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 repair. Group 2 innate lymphoid cells (ILC2s) produce type 2 cytokines, and although advances have been made in understanding the cytokine milieu that promotes ILC2 responses, 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). 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 G_αq 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.

Figure 1. ILC2s and neurons co-localize

Surface reconstruction of immunofluorescent staining from the intestinal submucosa shown in Fig. 1a. Scale bar 30 μm.

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. b–d, f, Histograms and dot plots show expression of Nmur1 as measured by conversion of the fluorescent LacZ substrate FDG. Histograms are gated on Lin−CD45+ cells and CD127+KLRG1+ (ILC2s), CD127+CCR6+ (ILC3s), CD127+CCR6−NKp46+NK1.1+ (ILC1s), CD3+ (T cells), CD19+ (B cells), CD3−CD19−CD11b+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 (j–m). Error bars, mean + s.d. Data are representative of two (e, g) or three independent experiments (b–f, j–m) with similar results. Data in a, h, i are based on the indicated number of biological replicates per group.

Figure 3. ILC2s and neurons co-localize

(a) Surface reconstruction of immunofluorescent staining from the intestinal submucosa shown Fig. 1i. Scale bar 50 μm. (b) Percentage (Mean + SD, 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 (Mean + SD, 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) (Mean + SD, n= 3) of ILC2s are shown. Data are representative of three independent experiments.

Figure 4. NMU activates ILC2s

(a) Gating strategy for flow cytometric analysis of bulk LPLs cytokine assays. Lineage1: CD11b, CD11c and B220 (all APC-eF780); Lineage2: CD3, CD5 (both PerCP-Cy5.5) and FcεRI PerCP-eF710. (b,c) Concentration (Mean + SD, n= 3) of IL-13 (b) or IL-5 (c) in the culture supernatant after 4h 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 4h in vitro. Percentage (Mean + SD, Il1rl1^+/+ n= 5, Il1rl1^−/− n= 4) of IL-5⁺ KLRG1⁺ cells. (e,f) LPLs from Nmur1^+/+ or Nmur1^−/− mice were analyzed by flow cytometry. Plots are gated on Lin⁻CD45⁺ lymphocytes (e). Percentage (Mean + SD, 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 4h. Percentage (Mean + SD, n=3) of IL-13 YFP⁺ KLRG1⁺ cells among all KLRG1⁺ cells. (h) Percentage (Mean + SD, n=4) of IL-5⁺ or IL-13⁺ KLRG1⁺ cells as determined by intracellular cytokine staining. (j-l) Overnight incubation of sort-purified intestinal ILC2s or C57BL/6 mice (k,l ; n=5). (i-k) Intestinal ILC2s from Il13^Yfp/+ mice were sort-purified and incubated in medium with or without NMU overnight in vitro. FSC (Mean + SD, n=3) (i), histogram overlay of IL-13 YFP (j) and percentage (Mean + SD ; 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 over night in vitro. Contour plots show intracellular flow cytometry analysis for IL-5 and IL-13. Data are representative of two (b-d, g,h) or three independent experiments (e, f, i-l) with similar results. Gating in (a) is representative for cytokine assays used in the whole study.

