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Neuropeptide regulation of non-redundant ILC2 responses at barrier surfaces

Abstract

Emerging studies indicate that cooperation between neurons and immune cells regulates antimicrobial immunity, inflammation and tissue homeostasis. For example, a neuronal rheostat provides excitatory or inhibitory signals that control the functions of tissue-resident group 2 innate lymphoid cells (ILC2s) at mucosal barrier surfaces1,2,3,4. ILC2s express NMUR1, a receptor for neuromedin U (NMU), which is a prominent cholinergic neuropeptide that promotes ILC2 responses5,6,7. However, many functions of ILC2s are shared with adaptive lymphocytes, including the production of type 2 cytokines8,9 and the release of tissue-protective amphiregulin (AREG)10,11,12. Consequently, there is controversy regarding whether innate lymphoid cells and adaptive lymphocytes perform redundant or non-redundant functions13,14,15. Here we generate a new genetic tool to target ILC2s for depletion or gene deletion in the presence of an intact adaptive immune system. Transgenic expression of iCre recombinase under the control of the mouse Nmur1 promoter enabled ILC2-specific deletion of AREG. This revealed that ILC2-derived AREG promotes non-redundant functions in the context of antiparasite immunity and tissue protection following intestinal damage and inflammation. Notably, NMU expression levels increased in inflamed intestinal tissues from both mice and humans, and NMU induced AREG production in mouse and human ILC2s. These results indicate that neuropeptide-mediated regulation of non-redundant functions of ILC2s is an evolutionarily conserved mechanism that integrates immunity and tissue protection.

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Fig. 1: A new mouse strain for efficient targeting of ILC2s.
Fig. 2: Nmur1iCre deletes AREG within ILC2s with high efficiency.
Fig. 3: ILC2-derived AREG is crucial for the clearance of the gut-dwelling helminth Trichuris and tissue protection following intestinal injury.
Fig. 4: NMU mediates the non-redundant tissue-protective functions of ILC2-derived AREG.

Data availability

Bulk RNA sequencing data generated for this study have been deposited at the NCBI Gene Expression Omnibus (GSE211735). The sequenced data were aligned against NCBI GRCm38/mm10 mouse genome information. The microarray data downloaded from E-GEOD-14580 were used for human intestinal NMU expression levels.  Source data are provided with this paper.

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Acknowledgements

We thank members of the Artis Laboratory for discussions and critical reading of the manuscript; members of A. Chavez’s laboratory at Columbia University for providing expertise in the RNA sequencing process; and members of the JRI IBD Live Cell Bank Consortium for patient recruitment and collection of human samples as well as processing of specimens for cryopreservation. Nmucre mice were generated by GENSAT and provided by the MMRRC Repository at UC Davis. This work was supported by the Crohn’s and Colitis Foundation Research Fellowship Award (award number 901000 to W.Z.); the Crohn’s and Colitis Foundation Research Fellowship Award (award number 527125 to A.M.T.); Crohn’s and Colitis Foundation Research Fellowship Award (award number 937437 to H.Y.), the Sackler Brain and Spine Institute Research (to C.C.); a WCM Department of Pediatrics Junior Faculty Pilot Award and The Jill Roberts Center Pilot Award for Research in IBD (all to A.M.T.); the National Heart, Lung, and Blood Institute (5T32HL134629) and Thomas C. King Pulmonary Fellowship (all to C.N.P.); a Crohn’s and Colitis Foundation Research Fellowship Award (award number 481087 to T.M.); and the National Institutes of Health (DK126871, AI151599, AI095466, AI095608, AI142213, AR070116, AI172027 and DK132244 to D. Artis; DK121009 and DK110352 to S.A.L.); the LEO foundation, the Cure for IBD, the Jill Roberts Institute, the Sanders Family, the Rosanne H. Silbermann Foundation (all to D. Artis). Work in the Klose Lab is supported by grants from the European Research Council Starting Grant (ERCEA; 803087), the German Research Foundation (DFG; Project-ID 259373024–CRC/TRR 167, FOR2599 project 5–KL 2963/5-2, SPP1937–KL 2963/2-1 and KL 2963/3-1) to C.S.N.K.

