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Tuft-cell-derived IL-25 regulates an intestinal ILC2–epithelial response circuit

Abstract

Parasitic helminths and allergens induce a type 2 immune response leading to profound changes in tissue physiology, including hyperplasia of mucus-secreting goblet cells1 and smooth muscle hypercontractility2. This response, known as ‘weep and sweep’, requires interleukin (IL)-13 production by tissue-resident group 2 innate lymphoid cells (ILC2s) and recruited type 2 helper T cells (TH2 cells)3. Experiments in mice and humans have demonstrated requirements for the epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP) and IL-25 in the activation of ILC2s4,5,6,7,8,9,10,11, but the sources and regulation of these signals remain poorly defined. In the small intestine, the epithelium consists of at least five distinct cellular lineages12, including the tuft cell, whose function is unclear. Here we show that tuft cells constitutively express IL-25 to sustain ILC2 homeostasis in the resting lamina propria in mice. After helminth infection, tuft-cell-derived IL-25 further activates ILC2s to secrete IL-13, which acts on epithelial crypt progenitors to promote differentiation of tuft and goblet cells, leading to increased frequencies of both. Tuft cells, ILC2s and epithelial progenitors therefore comprise a response circuit that mediates epithelial remodelling associated with type 2 immunity in the small intestine, and perhaps at other mucosal barriers populated by these cells.

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Figure 1: Intestinal tuft cells constitutively express Il25.
Figure 2: Worm infection induces IL-13-dependent tuft cell hyperplasia.
Figure 3: IL-13 signalling in epithelial progenitors gives rise to tuft cell hyperplasia.
Figure 4: Tuft cells regulate intestinal physiology through an ILC2–epithelium response circuit.

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Acknowledgements

We thank M. Consengco, R. Noyes, and Z. Wang for technical expertise, Y. Nusse for mice, members of the Locksley laboratory for helpful discussions, and R. Vance, M. Fontana, M. Anderson, and O. Klein for comments on the manuscript. This work was supported by the National Institutes of Health (AI026918, AI030663, HL107202), a Diabetes Endocrinology Research Center grant (DK063720), the Howard Hughes Medical Institute (HHMI), and the Sandler Asthma Basic Research Center at the University of California, San Francisco. J.v.M. is an HHMI Fellow of the Damon Runyon Cancer Research Foundation (DRG-2162-13).

Author information

Authors and Affiliations

Authors

Contributions

J.v.M. conceived the study, designed and performed experiments, analysed data, and wrote the paper with R.M.L. M.J. performed experiments. H.-E.L. cloned the Flare25 reporter cassette, performed the Flare25 embryonic stem cell work, and assisted with additional experiments. R.M.L. directed the study and wrote the paper with J.v.M.

Corresponding author

Correspondence to Richard M. Locksley.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Flare25 mouse and Vil1-cre-mediated Il25 deletion.

a, Gene-targeting strategy for the flox and reporter of Il25 (Flare25) mouse. b, PCR of genomic DNA isolated from the tail (lane 1, 2) or cells sorted from the small intestine (lane 3, 4) of indicated mice. c, Quantitative RT–PCR for Il25 on cDNA from EPCAM+ cells sorted from the small intestine of indicated mice. b, c, Data are representative of two experiments (n = 2). Frt, target site for FLIPASE recombinase; IRES, internal ribosomal entry site; loxP, target site for Cre recombinase; pA, bovine growth hormone poly(A) tail; tdRFP, tandem-dimer red fluorescent protein; UTR, untranslated region. For gel source data (b) see Supplementary Fig. 1.

Extended Data Figure 2 Il25 expression in epithelial surfaces.

a, b, Indicated tissues of Il25F25/F25 (a) and wild-type control (b) mice stained by immunohistochemistry for RFP (red), EPCAM (green), and DAPI (blue). Some data from Fig. 1a are repeated here to allow complete comparison. Scale bars, 50 μm. Images are representative of at least three independent experiments. n = 3.

Extended Data Figure 3 Flow cytometry gating strategies and organoid culture.

a, Flow cytometric analysis of indicated tissues in Il25F25/F25 and wild-type mice. b, Flow cytometric analysis of small intestine epithelial cells of Il25F25/F25 mice before and after fluorescence-activated cell sorting (FACS) into RFP+EPCAM+ and RFPEPCAM+ pools for analysis by quantitative RT–PCR. ce, Representative flow cytometric analysis of small-intestine-derived organoids from Il25F25/F25 (ce) and wild-type (c) mice cultured with or without recombinant protein (20 ng ml−1), as indicated (ce) or Notch signalling inhibitor DAPT (25 μM) (e). Single-cell suspensions of the organoids were stained for EPCAM (ce) and DCLK1 (d), and gated to quantify tuft cell (RFP+EPCAM+ or DCLK1+EPCAM+) frequency. f, Quantification of two technical replicates from experiment shown in e. d.p.i., days post-N. brasiliensis infection. Data in f are technical replicates. Data are representative of three (a, b, d) or two (c, e, f) independent experiments. In ad, n = 3; in e, f, n = 2. Error bars represent mean ± s.e.m.

