Inflammation early in life can prime the local immune milieu of peripheral tissues, which can cause lasting changes in immunological tone that confer disease protection or susceptibility1. The cellular and molecular mechanisms that prompt changes in immune tone in many nonlymphoid tissues remain largely unknown. Here we find that time-limited neonatal inflammation induced by a transient reduction in neonatal regulatory T cells causes a dysregulation of subcutaneous tissue in mouse skin. This is accompanied by the selective accumulation of type 2 helper T (TH2) cells within a distinct microanatomical niche. TH2 cells are maintained into adulthood through interactions with a fibroblast population in skin fascia that we refer to as TH2-interacting fascial fibroblasts (TIFFs), which expand in response to TH2 cytokines to form subcutaneous fibrous bands. Activation of the TH2–TIFF niche due to neonatal inflammation primes the skin for altered reparative responses to wounding. Furthermore, we identify fibroblasts in healthy human skin that express the TIFF transcriptional signature and detect these cells at high levels in eosinophilic fasciitis, an orphan disease characterized by inflammation and fibrosis of the skin fascia. Taken together, these data define a previously unidentified TH2 cell niche in skin and functionally characterize a disease-associated fibroblast population. The results also suggest a mechanism of immunological priming whereby inflammation early in life creates networks between adaptive immune cells and stromal cells to establish an immunological set-point in tissues that is maintained throughout life.
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Sequencing data are publicly available at the Gene Expression Omnibus under accession number GSE183031. Cross-tissue fibroblast atlas data9 were downloaded from https://www.fibroxplorer.com/download. Additional data presented here are available upon request from the corresponding author. Source data are provided with this paper.
Code used for analysis of scRNA-seq data is available upon request from the corresponding author.
Al Nabhani, Z. & Eberl, G. Imprinting of the immune system by the microbiota early in life. Mucosal Immunol. 13, 183–189 (2020).
Scharschmidt, T. C. et al. A wave of regulatory T cells into neonatal skin mediates tolerance to commensal microbes. Immunity 43, 1011–1021 (2015).
Scharschmidt, T. C. et al. Commensal microbes and hair follicle morphogenesis coordinately drive Treg migration into neonatal skin. Cell Host Microbe 21, 467–477 (2017).
Kim, J. M., Rasmussen, J. P. & Rudensky, A. Y. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat. Immunol. 8, 191–197 (2007).
Nussbaum, J. C. et al. Type 2 innate lymphoid cells control eosinophil homeostasis. Nature 502, 245–248 (2013).
Collins, N. et al. Skin CD4+ memory T cells exhibit combined cluster-mediated retention and equilibration with the circulation. Nat. Commun. 7, 11514 (2016).
Kobayashi, T. et al. Homeostatic control of sebaceous glands by innate lymphoid cells regulates commensal bacteria equilibrium. Cell 176, 982–997.e16 (2019).
Ali, N. et al. Regulatory T cells in skin facilitate epithelial stem cell differentiation. Cell 169, 1119–1129.e11 (2017).
Buechler, M. B. et al. Cross-tissue organization of the fibroblast lineage. Nature 593, 575–579 (2021).
Joost, S. et al. The molecular anatomy of mouse skin during hair growth and rest. Cell Stem Cell 26, 441–457.e7 (2020).
Zhang, L. et al. Diet-induced obesity promotes infection by impairment of the innate antimicrobial defense function of dermal adipocyte progenitors. Sci. Transl. Med. 13, eabb5280 (2021).
Driskell, R. R. et al. Distinct fibroblast lineages determine dermal architecture in skin development and repair. Nature 504, 277–281 (2013).
Kimura-Ueki, M. et al. Hair cycle resting phase is regulated by cyclic epithelial FGF18 signaling. J. Invest. Dermatol. 132, 1338–1345 (2012).
Kim, B. S. et al. TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation. Sci. Transl. Med. 5, 170ra16 (2013).
