The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation

Abstract

The type 2 cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13 have important roles in stimulating innate and adaptive immune responses that are required for resistance to helminth infection, promotion of allergic inflammation, metabolic homeostasis and tissue repair1,2,3. Group 2 innate lymphoid cells (ILC2s) produce type 2 cytokines, and although advances have been made in understanding the cytokine milieu that promotes ILC2 responses4,5,6,7,8,9, how ILC2 responses are regulated by other stimuli remains poorly understood. Here we demonstrate that ILC2s in the mouse gastrointestinal tract co-localize with cholinergic neurons that express the neuropeptide neuromedin U (NMU)10,11. In contrast to other haematopoietic cells, ILC2s selectively express the NMU receptor 1 (NMUR1). In vitro stimulation of ILC2s with NMU induced rapid cell activation, proliferation, and secretion of the type 2 cytokines IL-5, IL-9 and IL-13 that was dependent on cell-intrinsic expression of NMUR1 and Gαq protein. In vivo administration of NMU triggered potent type 2 cytokine responses characterized by ILC2 activation, proliferation and eosinophil recruitment that was associated with accelerated expulsion of the gastrointestinal nematode Nippostrongylus brasiliensis or induction of lung inflammation. Conversely, worm burden was higher in Nmur1−/− mice than in control mice. Furthermore, use of gene-deficient mice and adoptive cell transfer experiments revealed that ILC2s were necessary and sufficient to mount NMU-elicited type 2 cytokine responses. Together, these data indicate that the NMU–NMUR1 neuronal signalling circuit provides a selective mechanism through which the enteric nervous system and innate immune system integrate to promote rapid type 2 cytokine responses that can induce anti-microbial, inflammatory and tissue-protective type 2 responses at mucosal sites.

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Figure 1: A network of neuromedin-U-expressing neurons co-localizes with NMUR1+ ILC2s.
Figure 2: NMU stimulates ILC2s and activates a signalling pathway through NMUR1 and Gαq.
Figure 3: NMU stimulates ILC2s in vivo and promotes worm expulsion.
Figure 4: NMU induces ILC2-dependent lung inflammation.

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Acknowledgements

We thank I. Gabanyi and D. Mucida for help with the muscularis isolation, H.-R. Rodewald for providing Cpa3cre and G. Eberl for RorgtGFP mice. We thank the Epigenomics Core, the Imaging Core and the Mouse Genetics Core at Weill Cornell Medicine and MSKCC. Nmur1LacZ/+ mice were generated by Velocigene and NmuGFP by GENSAT and provided by the KOMP or MMRRC Repository at UC Davis. The work was supported by grants from the German Research Foundation (DFG; KL 2963/1-1 to C.S.N.K.; FOR2372 to E.K. and G.M.K.), the Australian National Health and Medical Research Comission (NHMRC) early career fellowship (to L.C.R.), the Novo Nordic Foundation (14052; to J.B.M.), the Weill Cornell Department of Medicine Pre-Career Award (to L.A.M.), the Naito Foundation (to S.M.), JSPS Overseas Research Fellowships (to S.M.), MSD Life Science Foundation (to H.K.), the Jill Roberts Institute (to G.G.P.), Defense Advanced Research Projects Agency (DARPA; HR0011-16-C-0138 to X.S.), the National Institutes of Health (NIH; F32AI134018 to L.A.M.; AI061570, AI087990, AI074878, AI083480, AI095466, AI095608, AI102942, AI106697 and AI097333 to D.A.; R01GM114254 and OT2-OD023849 to X.S.; AI106697 to D.L.F.), the Burroughs Wellcome Fund (to D.A.) and the Crohn’s & Colitis Foundation of America (to T.M. and D.A.).

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C.S.N.K. carried out most experiments and analysed the data. T.M., J.B.M., L.C.R., A.-L.F., H.K., L.A.M., S.M., N.R. and X.S. helped with experiments, and E.K. and G.M.K. provided the inhibitor FR900359 (previous commercial name UBO-QIC). G.G.P. performed RNA-seq analysis. T.S., D.C. and D.L.F. provided human tissue samples. D.A. and C.S.N.K. conceived the project, analysed data, and wrote the manuscript with input from all co-authors.

