelial cells, FB 1, FB 2: Fibroblast clusters 1&2, ILC: Innate lymphoid cells, LC: Langerhans cells, LEC: Lymphatic endothelial cells, MC: Mast cells, MEL: Melanocytes, NK: Natural killer cells, KC 1-7: Keratinocyte clusters 1-7, SMC: Smooth muscle cells, Treg cells: Regulatory T cells. c, Feature plots of the UMAP clusters in (b) showing the expression levels of cluster-specific transcripts used for cluster identification. Color intensity represents the level of normalized gene expression. d, Feature plots of the UMAP clusters in (b) showing expression levels of IL4R, IL13RA1, STAT6, KLF4, IL4 and IL13 transcripts. Color intensity represents the level of normalized gene expression.
Supplementary information
Supplementary Tables
Supplementary Table 1. Signature genes for Fig. 1g.
Supplementary Table 2
Transcription factor binding site analysis for Fig. 1h.
Supplementary Table 3
DEG list STAT6 KO versus C57BL/6, bulk RNA-seq, for Extended data Fig. 2g.
Supplementary Table 4
Top markers and DEGs for scRNA-seq (Fig. 2f).
Supplementary Table 5
Reactome Pathways Fig. 8a.
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Mayer, J.U., Hilligan, K.L., Chandler, J.S. et al. Homeostatic IL-13 in healthy skin directs dendritic cell differentiation to promote TH2 and inhibit TH17 cell polarization. Nat Immunol 22, 1538–1550 (2021). https://doi.org/10.1038/s41590-021-01067-0
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DOI: https://doi.org/10.1038/s41590-021-01067-0
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