Salimi, M. et al. A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis. J. Exp. Med. 210, 2939–2950 (2013).
Ricardo-Gonzalez, R. R. et al. Tissue signals imprint ILC2 identity with anticipatory function. Nat. Immunol. 19, 1093–1099 (2018).
Obata-Ninomiya, K. et al. The skin is an important bulwark of acquired immunity against intestinal helminths. J. Exp. Med. 210, 2583–2595 (2013).
Boothby, I. C., Cohen, J. N. & Rosenblum, M. D. Regulatory T cells in skin injury: at the crossroads of tolerance and tissue repair. Sci. Immunol. 5, eaaz9631 (2020).
Correa-Gallegos, D. et al. Patch repair of deep wounds by mobilized fascia. Nature 576, 287–292 (2019).
Naik, S. et al. Inflammatory memory sensitizes skin epithelial stem cells to tissue damage. Nature 550, 475–480 (2017).
Findley, T. W., Chaitow, L. & Huijing, P. (eds) Fascia: The Tensional Network of the Human Body (Churchill Livingstone, 2012).
Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902 (2019).
Ramachandran, P. et al. Resolving the fibrotic niche of human liver cirrhosis at single-cell level. Nature 575, 512–518 (2019).
Chan, K. K. et al. Eosinophilic fasciitis following checkpoint inhibitor therapy: four cases and a review of literature. Oncologist 25, 140–149 (2020).
Mortezavi, M., Barrett, M. & Edrissian, M. Successful treatment of refractory eosinophilic fasciitis with reslizumab. JAAD Case Rep. 6, 951–953 (2020).
Merrick, D. et al. Identification of a mesenchymal progenitor cell hierarchy in adipose tissue. Science 364, eaav2501 (2019).
Dahlgren, M. W. et al. Adventitial stromal cells define group 2 innate lymphoid cell tissue niches. Immunity 50, 707–722.e6 (2019).
Spallanzani, R. G. et al. Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose tissue immune and metabolic tenors. Sci. Immunol. 4, eaaw3658 (2019).
Schwalie, P. C. et al. A stromal cell population that inhibits adipogenesis in mammalian fat depots. Nature 559, 103–108 (2018).
Scott, R. W., Arostegui, M., Schweitzer, R., Rossi, F. M. V. & Underhill, T. M. Hic1 defines quiescent mesenchymal progenitor subpopulations with distinct functions and fates in skeletal muscle regeneration. Cell Stem Cell 25, 797–813.e9 (2019).
Dahlgren, M. W. & Molofsky, A. B. Adventitial cuffs: regional hubs for tissue immunity. Trends Immunol. 40, 877–887 (2019).
Vainchtein, I. D. et al. Astrocyte-derived interleukin-33 promotes microglial synapse engulfment and neural circuit development. Science 359, 1269–1273 (2018).
Hagan, A. S., Zhang, B. & Ornitz, D. M. Identification of a FGF18-expressing alveolar myofibroblast that is developmentally cleared during alveologenesis. Development 147, dev181032 (2020).
Camberis, M. et al. Evaluating the in vivo Th2 priming potential among common allergens. J. Immunol. Methods 394, 62–72 (2013).
Bankhead, P. et al. QuPath: open source software for digital pathology image analysis. Sci. Rep. 7, 16878 (2017).
Yang, B. et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158, 945–958 (2014).
Korotkevich, G. et al. Fast gene set enrichment analysis. Preprint at bioRxiv https://doi.org/10.1101/060012 (2021).
