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Neuroepithelial VIP–VIPR1 interactions differentially control enteric type 1 and type 2 immunity

Nature Immunology volume 26, pages 2244–2255 (2025)Cite this article

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Abstract

The nervous and immune systems cooperate to regulate mucosal barrier integrity. Nevertheless, whether enteric neurons establish neuroepithelial interactions to coordinate immunity remains elusive. Here, we identified neuroepithelial interactions that differentially control intestinal type 1 and type 2 immunity. Gut epithelial cells expressed vasoactive intestinal peptide (VIP) receptor 1 (VIPR1), and chemogenetic modulation of enteric VIPergic neurons led to altered epithelial-derived cytokines. Epithelial-intrinsic deletion of Vipr1 resulted in diminished type 1 immunity, including reduced type 1 alarmins and intraepithelial lymphocytes. In contrast, epithelial Vipr1 deficiency led to enhanced type 2 immunity, comprising increased type 2 alarmins, tuft cells and activated group 2 innate lymphoid cells. Disruption of neuroepithelial VIP–VIPR1 interactions resulted in increased susceptibility to invasive bacterial infection, which contrasted with enhanced resistance to parasite infection. Our work identifies a multi-tissue axis that controls type 1 and type 2 immunity, deciphering how neuroepithelial interactions distinctively set gut immunity programs.

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Fig. 1: Alarmins and neuroendocrine receptors are expressed by intestinal epithelial cells.
Fig. 2: Enteric VIPergic neurons control epithelial-derived alarmins.
Fig. 3: Epithelial-intrinsic VIPR1 deficiency reshapes tuft cells and alarmins.
Fig. 4: Neuroepithelial interactions shape intestinal IELs.
Fig. 5: Neuroepithelial interactions shape gut defense to bacterial infections.
Fig. 6: Neuroepithelial interactions shape intestinal ILC2.
Fig. 7: Neuroepithelial interactions shape gut defense to worm infections.

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Data availability

The datasets generated in this study are available from the corresponding author upon request. Bulk RNA-seq and single-cell RNA-seq data have been deposited in the NCBI Gene Expression Omnibus under accession numbers GSE308754 and GSE308755, respectively. Source data are provided with this paper.

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Acknowledgements

We thank the Vivarium, Flow Cytometry, Histopathology, Advanced BioImaging and BioOptics Experimental Platform, Molecular Biology, Hardware and Software Platform, Glass Wash, and Media platforms at Champalimaud Foundation. We thank Congento LISBOA-01-0145-FEDER-022170. We thank Mariana Monteiro (Histopathology platform, Champalimaud Foundation) for quantification of goblet, tuft and Ki67+ cells. We thank M. Patrício and S. Tehrani (Champalimaud Foundation, Portugal) for technical help with N. brasiliensis larvae. S. entericaserovar Typhimurium 14028 was kindly provided by I. Gordo, GIMM, Portugal. Infective (iL3) worms of N. brasiliensis were kindly provided by J. Allen, University of Manchester, UK. We thank C. Schneider (University of Zurich, Switzerland), M. Rao (Harvard Medical School, USA), P. Bastos, M. Martinez-Lopez, A. Rasteiro and M. Aliseychik (Champalimaud Foundation, Portugal) for helpful discussions. R.M.P. was supported by FCT (CEECIND/03601/2018; PTDC/MED-IMU/6381/2020), European Crohn’s and Colitis Organisation Grant, the European Foundation for the Study of Diabetes (EFSD)/Lilly Young Investigator award, EFSD/Novo Nordisk (NN) Rising Star Fellowship and the European Association for the Study of Obesity/NN grant. J.R. was supported by EU HORIZON-MISS-2021-CANCER-02-03 (GENIAL 101096312). C.G.-S. was supported FCT (2023.07506.CEECIND and PTDC/MED-IMU/2189/2021). M.R. was supported by FCT (PTDC/MED-IMU/2189/2021). C.W. was supported by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) under Germany’s Excellence Strategy – EXC2151 – 390873048 and WI 4554/6-1. C.S.N.K. was funded by DFG, under Germany’s Excellence Strategy – EXC 3118/1 – project 533770413. V.M.L was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases. M.F. and J.T. were supported by la Caixa Foundation (LCF/PR/HR22/52420007) and FCT (2022.06145.PTDC). C.M.M. and V.C. were supported by the European Research Council (101116335) and by the European Haematology Association. H.V.-F. was supported by the European Research Council (647274 and 101097830); Paul G. Allen Frontiers Group (12826); Chan Zuckerberg Initiative (INFL-0000000193); La Caixa (HR20-00841); EU HORIZON-MISS-2021-CANCER-02-03 (GENIAL 101096312) and FCT (PTDC/MED-IMU/6653/2020).