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 analyzed by flow cytometry for Ki67. Plots are gated on Lin⁻ CD45⁺ RORγt⁺ lymphocytes. Percentage (Mean + SD, 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 chimeras. One day later, ILC2s from the small intestine were analyzed by flow cytometry for KLRG1 and Ki67. Plots are gated on Lin⁻ CD127⁺ KLRG1⁺ lymphocytes and either CD45.1 or CD45.2 (b). KLRG1 MFI (Mean + SD, 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 chimeras. One day later, ILC2s from the small intestine were analyzed by flow cytometry for IL-5 and IL-13 expression. Percentage (Mean + SD, n= 7) of IL-5⁺ and IL-13⁺ ILC2s. Plots are gated on Lin⁻ CD127⁺ KLRG1⁺ lymphocytes and either CD45.1 or CD45.2. (e) PBS or NMU was injected once in Il13^Yfp/+ mice. FSC (Mean + SD, n= 3) of Lin⁻ CD45⁺ 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 Nmur1^LacZ/+ mice were infected with N. brasiliensis. Control Nmur1^+/+ mice were left untreated. On day 7, mice (n=6) were analyzed. Histogram overlay shows expression of Nmur1 (FDG) on ILC2s from the small intestine and are gated on Lin⁻ CD45⁺ KLRG1⁺ lymphocytes. (i) Nmur1^+/+ or Nmur1^LacZ/+ mice were infected with N. brasiliensis. Control mice were left untreated. On day 7, mice were analyzed and the percentage of Nmur1⁺ (FDG) determined by flow cytometry in the indicated subsets. Percentage (Mean + SD, n= 6 (lung subsets) or 8 (intestinal subsets) for infected Nmur1^LacZ/+ and n= 3 for control mice) of Nmur1⁺ (FDG) cells. Plots are gated on CD45⁺ lymphocytes and FcεRI⁺CD49b⁺c-Kit⁻ for basophils, FcεRI⁺CD49b⁺c-Kit⁺ for mast cells, CD11b⁺F4/80⁺ for MФ, CD3⁺CD5⁺ for T cells and Lin⁻ KLRG1⁺ for ILC2s. (j) Nmur1^LacZ/+ mice were infected with N. brasiliensis. On day 14, mice were analyzed and the percentage (Mean + SD, n= 7 or 9 (lung)) of Nmur1⁺ (FDG) CD3⁺CD5⁺ T cells or Lin⁻KLRG1⁺ ILC2s was determined by flow cytometry. (k,l) Il4^Gfp 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) (Mean + SD, 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 Xpa3^Xρε mice. One day later, ILC2s from the small intestine were analyzed by flow cytometry for KLRG1 and Ki67 expression. Plots are gated on Lin⁻ CD45⁺ GATA-3⁺ KLRG1⁺ cells (m). Percentage (Mean + SD, 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 analyzed by flow cytometry for KLRG1 and Ki67 expression. Percentage (Mean + SD, n=6 or 5 (PBS ΔdblGATA1) of Ki67⁺ KLRG1⁺ cells among all KLRG1⁺ cells. (p) Bone marrow chimeras reconstituted with Nmur1^+/+ or Nmur1^−/− bone marrow were infected s.c. with N. brasiliensis. On day 7, worm burden (Mean + SD, n=15) 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 (20μg) 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. Data are representative of two (b, c, j-q) or three independent experiments (a, e, h) with similar results and n=3-5 mice per group. The experiments in d, f, g, i are pooled data from two (d) or three (f, g, i) independent experiments.

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 analyzed by flow cytometry. Plots are gated on Lin⁻CD45⁺ 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 + SD, 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 analyzed 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 ILC2ps. One day later, ILC2s and eosinophils from the lung were analyzed 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 analyzed by flow cytometry. Plots are gated on Lin⁻ CD45⁺ CD25⁺ GATA-3⁺ lymphocytes. Data are representative of two (d,e) or three independent experiments (a-c) with similar results.

Figure 1. A network of Neuromedin U-expressing neurons co-localizes with NMUR1⁺ ILC2s

(a) Immunofluorescence staining of the intestinal submucosa. Scale bar 20 μM. (b) RNA-seq volcano plot of differential expression between ILC2s (positive log₂FC) and ILC3s (negative log₂FC). Genes belonging to the KEGG pathway ‘neuroactive receptor-ligand interaction’ (mmu04080) are shown in red and are enriched among the genes differentially expressed in ILC2s (corrected p value < 0.01). FC: fold change. (c) Heatmap showing expression Z-scores of the indicated genes in small intestine ILC2s and ILC3s, as measured by RNA-seq. For Nmur2, Z-scores of 0 indicate read counts of zero in all samples. (d) Expression of Nmur1 and Nmur2 in the indicated sort-purified lymphocyte populations as determined by qPCR analysis (Mean + SD, n=3). SI: small intestine; ILC2p: ILC2 progenitor; n.d. : not detectable . (e) Expression of NMUR1 in the indicated sorted lymphocyte populations from human intestine as determined by qPCR analysis (Mean + SD, n=7). (f) Dot plots show expression of Nmur1 as measured by conversion of the fluorescent LacZ substrate fluorescein di-β-D-galactopyranoside (FDG). Lineage1: CD11b, CD11c and B220 (all APC-eF780); Lineage2: CD3, CD5 (both PerCP-Cy5.5) and FcεRI PerCP-eF710. (g) CLARITY staining of the small intestine. Scale bar 100 μM. (h) Immunofluorescence staining of the intestinal muscularis mucosae. Scale bar 100 μM. (i) Immunofluorescence staining of the intestinal submucosa. Scale bar 50 μM. Data in a and f-i are representative of three independent experiments with similar results. Experiments in b-f are based on three (b-d) or seven (e) biological replicates per group.