We thank the staff at the UCSF Parnassus Flow Cytometry Core (RRID:SCR_018206) for assistance with flow cytometry analysis and cell sorting (supported in part by NIH P30 DK063720 and by the NIH S10 instrumentation grant 1S10OD021822-01); the UCSF Biological Imaging and Development Core for confocal microscopy and analysis; the UCSF Genomics CoLab and Institute for Human Genetics for scRNA-seq and the UCSF Mouse Pathology Core for histology; and T. Scharschmidt, C. Lowell, J. Cyster, M. Ansel, T. Peng and A. Abbas for comments and discussion; D. Ornitz and A. Hagan for the donation of Fgf18CreERT2 tissue; and R. Locksley and H.-E. Liang for N. brasiliensis larvae. Figs. 1a, b and Extended Data Figs. 8a, g, 9a, e, o and v were created using BioRender.com. I.C.B. was supported by NIH F30AI147364, NIH T32GM007618 and NIH T32AI007334. M.D.R. is supported by NIH R01AR077553 and R01AR071944.
Funding for scRNA-seq studies of healthy human skin was provided by LEO Pharmaceuticals. M.D.R. is a founder and consultant for TRex Bio., Sitryx Bio. and Mozart Therapeutics.
Peer review information Nature thanks Christopher Buckley, Laura Mackay and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
(a) Weights of PBS- and DT-treated FoxP3DTR mice from the time of treatment to adulthood. n = 3-7 mice per data point, 51 total. (b) Quantification of Tregs in skin and skin-draining lymph nodes (sdLN) following neoTreg depletion. n = 3-9 animals per data point (60 total), 1 experiment per time point. (c–f) Representative flow cytometry and quantifications of immune cell populations in skin and skin-draining lymph nodes during the inflammatory phase of neonatal Treg depletion at P25 and adult Treg depletion at P67 (both 10 days after DT). Gating: CD8 T cells (CD3+ CD8+); CD4 Teffs (CD3+ CD4+ FoxP3−); Ly6C monocytes (Ly6G− Siglec F− CD64+ CD11c− CD11b+ Ly6C+). n = 18 animals. (g) Representative histology of wildtype neonatal mice treated with PBS or DT and sacrificed 10 days post-injection. (h) Histology of selected organs in ΔneoTreg and control mice at P25. gWAT – perigonadal white adipose tissue. Data are displayed as mean +/- SD from one independent experiment, representative of 2-3 repeats. *p < 0.05, **p < 0.01, ***p < 0.001 (all two-sided); repeated measures two-way ANOVA (a); two-way ANOVA with Šídák multiple comparison test (b-c); one-way ANOVA with Tukey’s multiple comparisons test (c–f).
Extended Data Fig. 2 Resolution of inflammation and return to homeostasis following neonatal Treg reduction.
(a) Representative skin histology of control and neonatal Treg-depleted FoxP3DTR mice at P25, P35, and P50 (10, 20, and 35 days post-DT treatment). Fibrous bands are outlined with dotted lines and regenerating adipocytes are marked by arrows. (b) Abundance of selected inflammatory immune cell populations in skin 0-90 days after neoTreg depletion. n = 3-9 animals per data point (60 total), 1 experiment per time point. (c) T helper and CD8+ T cell cytokine production 0-35 days after neoTreg depletion, quantified by intracellular cytokine staining. n = 3-9 animals per data point (51 total), 1 experiment per time point. (d–f) Sample gating and quantification of dermal γδ T cells, ILC2s, and Tc2 cells 0-90 days after neoTreg depletion in Il5Red5/+; FoxP3DTR mice. n = 3-9 animals per data point (51 total, d-e); 3-6 animals per data point, (19 total, f), 1 experiment per time point. Data are displayed as mean +/- SD from one independent experiment, representative of 2-3 repeats. *p < 0.05, **p < 0.01, ***p < 0.001 (all two-sided); Student’s t test at selected time points during adulthood (b-e); two-way ANOVA with Šídák multiple comparison test (f) . All results were reproduced over 2-3 independent experiments.
Extended Data Fig. 3 CD49d marks subcutis-resident Th2 cells that are associated with subdermal eosinophilia and age-specific fibrous band formation.