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Authors and Affiliations

  1. Champalimaud Foundation. Champalimaud Centre for the Unknown. Champalimaud Research, Lisbon, Portugal

    Roksana M. Pirzgalska, Beatriz Henriques-Alves, Bruno Raposo, Eric de Sousa, Jaechan Ryu, Cristina Godinho-Silva, Miguel Rendas, Madalena Pereira, Inês Godinho, Hélder Ribeiro, Vasco Correia, Tânia Carvalho, Carlos M. Minutti & Henrique Veiga-Fernandes

  2. University of Coimbra, Center for Innovative Biomedicine and Biotechnology, Center for Neuroscience and Cell Biology, Coimbra, Portugal

    Júlio Torres & Manuela Ferreira

  3. Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Microbiology, Infectious Diseases and Immunology, Berlin, Germany

    Manuel O. Jakob, Patrycja M. Forster & Christoph S. N. Klose

  4. Department of Visceral Surgery and Medicine, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

    Manuel O. Jakob

  5. Institute of Clinical Chemistry and Clinical Pharmacology. University Hospital Bonn, Bonn, Germany

    Christoph Wilhelm

  6. Cluster of Excellence ImmunoPreCept, Charité - Universitätsmedizin Berlin, Berlin, Germany

    Christoph S. N. Klose

  7. Champalimaud Foundation. Champalimaud Centre for the Unknown. Champalimaud Clinical Center, Lisbon, Portugal

    Ricardo Rio-Tinto

  8. Metaorganism Immunity Section, Laboratory of Host Immunity and Microbiome, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Verena M. Link

Authors
  1. Roksana M. Pirzgalska
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  2. Beatriz Henriques-Alves
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  3. Bruno Raposo
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  7. Cristina Godinho-Silva
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  8. Miguel Rendas
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  11. Hélder Ribeiro
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  13. Manuel O. Jakob
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  20. Carlos M. Minutti
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  22. Henrique Veiga-Fernandes
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Contributions

Initial observations leading to the study: R.M.P.; conceptualization: R.M.P. and H.V.-F.; methodology: R.M.P., B.H.-A., B.R., E.d.S., J.T., C.G.-S., M.R., T.C., V.C., R.R.-T., C.W., C.S.N.K., T.C., V.M.L., C.M.M., M.F. and H.V.-F.; investigation: R.M.P., B.H.-A., B.R., E.d.S., J.T., J.R., C.G.-S., M.R., M.P., I.G., V.C., M.O.J. and P.M.F.; visualization: R.M.P., B.H.-A., E.d.S. and T.C.; funding acquisition: R.M.P. and H.V.-F.; project administration: H.R. and H.V.-F.; supervision: R.M.P. and H.V.-F.; and writing: R.M.P. and H.V.-F.

Corresponding author

Correspondence to Henrique Veiga-Fernandes.

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The authors declare no competing interests.

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Nature Immunology thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Epithelia-autonomous glucocorticoid signals are dispensable to IELs and ILC2 in the small intestine.

(a–c) Flow cytometry gating strategy. a, small intestinal epithelial cells. b, small intestinal IELs. c, small intestinal ILC2s in the lamina propria (d, e) Flow cytometry validation of ILC gating strategy with transcription factors. d, GATA2 for ILC2. e, RORγt for ILC3. f, Flow cytometry analysis of small intestinal IELs from Nr3c1fl (n = 7) and Nr3c1∆Villin (n = 6) mice. g, Flow cytometry analysis of small intestinal ILC2s from the lamina propria of Nr3c1fl (n = 5) and Nr3c1∆Villin (n = 5) mice. (f, g) Mean and error bars: s.e.m. n represents biologically independent animals. (f, g) Two-tailed unpaired t test with Welch correction.