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Contributions

A.M.T and H.Y. carried out most of the experiments and analysed the data. C.N.P., W.Z., C.C., K.J.J. and C.S.N.K. helped with CUBIC imaging or confocal immunofluorescence imaging. C.C. helped with the BM chimera experiments. G.G.P. helped with bioinformatics analysis. C.Z. and M.H. helped with various experiments. Members of the JRI IBD Live Cell Bank Consortium helped obtain, process and cryopreserve human samples. I.C.L., D. Andrew and P.B. helped develop the anti-NMUR1 antibody. Z.H., T.M. and S.A.L. generated the Aregfl/– mouse strain. D. Artis, A.T. and H.Y. 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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D. Artis has contributed to scientific advisory boards at Pfizer, Takeda, FARE, and the KRF. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 NMUR1-eGFP is highly expressed and enriched in ILC2s in lymphoid and non-lymphoid tissues.

a, Percentages of surface NMUR1+ ILC2s isolated from the indicated tissues from Nmur1+/+ WT mice (n = 5 mice). b, Schematic of targeting construct for generating Nmur1iCre-eGFP mice. c–k, Comprehensive analysis of NMUR1-eGFP expression in Nmur1iCre-eGFP mice. Representative gating strategy for live CD45+ cells (c), innate lymphoid cell (ILC) subsets (d), adaptive lymphocytes (e), and myeloid cells and granulocytes (g). Lin1: CD11b, CD11c, FcεRIα, B220. Lin2: CD3ε, CD5. Representative overlaid histograms depicting NMUR1-eGFP expression in adaptive lymphocytes (f) and myeloid cells and granulocytes (h) from Nmur1iCre-eGFP mice and Cre-negative littermate controls. Percentages of NMUR1-eGFP+ cells within the indicated immune cell subsets isolated from small intestines (i), mesenteric lymph nodes (MLN) (j), and spleens (k) of Nmur1iCre-eGFP mice (n = 3 mice). Data in a and i–k are representative of two independent experiments. Data are represented as means ± s.e.m.

Source data

Extended Data Fig. 2 ILC2-enriched eGFP expression and iCre-mediated induction of RFP expression.

a, Representative gating strategy. b, Representative histograms depicting NMUR1-eGFP expression in the indicated progenitor immune cell subsets in the bone marrow. c, Percentages of NMUR1-eGFP+ cells within the indicated progenitor cells isolated from Nmur1iCre-eGFP mice (n = 4 mice). Lin1: Ly6G (1A8), CD3ε, CD5, CD19. Lin2: Ly6G (Gr1), FcεRIα. d, Unbiased analysis of NMUR1-eGFP+ cells in the bone marrow by flow cytometry, depicting a representative of two independent experiments. Lin3: CD3ε, CD5, CD19. Lin4: Ly6G (Gr1), FcεRIα. e, Percentages of RFP+ ILC2s in indicated tissues from Nmur1iCre-eGFPROSA26LSL-RFP mice (n = 4 mice). f–m, Representative histograms showing iCre (RFP) expression in the indicated immune cell subsets isolated from the small intestine (f), colon (h), MLN (j), and spleen (l) of Nmur1iCre-eGFPROSA26LSL-RFP mice (n = 4 mice). Percentage of iCre (RFP)+ cells within the indicated immune cell subsets isolated from the small intestine (g), colon (i), MLN (k), and spleen (m). Data in c,e,g,i,k,m are pooled from of 2 independent experiments. Data are represented as means ± s.e.m. MLN - mesenteric lymph node, DC – dendritic cells.

Source data

Extended Data Fig. 3 Comparison of efficiency of ILC2-depletion and its impact on other immune cells in Nmur1iCre-eGFPROSA26LSL-DTR and Red5CreROSA26LSL-DTR mice.