(a-c) Expression of CD69, CD103 (αE integrin), and CD49d (α4 integrin) on skin lymphocytes in ΔneoTreg mice aged to adulthood. Bulk Teff defined as IL5Red5− FoxP3− CD4+ T cells. n = 4 animals. (d–f) ΔneoTreg mice were aged to adulthood, subcutis was separated from the dermis/epidermis, and the two skin fractions were analyzed separately by flow cytometry. Th2 localization over time (d), lymphocyte localization at P50 (e), and CD49d expression in dermal vs. subdermal lymphocytes (f) is shown. n = 4-5 animals per data point, 14 total (d); 4 animals (e); 5 animals (f). (g–i) Quantification of CD49d+ Th2 cells (g) and eosinophils (h) in ΔneoTreg and ΔadTreg mice during the inflammatory phase of Treg reduction at 10 days post-DT. Myeloid cell localization in ΔneoTreg mice (i) was quantified by flow cytometry of dissected skin layers. n = 19 animals (g-h); 5 animals (i). (j–l) ΔneoTreg mice were treated every other day with FTY720 or vehicle from P8 to P25. Histology (j), Th2 numbers (k), and eosinophil numbers (l) are shown at P25. n = 7 animals. (m–n) CD49d expression on IL5-Red5+ Th2 cells and frequency of CD49d+ Th2 cells in ΔneoTreg and ΔadTreg mice during (D10 / P25) and after (D35 / P50) inflammation. n = = 23 animals. Data are displayed as mean +/- SD from one independent experiment, representative of 2-3 repeats. *p < 0.05, **p < 0.01, ***p < 0.001 (all two-sided); repeated-measures ANOVA with Dunnett’s multiple comparison test (a-c, e); 2-way repeated-measures ANOVA with Sidak multiple comparison test (f); Welch’s ANOVA with Dunnett’s multiple comparison test (g-h); Student’s t-test (k-n) . All results were reproduced over 2-3 independent experiments.
Extended Data Fig. 4 Single-cell transcriptomic characterization of skin stroma in control and ΔneoTreg mice.
(a) Expression of marker genes for skin stromal clusters in control mice. (b) Expression of fibroblast markers and ECM genes in control mouse skin stroma. (c) Expression of immune-related genes in mouse skin stroma, split by control (PBS) and ΔneoTreg (DT) sample. (d) Differential gene expression analysis of Il13ra1+ FBs (TIFFs) in ΔneoTreg vs. control mice. (e) Expression of MHC class II-related transcripts in skin stromal clusters (ctrl and ΔneoTreg samples combined). (f) Expression of cell surface markers used to design the Il13ra1+ FB flow cytometry gating strategy in main figure 2d. (g) Representative flow cytometry of fibroblast markers PDPN and PDGFRα within subsets of Lin− skin stromal cells from P25 control mice.
Extended Data Fig. 5 Il13ra1+ FBs / TIFFs are transcriptomically similar to Pi16+ fibroblasts found across mouse organs.
Published data were downloaded from a mouse cross-tissue fibroblast atlas, containing twenty-eight 10X scRNAseq datasets across 16 murine tissues that were aligned, filtered, and analyzed with a standardized methodology to minimize batch effects.9. (a) Steady-state atlas of all 16 tissues was re-plotted, demonstrating similar clustering to published meta-analysis. (b) Expression of TIFF markers from control skin in cross-tissue clusters defined by the atlas. (c) Expression of the top 50 TIFF markers (ranked by log-fold change) in cross-tissue atlas clusters. (d) Cross-tissue atlas cluster representation in selected tissues. SAT – subcutaneous adipose tissue; VAT – visceral adipose tissue. (e) Enrichment of all skin TIFF markers with logFC > 0.25 (n = 313) among cross-tissue fibroblast atlas clusters, calculated using the Seurat AddModuleScore function. (f) Geneset enrichment analysis of the skin TIFF gene set among cross-tissue fibroblast atlas clusters.