Source data

Extended Data Fig. 2 VIPergic modulation of epithelial-derived alarmins.

a, Representative images of CD3+cells in the duodenum obtained by confocal microscopy. Scale bar, 20μm. VIPergic neurons (red); CD3+cells (green). b, Representative plots from flow cytometry analysis of small intestinal lamina propria of R26-TomatoVIP mice. c, Relative expression of type 1 and type 2 alarmins in purified intestinal epithelial cells from the duodenum of R26-hM3Dqfl (n = 3) and R26-hM3DqVIP (n = 3) mice under steady-state conditions, without CNO administration. (d–f) Systemic chemogenetic manipulation of VIP-expressing cells. d, Relative expression of type 2 alarmins in purified epithelial cells from the duodenum of R26-hM3Dqfl (n = 8) and R26-hM3DqVIP (n = 8) mice following systemic CNO-driven chemogenetic activation. e, Glucocorticoid levels in the serum of R26-hM3Dqfl (n = 5) and R26-hM3DqVIP (n = 6) mice following CNO-driven chemogenetic activation. Right panel depicts representative images of spleens from R26-hM3Dqfl and R26-hM3DqVIP mice following CNO-driven chemogenetic activation. f, Relative expression of type 2 alarmins in purified epithelial cells from the duodenum of R26-hM4Difl (n = 8) and R26-hM4DiVIP (n = 8) mice following CNO-driven chemogenetic inhibition. g, Validation of infection efficiency in duodenal chemogenetic activation (top) and inhibition (bottom) by confocal microscopy of duodenal sections. Scale bar, 20μm. AAV9-targetting construct (magenta), VIPergic neurons (yellow), nuclei (DAPI, blue). h, VIP levels in the duodenum from Vip-Cre mice (Vip-Cre.AAV9-hM3DqGut n = 6, represented in magenta) and control littermates (WT.AAV9-hM3DqGut n = 6, represented in dark gray) injected with AAV9-carrying activatory DREADDSs and after CNO-driven activation. i, Relative expression of mouse Vipr1 and Vipr2 (Left) (n = 9) and human VIPR1 and VIPR2 (Right) (n = 12) in small intestinal epithelial cells. (c–f, h, i) Mean and error bars: s.e.m. n represents biologically independent animals or samples. (c–f, h, i) Two-tailed unpaired t test with Welch correction.

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Extended Data Fig. 3 Vipr1 deletion in intestinal epithelial cells.

a, Graphical representation of Vipr1 gene targeting. Blue boxes represent exons; neo: neomycin resistance cassette; FRT: recognition sequence for flp recombinase; loxP: recognition sequence for Cre recombinase. b, Example of genotype PCR of Vipr1fl/fl mice. Primer sequences are provided in the Methods section. ntc-no template control. c, scRNA-seq analysis of Vipr1 and Vipr2 expression in small intestinal epithelial cells from Vipr1fl and Vipr1∆Villin mice. Data shown are representative of intestinal segments collected from two animals per group. d, Representative hematoxylin and eosin (H&E) staining of small intestinal tissue (duodenum-left; ileum-right) from Vipr1fl and Vipr1∆Villin mice. Original magnification 5x (upper row: scale bar, 200μm) and 40x (lower row: scale bar, 20μm). e, Ki-67 index in the duodenum (upper panel) and ileum (lower panel) from Vipr1fl (n = 6–7) and Vipr1∆Villin (n = 6) mice. f, Representative anti-Ki-67 staining in the duodenum (upper panels) and ileum (lower panels) from Vipr1fl and Vipr1∆Villin mice. Scale bar, 40μm (e) Mean and error bars: s.e.m. n represents biologically independent samples. Two-tailed unpaired t test with Welch correction.

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Extended Data Fig. 4 Profiling in Vipr1 conditional knockout mice and tuft cell gating.

a, VIP levels in the small intestine (duodenum) from Vipr1fl (n = 6) and Vipr1∆Villin (n = 5) mice. b, VIP levels in serum from Vipr1fl (n = 5) and Vipr1∆Villin (n = 5) mice. c, Intestinal transit time measured in Vipr1fl (n = 6) and Vipr1∆Villin (n = 6) mice. d, scRNA-seq analysis of Cftr and Slc12a2 expression in small intestinal epithelial cells from Vipr1fl and Vipr1∆Villin mice. Data shown are representative of intestinal segments collected from two animals per group. e, Fecal water content measured as wet-to-dry weight in Vipr1fl (n = 6) and Vipr1∆Villin (n = 6) mice. f, Representative plots of flow cytometry gating for small intestinal tuft cells. g, Relative expression of Vipr1 in purified epithelial cells from the duodenum and ileum of Vipr1fl (n = 6) and Vipr1∆VillinERT2 (n = 5) mice following tamoxifen-driven recombination. Expression was assessed using primers targeting exon 2 of the Vipr1 gene. (a–c, e, g) Mean and error bars: s.e.m. n represents biologically independent animals. (a–c, e) Two-tailed unpaired t test with Welch correction. (d) Two-tailed Wilcoxon rank-sum test. n.d. not detected.