Extended Data Figure 4 Il25 is expressed constitutively in tuft cells.

ac, Jejunum (a, b) or trachea (c) of Il25F25/F25 (a, c) and wild-type control (b) mice stained by immunohistochemistry for RFP (red), indicated lineage markers (green), and DAPI (blue). Scale bars, 50 μm. Images are representative of one (c) or two (a, b) independent experiments. n = 2.

Extended Data Figure 5 Tuft cells are not a major source of intestinal TSLP or IL-33.

a, Jejunum of Il25F25/F25 mice stained for RFP (red), IL-33 (green), and DAPI (blue). bd, Quantitative RT–PCR on indicated (b, c) or RFP+EPCAM+ (d) cells sorted from untreated (b, c) mice or mice treated as indicated (d). RNA isolated from whole lung 8 days post-N. brasiliensis infection is used as a positive control for Tslp expression in c. Expression of Tslp in sorted Tslp-expressing cells of the lung would probably be higher. Scale bars, 50 μm. Data are representative of two independent experiments. In a, n = 3; in bd, n = 2.

Extended Data Figure 6 N. brasiliensis induces tuft cell hyperplasia throughout the small intestine but not in stomach and colon.

a, b, Indicated tissues of Il25F25/F25 (a) and wild-type control (b) mice treated as indicated and stained by immunohistochemistry for RFP (a) or DCLK1 (b) (red), EPCAM (green), and DAPI (blue). d.p.i., days post-N. brasiliensis infection. Scale bars, 50 μm. Data are representative of two (stomach and colon) or at least three (all others) independent experiments. In a, stomach and colon: n = 2; all others: n > 5.

Extended Data Figure 7 Il25 is expressed only in tuft cells during worm infection and H. polygyrus infection also induces tuft cell hyperplasia.

a, b, Jejunum of Il25F25/F25 (a) and wild-type control (b) mice infected for 7 days with N. brasiliensis stained by immunohistochemistry for RFP (red), indicated lineage markers (green), and DAPI (blue). c, Jejunum of indicated mice left untreated or infected 14 days with H. polygyrus and stained by immunohistochemistry for DAPI (blue), EPCAM (green) and DCLK1 (red). Scale bars, 50 μm. d.p.i., days post-H. polygyrus infection. Images are representative of one (c) or two (a, b) independent experiments. In a, b, n = 2; in c, n = 1 (uninfected) or n = 2 (infected).

Extended Data Figure 8 Absence of Paneth and CHGA+ cell hyperplasia after N. brasiliensis infection and model of ILC2–epithelial signalling circuit.

a, b, Jejunum of indicated mice stained for DAPI (blue) and LYZ1/2 (a) or CHGA (b) (green). c, Quantification of CHGA+ cells from imaging in (b). d, During homeostasis, rare epithelial tuft cells of the small intestine constitutively express Il25, which maintains low levels of IL-13 production in lamina propria ILC2s. IL-13 in turn signals uncommitted epithelial progenitors to promote emergence of tuft and goblet cells. In the absence of infection, this feed-forward ILC2–epithelial circuit is restrained by as yet unknown mechanisms. After N. brasiliensis (N.b.) infection, a helminth-derived signal or a change in host physiology activates the ILC2–epithelial circuit leading to tuft and goblet cell hyperplasia and enhanced IL-13 production by ILC2s. Adaptive TH2 cells probably also provide IL-13 and/or support ILC2 activation, especially when infection or inflammation lasts more than a week. Recombinant proteins are sufficient to induce tuft cell hyperplasia, either by inducing IL-13 production in lymphoid cells (IL-25 or IL-33) or by directly binding epithelial progenitors (IL-4). Scale bars, 50 μm. d.p.i., days post-N. brasiliensis infection. Data in c are biological replicates. Data are representative of two (a) or three (b) independent experiments or pooled from multiple experiments (c). In a, n = 2; in b, c, n is as shown in c. Error bars represent mean ± s.e.m.

Extended Data Figure 9 IL-13 production by lamina propria ILC2 and CD4+ cells.

a, b, Lamina propria cells from Il25−/−;Il13Smart/+, Il13Smart/+, and wild-type control mice analysed by flow cytometry and gated on ILC2 (a, LinCD45+GATA3+) or CD45+CD4+ (b) cells. IL-13 secretion was quantified by measuring surface expression of human CD4, which is expressed from the Il13 locus in Il13Smart reporter mice. c, Frequency of lamina propria CD4+ cells as a percentage of total CD45+ cells as assessed by flow cytometry. d.p.i., days post-N. brasiliensis infection. Data in b, c are biological replicates. Data are representative of at least three (a) independent experiments, or pooled from multiple experiments (b, c). In a, n = 5; in b, c, n is as shown. Error bars represent mean ± s.e.m.

Extended Data Table 1 Antibodies and quantitative RT–PCR primers used in this study

Supplementary information

Supplementary Figure 1

This file contains the gel source data. (PDF 123 kb)

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von Moltke, J., Ji, M., Liang, HE. et al. Tuft-cell-derived IL-25 regulates an intestinal ILC2–epithelial response circuit. Nature 529, 221–225 (2016). https://doi.org/10.1038/nature16161

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