a–c, ROSA26LSL-DTR (n = 11 mice) and Nmur1iCre-eGFPROSA26LSL-DTR (n = 7 mice) were treated with 2 daily injections of DT. The small intestine (SI), colon, mesenteric lymph nodes (MLN), and spleen were harvested for analysis after resting the mice for 2 days. Abundances of non-ILC2 innate lymphoid cell subsets (a), adaptive lymphocytes (b) or myeloid cells and granulocytes (c) within the indicated tissues. d–g, ROSA26LSL-DTR (n = 7 mice) and Red5CreROSA26LSL-DTR (n = 6 mice) were treated with 2 daily injections of DT and rested for 2 days. Representative flow cytometry analysis of ILC2s in the colon, pre-gated on total ILCs (d) (Total ILCs gated as live CD45+Lin-CD90+CD127+ events. Lin: CD3ε, CD5, CD11b, CD11c, FcεRIα, B220). Percentages of ILC2s out of total ILCs in the SI, colon, MLN, and spleen (e). Abundances of innate lymphoid cell subsets (f), adaptive lymphocytes (g). Data in a–c and e–g are pooled from two independent experiments. Two-way ANOVA with Šídák multiple comparisons test (a–c, e–g). P values are presented where appropriate. NS, not significant. Data are represented as means ± s.e.m.

Source data

Extended Data Fig. 4 Nmur1iCre-eGFP-mediated AREG deletion does not impact AREG production in CD4+ T cells or intrinsic ILC2 homeostasis.

a–c, Representative flow cytometry analysis of AREG and NMUR1 co-expression within ST2 and ST2+ CD4+ T cells (a) and ILC2s (b) at steady state in the MLN of WT mice. Percentages of NMUR1+ cells within AREG-producing cells isolated from WT mice (n = 3 mice) (c). d, Schematic of Aregfl/– mouse generation using CRISPR/Cas9n. e, f, Representative flow cytometry analysis of ST2 and ST2+ CD4+ T cells (e) and percentages of AREG-producing cells (f) in the MLN of Aregfl/– (n = 7 mice), AregΔRed5 (n = 5 mice), and AregΔILC2 (n = 5 mice). g–l, Small intestine and MLN from Aregfl/– (n = 4 mice) and AregΔILC2 (n = 5 mice) were analyzed at steady state for ILC2 abundance and their cytokine production capacity. Percentages of ILC2s within total ILCs in the small intestine (g) and MLN (i). Absolute cell numbers of ILC2s in the small intestine (h) and MLN (j). Percentages of IL-5+IL-13+ DP, total IL-5+ and total IL-13+ ILC2s isolated from the small intestine (k) and MLN (l). Data in c, f, g-l are representative of two independent experiments. One-way ANOVA with Tukey multiple comparisons test (c, f). Unpaired two-sided t-test (g-l). P values are presented where appropriate. NS, not significant. Data are represented as means ± s.e.m. MLN – mesenteric lymph node. DP – double positive.

Source data

Extended Data Fig. 5 Deletion of ILC2-derived AREG does not impact the priming of adaptive immune responses.

a-e, Analysis of MLN isolated from naïve Aregfl/– (n = 3 mice) and Trichuris-infected Aregfl/– (n = 7 mice) and AregΔILC2 (n = 8 mice) at day 19 post-infection. Cecal patches (CP) were pooled as one sample when necessary due to cell yield for flow cytometry analysis (Aregfl/– (n = 2 pooled CP) and Trichuris-infected Aregfl/– (n = 3 pooled CP) and AregΔILC2 (n = 3 pooled CP)). Representative flow cytometry analysis of CD4+ T cell subsets (a, b) and percentages of Foxp3+ Tregs, T-bet+ TH1 and GATA3+ TH2 CD4+ T cells (c). Representative flow cytometry analysis of pathogen-specific cytokine production in MLN CD4+ T cells after stimulation with Trichuris antigen for 3 days ex vivo (d). Percentage of CD4+ T cells producing TNFα, IFNγ, IL-4, IL-5, and IL-13 (e). f, Detection of Trichuris antigen-specific IgG1 and IgG2c in serum from naïve Aregfl/– (n = 4 mice) and Trichuris-infected Aregfl/– (n = 12 mice) and AregΔILC2 (n = 13 mice) at day 19 post-infection by serial dilution ELISA. Data in c and e are pooled from two independent experiments. Data in f are pooled from three independent experiments. One-way ANOVA with Tukey multiple comparisons test (c, e). Two-way ANOVA with Šídák multiple comparisons test (f). NS, not significant. Data are represented as means ± s.e.m.

