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Neuropilin-1 mediates lung tissue-specific control of ILC2 function in type 2 immunity

Nature Immunology volume 23, pages 237–250 (2022)Cite this article

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Abstract

Group 2 innate lymphoid cells (ILC2s) are highly heterogeneous tissue-resident lymphocytes that regulate inflammation and tissue homeostasis in health and disease. However, how these cells integrate into the tissue microenvironment to perform tissue-specific functions is unclear. Here, we show neuropilin-1 (Nrp1), which is induced postnatally and sustained by lung-derived transforming growth factor beta-1 (TGFβ1), is a tissue-specific marker of lung ILC2s. Genetic ablation or pharmacological inhibition of Nrp1 suppresses IL-5 and IL-13 production by ILC2s and protects mice from the development of pulmonary fibrosis. Mechanistically, TGFβ1–Nrp1 signaling enhances ILC2 function and type 2 immunity by upregulating IL-33 receptor ST2 expression. These findings identify Nrp1 as a tissue-specific regulator of lung-resident ILC2s and highlight Nrp1 as a potential therapeutic target for pulmonary fibrosis.

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Fig. 1: Nrp1 is a tissue-specific marker of lung ILC2s.
Fig. 2: Lung-derived signals are required for Nrp1 expression by ILC2s.
Fig. 3: TGFβ1 induces and maintains Nrp1 expression by lung ILC2s.
Fig. 4: Ablation of Nrp1 in ILC2s ameliorates pulmonary fibrosis.
Fig. 5: Nrp1 regulates ILC2s function in a cell-intrinsic manner during fibrosis.
Fig. 6: Nrp1 promotes ILC2 function through upregulation of ST2.
Fig. 7: Nrp1 inhibitor EG00229 alleviates pulmonary fibrosis.

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Data availability

RNA-sequencing data have been deposited in GEO under the primary accession code GSE168809. Published ILC2 scRNA-seq datasets were accessed in GEO under the accession code GSE117568. Published ILC2 bulk RNA-seq dataset were accessed in GEO under the accession code GSE126107. The raw reads of RNA-seq were aligned to the mouse reference genome (version mm10). All other data are available in the article and supplementary files or from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank members of L. Shen’s laboratory for their help and suggestions on this project. We thank the National Institute of Parasitic Diseases at Chinese Center for Disease Control and Prevention for help with N. brasiliensis infection experiments. We thank the Flow Cytometry Facility and Animal Facility at Shanghai Jiao Tong University School of Medicine for service and assistance. We thank J. Zhou and T. Hong for helping with human samples collection. We thank E. Engleman for editing the manuscript. This study was supported by grant 2020YFA0509200 (to L.S.) and 2020YFA0509103 (to J.Q.) from the Ministry of Science and Technology of China, grant 81971487 (to L.S.), 32022027 and 31970860 (to J.Q.) from the National Natural Science Foundation of China, grant 20ZR1430200, 20142202300 (to L.S.) and 20ZR1466900 (to J.Q.) from Science and Technology Commission of Shanghai Municipality, and Shanghai Jiao Tong University School of Medicine Innovation Team on Pediatric Research (to L.S.).

Author information

Author notes

  1. These authors contributed equally Jingjing Zhang, Jinxin Qiu.

Authors and Affiliations

  1. Shanghai Institute of Immunology, Department of Immunology and Microbiology, and Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Jingjing Zhang, Bing Su & Lei Shen

  2. Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Jingjing Zhang & Lei Shen

  3. CAS Key Laboratory of Tissue Microenvironment and Tumor, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China

    Jinxin Qiu & Ju Qiu

  4. Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China

    Wenyong Zhou

  5. NHC Key Laboratory of Parasite and Vector Biology, National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, China

    Jianping Cao

  6. The School of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Jianping Cao

  7. Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China

    Xuefei Hu

  8. Department of Integrative Medicine and Neurobiology, School of Basic Medical Science; Institutes of Integrative Medicine, Shanghai Medical College, Fudan University, Shanghai, China

    Wenli Mi

  9. Department of Critical Care Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China

    Bin He

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Contributions

J.Z. and J.X.Q. performed the experiments and analyzed the data. W.Z. and X.H. contributed to human samples collection and experiment design. J.C. supervised worm infection experiments. W.M. and B.S. contributed to discussion. J.Z. performed bioinformatics analyses. L.S., J.Q., and J.Z. wrote the manuscript. L.S., J.Q., and B.H. conceived, designed, and supervised the project.

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Correspondence to Bin He, Ju Qiu or Lei Shen.