Figure 2. NMU stimulates ILC2s and activates a signaling pathway through NMUR1 and Gαq

Bulk LPLs were incubated in medium without or with NMU and/or the indicated molecules for 4 h in vitro. (a,b) Representative flow cytometry analysis (a). Percentage (Mean + SD, n= 3) of IL-13 YFP⁺ KLRG1⁺ cells (b). Gating is shown in ED 4a. (c,d) Percentage (Mean + SD, n=5) of IL-13⁺ (c) or IL-5⁺ (d) KLRG1⁺ cells as determined by intracellular cytokine staining. (e,f) Percentage (Mean + SD, n= 3) of IL-13 YFP⁺ (e) or IL-5⁺ (f) KLRG1⁺ cells. (g) Percentage (Mean + SD, n=6) of IL-5⁺ KLRG1⁺ cells. M: Medium; N: NMU; P: PMA/ionomycin. (h) Bulk LPLs were incubated for 30 min with DMSO or the inhibitor of Gαq proteins FR900359 before incubation with medium or NMU. Percentage (Mean + SD, n=3) of IL-13 YFP⁺ KLRG1⁺ cells. (i,j) Overnight incubation of sort-purified intestinal ILC2s. Percentage (Mean + SD; n=5) of IL-5⁺ (i) and IL-13⁺ (j) ILC2s. (k-m) 7 days incubation of sort-purified intestinal ILC2s. Concentration (Mean + SD, n=6) of IL-5 (k), IL-9 (l), IL-13 (m) in the supernatant. Data are representative of three (a-f, h-j) independent experiments with similar results. The data in g and k-l are pooled data from two independent experiments and representative for a total of four independent experiments.