Source data

Extended Data Fig. 6 Bone marrow chimera mice recapitulated the phenotype of AregΔILC2 mice during Trichuris muris infection.

Irradiated CD45.1 WT mice received BM from Aregfl/– or AregΔILC2 donor mice. After 8 weeks, chimera mice were infected with 200 Trichuris eggs by oral gavage and analyzed 19 days post-infection. a, Experimental schematic. b-d, AREG deletion in ILC2s (b), ST2 CD4+ T cells (c), and ST2+ CD4+ T cells (d) in Trichuris-infected Aregfl/– BM (n = 5 mice) and AregΔILC2 BM (n = 5 mice). e-g, Representative AB-PAS-stained images of cecal tips (bars = 100 µm) (e). Enumeration of cecal tip goblet cells (f) and crypt length (g) of naïve Aregfl/– BM (n = 4 mice) or AregΔILC2 BM (n = 3 mice) and Trichuris-infected Aregfl/– BM (n = 5 mice) or AregΔILC2 BM (n = 5 mice). h, Worm burden in Aregfl/– BM (n = 5 mice) or AregΔILC2 BM (n = 5 mice). i-l, Intestinal immune responses in cecum of Trichuris-infected Aregfl/– BM (n = 4 mice) and AregΔILC2 BM (n = 5 mice). Frequencies of ILC2s (i), ST2+ Th2 cells (j), and eosinophils (k), and percentages of IL-4+ and IL-13+ cecal ILC2s and CD4+ T cells (l). m-o, Gene expression analysis of proximal colons isolated from naïve Aregfl/– BM (n = 4 mice) and AregΔILC2 BM (n = 3 mice) and Trichuris-infected Aregfl/– BM (n = 10 mice) and AregΔILC2 BM (n = 10 mice). Normalized to Actb. Relative expression levels of pro-inflammatory genes (Il4, Il5, Il13, Ifng) and immunoregulatory responses (Il10 and Tgfb1) (m). Ratio of type 1 gene (Ifng) over type 2 gene (Il13) expression (n). Expression levels of goblet cell functional marker gene Retnlb (o). Data in b-d, f-h, i-l are representative of two independent experiments. Data in m-o are pooled from two independent experiments. Unpaired two-sided t-test (b-d, h-l). One-way ANOVA with Tukey multiple comparisons test (f, g, m-o). P values are presented where appropriate. NS, not significant. Data are represented as means ± s.e.m.

Source data

Extended Data Fig. 7 Specific and efficient deletion of Areg within T cells in AregΔCD4 mice.

a, WT mice were infected with 200 Trichuris eggs by oral gavage, and the cecum was analyzed by flow cytometry for AREG+ cells among ILC2s, Foxp3+ Tregs and Foxp3 CD4+ T effectors at day 0 (n = 6 mice), day 3 (n = 9 mice), day 9 (n = 5 mice), and day 16 (n = 4 mice) post-infection. b-e, Aregfl/– (n = 6 mice) and AregΔCD4 (n = 8 mice) were infected with 200 Trichuris eggs by oral gavage and analyzed 19 days post-infection. Representative flow cytometry analysis of ILC2s (b) and CD4+ T cells (d) for AREG+ cells. Percentage of AREG-producing cells within ILC2s (c) and CD4+ T cells (e). Data in a are pooled from two independent experiments. Data in c and e are representative of two independent experiments. Unpaired two-sided t-test (c, e). P values are presented where appropriate. NS, not significant. Data are represented as means ± s.e.m.

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Extended Data Fig. 8 Bone marrow chimera mice recapitulated the phenotype of AregΔILC2 mice in the context of DSS-induced intestinal damage and inflammation.