(a) IF microscopy of mouse back, ear, and tail skin to identify fascia (CD26), adipocytes (PLIN1), and skeletal muscle (MyoIV). (b) Variable layering of fascia, adipocytes, and skeletal muscle at two different back skin locations at in P22 control mice. (c) Control skin was dissected from P22 mice and the subcutis was manually separated from the dermis and epidermis. Il13ra1+ FBs (TIFFs) were quantified in each fraction. (d) Fgf18 expression by scRNAseq in control Lin− skin stromal cells. (e) Confocal microscopy of adult skin from Fgf18CreRET2; Rosa26tdTomato mice injected with tamoxifen for five days prior to harvest. Top row: Z-projection with tdTomato signal thresholded for visualization. Bottom row: inset of fascia with original tdTomato fluorescence. All results were reproduced over 2-3 independent experiments.
(a) IF microscopy of skin from wildtype mice injected for five days with IL-13 or IL-33 starting at P21 with quantification of fascial proliferation by Ki67. n = 9 animals. (b) Skin histology from ΔneoTreg mice crossed to IL4/13- or IL33-deficient strains at P25 (10 days post-DT). (c) Expression of IL4RA on TIFFs from wildtype neonate (P25) and adult (P50) mice. n = 10 animals. (d–f) Adult mice were injected for 7 days with type 2 cytokines and the indicated cell populations in skin were quantified by flow cytometry. n = 13 animals (d); 7 animals (e-f). (g–h) TIFFs and dermal fibroblasts were sorted from P21 mouse skin and co-cultured with sorted IL-5Red5+ skin Th2 cells from ΔneoTreg mice for four days. n = 12 samples (h). (i–j) IL-18R1 and ST2 expression in skin lymphocyte subsets with quantification of IL-18R1 expression (see main fig. 3g for ST2). n = 4 animals. (k–l) ΔneoTreg mice were aged to adulthood, the subcutis was separated from the dermis/epidermis, and expression of alarmin receptors was quantified across lymphocyte subsets. n = 5 animals. Data are displayed as mean +/- SD from one independent experiment, representative of 2-3 repeats. *p < 0.05, **p < 0.01, ***p < 0.001 (all two-sided); Welch’s ANOVA with Dunnett Multiple Comparisons Test (a, d, h, j); Student’s t-test (c, e-f); two-way ANOVA with Šídák multiple comparison test (l).
Extended Data Fig. 8 Th2-TIFF interactions and niche priming across multiple models of early life subcutaneous inflammation.
(a-d) Mice were immunized subcutaneously (s.c.) with ovalbumin (OVA) and papain. CD49d+ Th2 cells (b, c) and TIFFs (d) were quantified at indicated timepoints. n = 25 animals I; 10 animals (d). (e–f) PdgfraCre; Il4raf/f mice and Cre− controls were injected with OVA-papain at P8 and P15. IL4RA expression (e) and TIFF frequency (f) were quantified at P25. n = 13 mice. (g–j) Mice were infected s.c. with Nippostrongylus brasiliensis at P8 and boosted with s.c. N. brasiliensis allergen at P15. CD49d+ Th2 cells (g, i) and TIFFs (j) were quantified at indicated timepoints. n = 13 mice (h); 12 mice (j). Data are displayed as mean +/- SD from one independent experiment; each experiment was reproduced 2-3 times. *p < 0.05, **p < 0.01, ***p < 0.001 (all two-sided); Student’s t-test (c-d, f, i-j); two-way ANOVA with Šídák multiple comparison test (e).
Extended Data Fig. 9 Neonatal Treg reduction primes skin for Th2-driven tissue reparative responses during adulthood.