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Correspondence to David Artis.

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Reviewer Information Nature thanks W. J. de Jonge and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 ILC2s and neurons co-localize.

Surface reconstruction of immunofluorescence staining from the intestinal submucosa shown in Fig. 1a. Scale bar, 30 μm. Data are representative of three independent experiments with similar results.

Extended Data Figure 2 ILC2s selectively express NMUR1.

a, Expression of Nmur1 in the indicated sorted cell populations as determined by qPCR analysis (n = 5). MC, mast cells; MΦ, macrophages; PMΦ, peritoneal macrophages. MC and PMΦ were obtained by peritoneal lavage; MΦ and ILC2s were purified from the small intestine. bd, f, Histograms and dot plots show expression of Nmur1 as measured by conversion of the fluorescent LacZ substrate FDG. Histograms are gated on LinCD45+ cells and CD127+KLRG1+ (ILC2s), CD127+CCR6+ (ILC3s), CD127+CCR6NKp46+NK1.1+ (ILC1s), CD3+ (T cells), CD19+ (B cells), CD3CD19CD11b+SiglecF+ (Eosinophils) from the small intestine (b, c). Gating for mast cells and basophils from the lung is shown (d). Percentage (n = 3) of FDG+ cells from the indicated population of the small intestine. Eos, eosinophils (f). e, g, Flow cytometry analysis of the indicated immune cell populations (top row) for Nmur1 (bottom row). MC and PMΦ were obtained by peritoneal lavage; MΦ and ILC2 were purified from the small intestine (e). Percentage (n = 3) of FDG+ cells (g). h, Expression of Nmu (n = 3 (LPL), n = 5 (all others)), as determined by qPCR from the indicated fractions of the murine small intestine. i, Expression of NMU (n = 9 (epithelium), n = 8 (whole jejunum and LPL), n = 7 (IEL), n = 6 (parenchyma)) as determined by qPCR from the indicated fractions of the human jejunum. j, k, CLARITY staining of the small intestine (j) or colon (k) for NMU. l, Image of the intestinal muscularis mucosae from NmuGFP mice. m, Immunofluorescence staining of the intestinal mucosa from Chatcre × Ai14 mice for NMU. Scale bar, 100 μm (jm). Error bars, mean + s.d. Data are representative of two (e, g) or three independent experiments (bf, jm) with similar results. Data in a, h, i are based on the indicated number of biological replicates per group. Source data

Extended Data Figure 3 ILC2s and NMU+ neurons co-localize.

a, Surface reconstruction of immunofluorescent staining from the intestinal submucosa shown Fig. 1i. Scale bar, 50 μm. b, Percentage (n = 4) of ILC2s, that have overlapping pixels with neurons. A total of 348 cells were counted and 236 cells exhibited pixels overlapping with NMU staining. c, Expression of Nmu as determined by qPCR in enteric neuron (n = 5, pooled from two independent experiments) cultures and compared to the epithelial fraction or parenchyma of the small intestine (n = 3). d, e, Sort-purified ILC2s (3 × 104) were cultured with or without enteric neurons for 5 days. Absolute number (d) and FSC (e) (n = 3) of ILC2s are shown. Error bars, mean + s.d. Data are representative of three independent experiments (a, d, e) or on the indicated number of biological replicates per group (b, c). Source data

Extended Data Figure 4 NMU activates ILC2s.