Irradiated CD45.1 WT mice received BM from Aregfl/– or AregΔILC2 donor mice. After 8 weeks, chimera mice were exposed to 3% DSS for 5 days and then allowed to recover for 5 days on regular drinking water. a, Experimental schematic. b-d, Examination of AREG deletion in ILC2s (b), ST2 CD4+ T cells (c), and ST2+ CD4+ T cells (d) in DSS-exposed Aregfl/– BM (n = 4 mice) and AregΔILC2 BM (n = 5 mice) by flow cytometry. e-g, Disease severity of DSS-exposed Aregfl/– BM (n = 7 mice) or AregΔILC2 BM (n = 8 mice) as determined by weight loss (e) and colon length (f, g). Data in b-d are representative of three independent experiments. Data in e and g are pooled from 2 independent experiments. Unpaired two-sided t-test (b-d and g). Two-way ANOVA with Šídák multiple comparisons test (e). P values are presented where appropriate. NS, not significant. Data are represented as means ± s.e.m.

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Extended Data Fig. 9 Neuronal production of NMU is upregulated in the context of intestinal inflammation.

a, Nmu expression in the proximal colon of naïve (n = 4 mice) or Trichuris-infected (n = 5 mice) determined by qRT-PCR. Normalized to Actb housekeeping gene. b, c Muscularis propria of naïve water control (n = 4 mice) or DSS-exposed inflamed colons (n = 4 mice) from WT mice were stained for β3-tubulin (white), HuC/D (green), and NMU protein (red). Representative images (bars = 50 µm) (b). Neuronal NMU intensity was quantified by normalizing fluorescent intensity of NMU staining to the number of HuC/D+ nuclei in two fields per sample (c). d, e, CUBIC imaging of naïve water control (n = 3 mice) or DSS-exposed (n = 3 mice) colon from WT mice stained for β3-tubulin (green) and NMU protein (red). Representative images (bars = 50 µm) (d) and quantification of NMU+ pixels (e). f, Representative images of swiss rolls of naïve water or DSS-exposed colons stained for DAPI (white), KLRG1 (cyan), NMU (red) and β3-tubulin (green) (bars = 50 µm). Arrows depicting increased NMU staining in DSS-exposed colons. Images are representative of two independent experiments. g, Representative images of swiss rolls of naïve water or DSS-exposed condition colons stained for DAPI (white), Epcam (cyan), NMU (red) and β3-tubulin (green) (bars = 50 µm). Images are representative of two independent experiments. Data in a and e are representative of two independent experiments. Data in c are pooled from two independent experiments. Unpaired two-sided t-test (a, c, e). P values are presented where appropriate. Data are represented as means ± S.E.M.

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Extended Data Fig. 10 NMU promotes AREG expression in ILC2s in both mice and humans.

a, Experimental schematic of RNA sequencing (RNA-seq) analysis of ILC2s and CD4+ T cells in the context of intestinal inflammation. NMUR1-eGFP+ ILC2s, NMUR1-eGFP+ CD4+ T cells and NMUR1-eGFP CD4+ T cells were sort-purified from colonic LPLs (n = 3 mice) and subjected to bulk RNA-seq. b, c, mRNA expression levels of Nmur1 (b) and Areg (c) as determined by comparing the normalized read counts of each gene. d, e, Representative flow cytometry analysis of AREG+ CD4+ T cells (d) and percentage of AREG+ CD4+ T cells (e) in the colons of WT mice injected with PBS (n = 8 mice) or NMU (n = 9 mice). f, Gating strategy for analyzing human colonic LPLs isolated from surgical biopsy samples from ulcerative colitis (UC) patients. g, h, AREG production by human CD4+ T cells after stimulation of human colonic LPLs from UC patients with or without NMU in vitro overnight. Representative flow cytometry analysis of human CD4+ T cells (g). Percentage of AREG+ CD4+ T cells (n = 5 specimens) (h). Data in b, c represent sequenced data from three independent biological replicates. Data in e are pooled from two independent experiments. Data in h contain 5 independent human specimens, with each pair of dots representing an individual specimen. Two-way ANOVA with Šídák multiple comparisons test (b, c). Unpaired two-sided t-test (e). Paired two-sided t-test (h). P values are presented where appropriate. NS, not significant. Data are represented as means ± s.e.m.

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Supplementary information

Reporting Summary

Supplementary Table 1

Clinical metadata for patients with IBD.

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Tsou, A.M., Yano, H., Parkhurst, C.N. et al. Neuropeptide regulation of non-redundant ILC2 responses at barrier surfaces. Nature 611, 787–793 (2022). https://doi.org/10.1038/s41586-022-05297-6

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