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Nature Immunology thanks Ralph Stadhouders and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. N. Bernard was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Nrp1 is a tissue-specific marker of lung ILC2s.

(a) Box plot compared the expression of NRP1 in human ILC2s from lung (n = 6) and spleen (n = 5). The bulk RNA-seq data were obtained from Gene Expression Omnibus (GEO) under accession code GSE126107. The center line, lower whisker, and upper whisker stand for median, minima, and maxima, respectively. Bounds of box are 25-75th percentile. (b) Gating strategy of ILC2s. ILC2s were gated on live and single cells with lin−(CD3−B220−CD11b−CD11c−CD5−) CD45+CD127+GATA3+. (c) The compensation of 15-color (including 19 markers) flow cytometric panel was shown by 14×14 view. Cells were gated on single lymphocytes. (d) ILC2s sorted from large intestine were cultured with TGFβ1 at 1 ng/ml for 48 h. mRNA expression level of Nrp1 was analyzed by quantitative RT-PCR (n = 3/group). Each symbol represents an individual mouse (d). Data are representative of 3 independent experiments. Two-tailed unpaired Student’s t test.

Source data

Extended Data Fig. 2 Loss of Nrp1 does not alter lung ILC2 homeostasis in Id2cre-ERT2Nrp1f/f mice under steady state.

a-m Id2cre-ERT2Nrp1f/+ and Id2cre-ERT2Nrp1f/f mice were injected intraperitoneally with tamoxifen (2 mg/day per mouse) for 5 days to knock out Nrp1 expression in the ILC lineage. (a-c) The expression level of Nrp1 on Treg, DCs, and neutrophils from lung were analyzed by flow cytometry. (a) The top row was gated on CD45+ live cells. (b) The top row was gated on CD45+CD11b+F4/80−Ly6G− live cells. (c) The top row was gated on CD45+ live cells. (d-f) Absolute cell counts of lung Treg, DCs, and neutrophils (n = 3/group). (g) The expression level of Nrp1 on lung ILC2s were analyzed by flow cytometry. Cells were gated on lin−CD45+CD127+ lymphocytes. (h, i) H&E staining and Masson’s trichrome staining of lung tissue. (j) Absolute cell counts of lung ILC2s (n = 11/group). (k) Percentage of IL-5+ ILC2s and IL-13+ ILC2s. Cells were gated on total ILC2s. (l, m) Frequency and total number of IL-5+ ILC2s (n = 10/group) and IL-13+ ILC2s (n = 7/group) Each symbol represents an individual mouse (d-f, j, l, and m). Data are representative of 3-4 independent experiments and are presented as mean ± SEM.

Source data

Extended Data Fig. 3 Ablation of Nrp1 impairs anti-helminth immunity in Id2cre-ERT2Nrp1f/f mice.

a-i Id2cre-ERT2Nrp1f/+ and Id2cre-ERT2Nrp1f/f mice were injected intraperitoneally with tamoxifen (2 mg/day per mouse) for 5 days to knock out Nrp1 expression in the ILC lineage. 3 days later, these mice were infected with 500 L3 N brasiliensis. Lung tissues were collected and analyzed 5 days after worm infection. (a) Intracellular staining of IL-5 and IL-13 in lung ILC2s. (b, c) Frequency and absolute account of IL-5+ ILC2s in lung. (d, e) Frequency and absolute account of IL-13+ ILC2s in lung. (f) Total number of lung ILC2s. (n = 7 for black group and n = 8 for red group in b-f) (g) Flow cytometric analysis of eosinophils (CD45+Ly6G−CD11b+SiglecF+) in lung. Cells were gated on CD45+Ly6G− live single cells. (h) Absolute count of lung eosinophils. (i) Worm burden in the small intestine collected on day 7 post infection. (n = 8 for black group and n = 9 for red group in h-i) Each symbol represents an individual mouse (b-f, h, and i). Data are representative of 3 independent experiments and are presented as mean ± SEM. Two-tailed unpaired Student’s t test.

Source data

Extended Data Fig. 4 Nrp1 deficiency does not affect ILC2 compartment in R5/ + Nrp1f/f mice under steady state.

(a)The expression of Nrp1 on lung ILC2s in R5/ + and R5/ + Nrp1f/f mice was analyzed by flow cytometry. Cells was gated on lin-CD45 + CD127 + lymphocytes. (b, c) Frequency and absolute number of IL-5+ILC2s in lung (n = 4 for black group and n = 7 for red group). (d, e) Frequency and absolute number of IL-13+ILC2s in lung. (f) Absolute count of total ILC2s in lung. (n = 5 for black group and n = 7 for red group in d-f) Each symbol represents an individual mouse (b-f). Data are representative of 3 independent experiments and are presented as mean ± SEM.