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Extended Data Fig. 5 Epithelial-intrinsic Vipr1 deletion shape intestinal IELs and cell-intrinsic Vipr2 signaling is dispensable to IEL homeostasis.

a, Flow cytometry analysis of small intestinal IELs collected from the duodenum (duo) and ileum of Vipr1fl (n = 6) and Vipr1∆Villin (n = 4) mice after microbiota depletion with antibiotics. b, Flow cytometry analysis of small intestinal IELs collected from the duodenum (duo) and ileum of Vipr1fl (n = 5) and Vipr1∆VillinERT2 (n = 5) after antibiotic treatment. c, d, Flow cytometry analysis of small intestinal IELs collected from the duodenum (duo) and ileum of Vipr2fl (n = 5) and Vipr2∆CD8a (n = 5) mice. e, Relative expression of type 1 (Il15, Il18) and type 2 (Il33, Il25) alarmins in murine organoids stimulated with VIP or vehicle. Vehicle n = 5; VIP n = 5. (a–e) Mean and error bars: s.e.m. n represents biologically independent animals. (a–d) Ordinary one-way ANOVA with Šídák multiple comparisons test. (e) Two-tailed unpaired t test with Welch correction.

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Extended Data Fig. 6 Neuroepithelial interactions modulate gut defense against bacteria.

(a–d) Co-housed Vipr1fl and Vipr1∆Villin mice were orally infected with Salmonella enterica. a, Relative expression of type 1 (Il15, Il18) and type 2 (Il33) alarmins in purified epithelial crypts from the duodenum of Vipr1fl (n = 5) and Vipr1∆Villin (n = 5) mice at day 1 post-infection. b, GFP immunostaining in Salmonella infected mice. Peyer’s Patches (PPs) from the Vipr1fl mice (left) displayed preserved morphological features of the follicle-associated epithelium (arrow) and lack of GFP+ staining, whereas PPs from Vipr1∆Villin mice showed numerous GFP+ Salmonella (brown), erosion and necrosis of the epithelium (asterisk; right panel). DAB counterstained with Harris’ hematoxylin. Original magnification: 40x, Scale bar, 40 µm (first two panels); 100x, Scale bar, 20 µm (magnified panels). c, Histopathology of the small intestine (ileum) at day 1 post-infection. Pathological changes include moderate to severe enteritis characterized by a mononuclear-rich inflammatory infiltrate in the mucosa (black arrows). Necrotic cell debris in the lumen (*) and necrosis of epithelial cells (open arrow); pronounced villous atrophy (open arrowhead) and villous blunting are more prominent in the Vipr1∆Villin mice. Hematoxylin and eosin (H&E). Scale bar, 200 μm (top); 25 μm (bottom). d, Histopathology of the small intestine (jejunum) at day 2 post-infection. Pathological changes in Vipr1∆Villin mice include mild enteritis (black arrows), abundant necrotic cell debris in the lumen (*), and necrosis of epithelial cells (white arrows). Hematoxylin and eosin (H&E). Scale bar, 400 μm (top); 50 μm (bottom). (a) Mean and error bars: s.e.m. n represents biologically independent animals. Two-tailed unpaired t test with Welch correction.

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Extended Data Fig. 7 Epithelial-intrinsic Vipr1 deletion and intestinal immune cells.

a, Flow cytometry analysis of immune cell populations in the small intestinal lamina propria from Vipr1fl and Vipr1∆Villin mice. Absolute numbers of CD4 T cells, TCRαβ CD8 T cells, TCRγδ CD8 T cells, B cells, cDC1, cDC2, macrophages, and neutrophils were determined in Vipr1fl (n = 7) and Vipr1∆Villin (n = 6) mice, while ILC2 and ILC3 were analyzed in Vipr1fl (n = 11) and Vipr1∆Villin (n = 10) mice, and ILC1 and NK cells were assessed in Vipr1fl (n = 4) and Vipr1∆Villin (n = 4) mice. b, Flow cytometry gating strategy for small intestinal lamina propria immune cells. (a) Mean and error bars: s.e.m. n represents biologically independent animals. Two-tailed unpaired t test with Welch correction.