Figure 3. NMU stimulates ILC2s in vivo and promotes worm expulsion

(a-c) RNA was extracted from sort-purified ILC2s one day after PBS or NMU administration and sequenced (n= 3). Principal component (PC) analysis. Ellipses show, for each group, the curve at which the fitted bivariate normal distribution equals 0.68 (a). Heatmap showing level of significance of GO enrichment tests, as measured by -log₁₀(p_corrected). Blue = not significant (p_corrected > 0.01) (b). Heatmap showing expression Z-scores of selected differentially expressed genes between PBS- or NMU-treated mice (c). (d,e,f) PBS or NMU was injected daily for two days in C57BL/6 mice (d,e) or in CD45.1 Nmur1^+/+ : CD45.2 Nmur1^−/− chimeras (f). One day later, ILC2s from the small intestine were analyzed by flow cytometry for KLRG1 and Ki67 expression. Plots are gated on Lin⁻ CD45⁺ CD25⁺ KLRG1⁺ lymphocytes (d). Percentage (Mean + SD, n= 3 (e), n=4 (PBS) n=5 (NMU) (f)) of Ki67⁺ KLRG1⁺ cells (e,f). (g-i) PBS or NMU was injected in Il13^Yfp/+ mice. MFI of KLRG1 expression (g). Histogram overlay for IL-13 (h) is gated on Lin⁻ CD45⁺ CD127⁺ CD25⁺ KLRG1⁺ lymphocytes and percentage (Mean + SD, n= 3) of IL-13 YFP⁺ KLRG1⁺ cells among all KLRG1⁺ cells (i) one day after injection. (j,k) PAS staining after PBS or NMU administration (j). Scale bar 100 μm. Goblet cell number (Mean + SD, n=4 (PBS) n=5 (NMU)) per villus (k). (l) C57BL/6 mice were infected with N. brasiliensis or left untreated. Nmu expression was determined by qPCR in the small intestine on day 7 (Mean + SD, uninfected: n= 3, N.b. : Nippostrongylus brasiliensis n=5). (m) Flow cytometry analysis of Nmur1 (FDG) in LPLs of N. brasiliensis-infected Nmur1^LacZ/+ mice. Lineage1: CD11c and B220 (both APC-eF780); Lineage2: CD3, CD5 (both PerCP-Cy5.5). (n,o) C57BL/6 mice were infected with N. brasiliensis and treated with PBS or NMU and analyzed on day 7. Percentage (Mean + SD, n= 3) of CD11b⁺ SiglecF⁺ eosinophils in the mesenteric lymph node (n) Worm burden (Mean + SD, PBS n=8, NMU n=9) in the small intestine (o). (p,q) Worm burden (Mean + SD, Nmur1^+/+ n=16 and Nmur1^−/− n=19) was determined on day 7 (p) and percentage (Mean + SD, n=6) of IL-13⁺ ILC2s in the small intestine on day 6 (q). (r,s) Worm burden (Mean + SD, Nmur1^+/+ n=6, Nmur1^−/− n=8) of ILC2-reconstituted Rag2^−/− Il2rg^−/− mice (r) and percentage of KLRG1⁺ ST2⁺ was measured by flow cytometry in the lung (s). (t-v) Worm burden (Mean + SD, Nmur1^+/+ n=7, Nmur1^−/− n=8) of ILC2- reconstituted and NMU injected Rag2^−/− Il2rg^−/− mice (t) or percentage of KLRG1⁺ ST2⁺ (u) or eosinophils (v) was measured by flow cytometry in the lung. Data are representative of two (q) or three independent experiments (d-m) with similar results. Experiments in (o, q-v) are pooled data from two (q-v) or three (o) independent experiments. RNA-seq data in a-c are based on three biological replicates per group.

Figure 4. NMU induces ILC2-dependent lung inflammation

(a-c) PBS or NMU was intranasally administered to Il13^Yfp/+ (a,b) or C57BL/6 (c) mice daily for four days. One day later, ILC2s from the lung were analyzed by flow cytometry. MFI (Mean + SD, n= 3) of KLRG1 expression (a). Percentage (Mean + SD, n= 3) of IL-13 YFP⁺ ILC2s (b). Percentage (Mean + SD, n= 5) of Ki67⁺ KLRG1⁺ cells (c). (d,e) PBS or NMU was delivered intranasally to C57BL/6 mice daily for five days and lung infiltration was examined three days later. Percentage (Mean + SD, n= 5) of CD11b⁺ SiglecF⁺ CD11c⁻ eosinophils in the lung (d). PAS staining of lung sections (left side: scale bar 100 μm; right side: scale bar 50 μm. Electronically magnified images from the left are shown on the right (e). (f-k) PBS or NMU was intranasally administered to Rag2^−/− or Rag2^−/− Il2rg^−/− mice (f,g), ILC2-reconstituted Rag2^−/− Il2rg^−/− mice (h,i), or Nmur1^+/+ or Nmur1^−/− mice (j,k) for four days. One day later, ILC2s and eosinophils from the lung were analyzed by flow cytometry. Percentage (Mean + SD, n= 4 (f,g), n= 4 (Rag2^−/− Il2rg^−/−), n=5 (Rag2^−/− Il2rg^−/− + ILC2p) (h,i), n=3 (PBS), n=5 (NMU) n=4 (NMU Nmur1^−/−) (j,k) of Ki67⁺ KLRG1⁺ cells (f,h,j) or percentage of CD11b⁺ SiglecF⁺ CD11c⁻eosinophils (g, i, k) (n.a. : not applicable). Data are representative of two (h, i) or three independent (a-g and j,k), experiments with similar results.