(a–b) Control and ΔneoTreg mice were aged to adulthood and then treated with two shots DT (identical to neonatal dosing regimen). Representative histology is shown with fibrous bands outlined. (c) Th2 and ILC2 cell numbers in adult ctrl (Il5Red5/+; FoxP3DTR + PBS), ΔneoTreg (Il5Red5/+; FoxP3DTR + DT), and ΔneoTreg/ΔTh2 (Il5Red5/+; Rosa26DTA/+; FoxP3DTR) mice. n = 16 animals. (d) Wound bed ILC2 numbers (gated as CD45+ CD3− CD4− CD8− Thy1+ IL5Red5+). n = 7-10 animals per data point in 2 pooled experiments per time point (108 total). (e–n) Control, ΔneoTreg, and ΔneoTreg/ΔTh2 mice were aged to adulthood and subjected to full-thickness cutaneous wounding. (f) Th2 numbers in skin 0 − 10 days post wounding (dpw). n = 4-11 animals per data point in 2 pooled experiments per time point (89 total). (g) Th2 frequency in paired skin biopsies taken at dpw0 and dpw7. n = 22 animals in one experiment. (h–i) Wound area was quantified daily, fit to a one-phase exponential decay model, and tested for equivalence of the rate constant (h). Rate constants of curves fit to each biological replicate are shown in (i). (j–k) Alternatively activated macrophage (AAM) and eosinophil frequency in dpw10 wounded skin. n = 42 animals in 2 pooled experiments. (l) TIFF abundance in wound beds at 0 −10 dpw. n = 4-16 animals in 2 pooled experiments (97 total). (m–n) Flow cytometric quantification (m) and IF microscopy (n) of CD26hi TIFFs in wounds at dpw10. n = 39 animals in 2 pooled experiments. (o–u) ΔneoTreg mice were aged to adulthood, wounded, and treated with FTY720 every other day. Wound closure (p-q) and flow cytometric quantifications of Th2 cells, alternatively activated macrophages (AAMs), eosinophils, and TIFFs are shown (r-u) are shown. n = 11 animals. (v-ab) Neonatal mice were immunized with OVA/papain, aged to adulthood, and wounded. Wound closure (w-x) and flow cytometric quantifications of Th2 cells, alternatively activated macrophages (AAMs), eosinophils, and TIFFs are shown (y-ab). n = 22 animals. Data are displayed as mean +/- SD (c-d, f, i-m, q-u, x-ab) or SEM (h, p, w). Results were reproduced over 2 independent experiments (a-d; o-ab) or 4 independent experiments pooled into two separate analyses (e-n). *p < 0.05, **p < 0.01, ***p < 0.001 (all two-sided); ANOVA with Dunnett Multiple Comparisons Test (c, i-k, m, x-ab); least-squares quadratic regression with extra sum-of-squares F test (f, l); mixed-effects analysis with Šídák multiple comparison test (g); nonlinear one-phase exponential decay regression (h, p, w); Student’s t-test (q-u).
(a) Gating strategy used to FACS-purify human Lin− stromal cells (CD45− CD31− Ecad−, CD235a−). (b) Expression of top 50 murine TIFF (mTIFF) orthologs in human skin stroma, ranked by fold-change. (c) Expression of mouse TIFFs and Thbs4+ FB markers in healthy human stromal clusters. (d) Human and ortholog-converted mouse scRNAseq data were integrated and co-clustered. Cluster identities from single-species analyses (main fig. 4a-b) are shown projected onto the cross-species UMAP. (e) Sample histology of healthy and eosinophilic fasciitis lesional skin with IHC staining for GATA3 and CD4.
Representative flow cytometry gating.
Metadata and cell counts by cluster for all human and mouse scRNA-seq experiments.
List of marker genes for each cluster of murine stromal cells (control and ΔneoTreg cell integrated analysis).
List of marker genes for each cluster of human stromal cells (analysis of healthy samples only).
Orthologues of mTIFF signature genes used for enrichment analyses of human data.
Patient characteristics for skin samples used in immunohistochemistry analysis.
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Boothby, I.C., Kinet, M.J., Boda, D.P. et al. Early-life inflammation primes a T helper 2 cell–fibroblast niche in skin. Nature 599, 667–672 (2021). https://doi.org/10.1038/s41586-021-04044-7
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