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Extended Data Fig. 8 Epithelial-intrinsic Vipr1 deletion amplifies type 2 responses independently of microbiota.

(a–d) Flow cytometry analysis after microbiota depletion. a, Small intestinal PD-1+ ILC2 from the lamina propria of Vipr1fl (n = 6) and Vipr1∆Villin (n = 4) mice. b, Small intestinal eosinophils from the lamina propria of Vipr1fl (n = 6) and Vipr1∆Villin (n = 4). c, Small intestinal PD-1+ ILC2 from the lamina propria of Vipr1fl (n = 5) and Vipr1∆VillinERT2 (n = 5) mice. d, Small intestinal eosinophils from the lamina propria of Vipr1fl (n = 5) and Vipr1∆VillinERT2 (n = 6) mice. (e, f) Vipr2 deletion in IL-5–producing cells. e, Flow cytometry analysis of small intestinal ILC2 from the lamina propria of Vipr2WTIl5 (n = 4) and Vipr2∆Il5 (n = 5) mice. f, Flow cytometry analysis of cytokine production by small intestinal ILC2 from the lamina propria of Vipr2WTIl5 (n = 4) and Vipr2∆Il5 (n = 5) mice. g, Flow cytometry analysis of PD-1+ ILC2 from the mesenteric lymph nodes (mLN) from Vipr1fl (n = 6) and Vipr1∆Villin (n = 6). h, Small intestinal PD-1+ ILC2 and eosinophils isolated from the lamina propria after duodenal chemogenetic inhibition of VIPergic neurons. WT.AAV9-hM4DiGut n = 6; Vip-Cre.AAV9-hM4DiGut n = 5. i, Representative images of PAS staining in the ileum of Rag1−/−.Vipr1fl and Rag1−/−.Vipr1∆VillinERT2 mice. Periodic acid-Schiff (PAS). Scale bar, 200 μm (top panels) and 20 μm (bottom panels). j, Small intestinal PD-1+ ILC2 isolated from the lamina propria of Rag1−/−.Vipr1fl (n = 5) and Rag1−/−.Vipr1∆VillinERT2 (n = 4) mice after antibiotic treatment. k, Small intestinal eosinophils isolated from the lamina propria of Rag1−/−.Vipr1fl (n = 5) and Rag1−/−.Vipr1∆VillinERT2 (n = 4) mice after antibiotic treatment. (a–h, j, k) Mean and error bars: s.e.m. n represents biologically independent animals. (a, b) Two-tailed unpaired Mann–Whitney U-test. (c–g, j, k) Two-tailed unpaired t test with Welch correction. (h) Ordinary one-way ANOVA with Šídák multiple comparisons test.

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Extended Data Fig. 9 Neuroepithelial interactions modulate gut defense against worm infections.

(a, b) Co-housed Vipr1fl and Vipr1∆Villin mice were infected with Nippostrongylus brasiliensis. a, Relative expression of type 1 (Il15, Il18) and type 2 (Il33) alarmins in purified epithelial crypts from the duodenum of Vipr1fl (n = 6) and Vipr1∆Villin (n = 5) mice at day 5 post-infection. b, Representative H&E images from the duodenum and ileum of the Vipr1fl and Vipr1∆Villin mice showing a mild increase in inflammatory cell infiltration in the mucosa of some villi (asterisks), predominantly composed of mononuclear cells. Hematoxylin and eosin (H&E). Scale bar, 200 μm (top panels) and 50 μm (bottom panels). (a) Mean and error bars: s.e.m. n represents biologically independent animals. Two-tailed unpaired Mann-Whitney U test.

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Supplementary information

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Unprocessed PCR gel from Extended Data Fig. 3b.

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Pirzgalska, R.M., Henriques-Alves, B., Raposo, B. et al. Neuroepithelial VIP–VIPR1 interactions differentially control enteric type 1 and type 2 immunity. Nat Immunol 26, 2244–2255 (2025). https://doi.org/10.1038/s41590-025-02326-0

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