a, Gating strategy for flow cytometric analysis of bulk LPLs cytokine assays. Lineage 1: CD11b, CD11c and B220 (all APC-eF780); lineage 2: CD3, CD5 (both PerCP-Cy5.5) and FcεRI PerCP-eF710. b, c, Concentration (n = 3) of IL-13 (b) or IL-5 (c) in the culture supernatant after 4 h stimulation of bulk LPLs with a control peptide or NMU as determined by ELISA (n.d., not detectable). d, Bulk LPLs from Il1rl1+/+ and Il1rl1−/− mice were incubated in medium with or without NMU for 4 h in vitro. Percentage (Il1rl1+/+, n = 5 and Il1rl1−/−, n = 4) of IL-5+KLRG1+ cells. e, f, LPLs from Nmur1+/+ or Nmur1−/− mice were analysed by flow cytometry. Plots are gated on LinCD45+ lymphocytes (e). Percentage (n = 3) of GATA-3+KLRG1+ cells (f). g, Bulk LPLs were incubated for 30 min with DMSO or the inhibitor of Gαq proteins FR900359 in vitro. Medium, NMU or the indicated cytokine cocktail was then added and the assay was incubated for another 4 h. Percentage (n = 3) of IL-13 YFP+KLRG1+ cells among all KLRG1+ cells. h, Percentage (n = 4) of IL-5+KLRG1+ or IL-13+KLRG1+ cells as determined by intracellular cytokine staining. ik, Overnight incubation of sort-purified intestinal ILC2s from Il13YFP/+ mice in medium with or without NMU. FSC (n = 3) (i), histogram overlay of IL-13 YFP (j) and percentage (n = 3) of IL-13 YFP+ ILC2s (k). l, ILC2s from the small intestine were sort-purified and incubated in medium without or with NMU overnight in vitro. Contour plots show intracellular flow cytometry analysis for IL-5 and IL-13. Error bars, mean + s.d. Data are representative of two (bd, g, h) or three independent experiments (e, f, il) with similar results. Gating in a is representative for cytokine assays used in the whole study. Source data

Extended Data Figure 5 Nmu stimulates ILC2s in vivo.

a, PBS or NMU was injected daily in C57BL/6 mice. After two days, ILC3s from the small intestine were analysed by flow cytometry for Ki67. Plots are gated on LinCD45+RORγt+ lymphocytes. Percentage (n = 3) of Ki67+ cells. b, c, PBS or NMU was injected daily for two days in CD45.1:Nmur1+/+/CD45.2:Nmur1−/− mixed bone marrow chimaeras. One day later, ILC2s from the small intestine were analysed by flow cytometry for KLRG1 and Ki67. Plots are gated on LinCD127+KLRG1+ lymphocytes and either CD45.1 or CD45.2 (b). KLRG1 mean fluorescence intensity (PBS n = 4, NMU n = 5) (c). d, PBS or NMU (100 μg) was injected in CD45.1:Nmur1+/+/CD45.2:Nmur1−/− mixed bone marrow chimaeras. One day later, ILC2s from the small intestine were analysed by flow cytometry for IL-5 and IL-13 expression. Percentage (n = 7 (PBS), n = 8 (NMU)) of IL-5+ and IL-13+ ILC2s. Plots are gated on LinCD127+KLRG1+ lymphocytes and either CD45.1 or CD45.2. e, PBS or NMU was injected once in Il13YFP/+ mice. FSC (n = 3) of LinCD45+CD127+CD25+KLRG1+ LPLs one day after injection. f, C57BL/6 mice were infected with T. muris (n = 11) or left untreated (n = 6). On day 18, Nmu expression was determined by qPCR in a piece of the proximal colon. g, C57BL/6 mice were infected with H. polygyrus (n = 18). Control C57BL/6 (n = 4) mice were left untreated. On day 18, Nmu expression was determined by qPCR in a piece of the duodenum. h, Nmur1+/+ or Nmur1LacZ/+ mice were infected with N. brasiliensis. Control Nmur1+/+ mice were left untreated. On day 7, mice (n = 6) were analysed. Histogram overlay shows expression of Nmur1 (FDG) on ILC2s from the small intestine and are gated on LinCD45+KLRG1+ lymphocytes. i, Nmur1+/+ or Nmur1LacZ/+ mice were infected with N. brasiliensis. Control mice were left untreated. On day 7, mice were analysed and the percentage of Nmur1+ (FDG) determined by flow cytometry in the indicated subsets. Percentage (n = 6 (intestinal subsets) or 8 (lung subsets) for infected Nmur1LacZ/+ and n = 3 for control mice) of Nmur1+ (FDG) cells. Plots are gated on CD45+ cells and FcεRI+CD49b+c-Kit for basophils (BF), FcεRI+CD49b+c-Kit+ for mast cells, CD11b+F4/80+ for MΦ, CD3+CD5+ for T cells and LinKLRG1+ for ILC2s. j, Nmur1LacZ/+ mice were infected with N. brasiliensis. On day 14, mice were analysed and the percentage (n = 7 or 9 (lung)) of Nmur1+ (FDG) CD3+CD5+ T cells or LinKLRG1+ ILC2s was determined by flow cytometry. k, l, Il4GFP mice were infected with N. brasiliensis. On day 14, CD4+ T cells (gated on CD3+CD5+ lymphocytes) were sort-purified in IL-4-positive and -negative populations based on GFP expression (k) and Nmur1 expression was determined by qPCR (l) (n = 3 (ILC2), n = 4 (IL-4+CD4+ lung) or n = 5). m, n, PBS or NMU was injected daily for two days in C57BL/6 or Cpa3cre mice. One day later, ILC2s from the small intestine were analysed by flow cytometry for KLRG1 and Ki67 expression. Plots are gated on LinCD45+GATA-3+KLRG1+ cells (m). Percentage (n = 5 or 3 (PBS)) of Ki67+KLRG1+ cells among all KLRG1+ cells (n). o, PBS or NMU was injected daily for two days in BALB/c or ΔdblGATA1 mice. One day later, ILC2s from the small intestine were analysed by flow cytometry for KLRG1 and Ki67 expression. Percentage (n = 6 or 5 (PBS ΔdblGATA1)) of Ki67+KLRG1+ cells among all KLRG1+ cells. p, Bone marrow chimaeras reconstituted with Nmur1+/+ or Nmur1−/− bone marrow were infected subcutaneously with N. brasiliensis. On day 7, worm burden (n = 15 (Nmur1+/+), n = 14 (Nmur1−/−)) in the small intestine was quantified. q, Rag2−/−Il2rg−/− mice were reconstituted with ILC2 precursors from Nmur1+/+ or Nmur1−/− mice. After reconstitution, mice were infected with N. brasiliensis and NMU (20 μg) was injected i.p. on day 2, 4 and 6. Plots show flow cytometry analysis of cells from the lung and are gated on CD45+CD11c cells. Error bars, mean + s.d. Data are representative of two (b, c, ko, q) or three independent experiments (a, e, h) with similar results. The data are pooled from two (d) or three (f, g, i) independent experiments. The data in j and p are representative of the indicated number of biological replicates per group. Source data

Extended Data Figure 6 ILC2 are required for NMU-induced lung inflammation.

a, PBS or NMU was intranasally administered to C57BL/6 mice daily for four days. One day later, ILC2s from the lung were analysed by flow cytometry. Plots are gated on LinCD45+GATA-3+CD25+ lymphocytes. b, PBS or NMU was delivered intranasally to C57BL/6 mice daily for five days. Three days later, eosinophil infiltration was determined in BAL by flow cytometry. Percentage (mean + s.d., n = 5) of CD11b+SiglecF+CD11c eosinophils in the BAL. c, PBS or NMU was intranasally administered to Rag2−/− or Rag2−/−Il2rg−/− mice daily for four days. One day later, ILC2s and eosinophils from the lung were analysed by flow cytometry. d, PBS or NMU was intranasally administered daily for four days to Rag2−/−Il2rg−/− mice or Rag2−/−Il2rg−/− that were reconstituted with ILC2 progenitors. One day later, ILC2s and eosinophils from the lung were analysed by flow cytometry. e, PBS or NMU was intranasally administered to Nmur1+/+ or Nmur1−/− mice daily for four days. One day later, ILC2s and eosinophils from the lung were analysed by flow cytometry. Plots are gated on LinCD45+CD25+GATA-3+ lymphocytes. Data are representative of two (d, e) or three independent experiments (ac) with similar results. Source data

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Klose, C., Mahlakõiv, T., Moeller, J. et al. The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation. Nature 549, 282–286 (2017). https://doi.org/10.1038/nature23676

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