Source data

Extended Data Fig. 5 Loss of Nrp1 reduces ILC2 function and anti-helminth immunity in R5/ + Nrp1f/f mice.

a-i R5/ + and R5/ + Nrp1f/f mice were infected with 500 L3 N brasiliensis subcutaneously. Lung tissues were collected and analyzed 5 days after worm infection. (a) Intracellular staining of IL-5 and IL-13 in lung ILC2s. (b, c) Frequency and absolute account of IL-5+ ILC2s in lung (n = 5/group). (d, e) Frequency and absolute account of IL-13+ ILC2s in lung (n = 5/group). (f) Total number of lung ILC2s (n = 5/group). (g) Flow cytometric analysis of eosinophils in lung. Cells were gated on CD45+ CD11b+Ly6G− live single cells. (h) Absolute count of lung eosinophils (n = 5/group). (i) Worm burden in the small intestine collected on day 7 post infection (n = 5/group). Each symbol represents an individual mouse (b-f, h, and i). Data are representative of 3 independent experiments and are presented as mean ± SEM. Two-tailed unpaired Student’s t test.

Source data

Extended Data Fig. 6 Nrp1 deficiency leads to reduced ILC2 function during pulmonary fibrosis.

(a) R5/ + and R5/ + Nrp1f/f mice were challenged with bleomycin to induce pulmonary fibrosis. Mice were sacrificed on day 21 and lung tissues were collected. Flow cytometric analysis of IL-5+ lymphocytes. Cells were gated on live CD45+ lymphocytes. b-e ILC2s isolated from lung and large intestine of R5/ + or R5/ + Nrp1f/f mice were transferred intravenously into Rag2–/–Il2rg–/– mice, respectively. After 7 days, the recipient mice were given bleomycin intranasally. Lung tissues were collected and analyzed on day 21 of fibrosis. (b, c) Frequency and absolute number of lung ILC2s (n = 4/group). (d, e) Frequency and absolute number of IL-5+ ILC2s in lung (n = 4/group). Each symbol represents an individual mouse (b-e). Data are representative of 2-3 independent experiments and are presented as mean ± SEM. Two-tailed unpaired Student’s t test.

Source data

Extended Data Fig. 7 The expression profile of Nrp1 in lung immune cells.

a, c-e Wild-type mice were challenged with bleomycin (2 mg/kg body weight) intranasally to induce fibrosis, and subsequently were treated with EG00229 or DMSO control for three weeks. (a) H&E staining of the representative lung tissue. (b) Nrp1 expression in lung immune cells from WT mice was analyzed by flow cytometry. (c-e) Absolute numbers of lung macrophages (n = 14 for DMSO, n = 11 for EG00229 in c), DCs and Treg cells (n = 5 for DMSO, n = 4 for EG00229 in d-e) in each group of mice. Each symbol represents an individual mouse (c-e). Data are representative of at least 3 independent experiments and are presented as mean ± SEM.

Source data

Extended Data Fig. 8 Treatment with EG00229 intranasally alleviates pulmonary fibrosis.

a-i Bleomycin-challenged mice were treated with EG00229 (2.5 mg/kg body weight in 20μl PBS) or DMSO control intranasally three times weekly for three weeks. (a) The survival rate of mice treated with EG00229 or DMSO control was observed for a period of 21 days (n = 16/group). (b, c) H&E staining and Masson’s trichrome staining of lung tissues. (d) mRNA expression of the fibrotic genes in lung (n = 3, 6, 6 for PBS, DMSO, and EG00229 group). (e, f) Frequency and absolute count of IL-13+ ILC2s (n = 6/group). (g, h) Frequency and absolute count of IL-5+ ILC2s (n = 6/group). (i) Total number of α-SMA+ myofibroblast (n = 6/group). Each symbol represents an individual mouse (d-i). Data are representative of at least 3 independent experiments and are presented as mean ± SEM. (a) Log-rank test. (d-i) Two-tailed unpaired Student’s t test.

Source data

Extended Data Fig. 9 The therapeutic effect of EG00229 on pulmonary fibrosis is dependent on lung ILC2s.

(a) Bleomycin-challenged Rag1−/− mice were treated with EG00229 (10 mg/kg body weight) or DMSO control intraperitoneally for three weeks. Each group of mice were also received 200 μg of anti-Thy1.2 or isotype control antibodies every 3 days throughout the treatment. Masson’s trichrome staining for each group was shown. Data are representative of 3 independent experiments.

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Zhang, J., Qiu, J., Zhou, W. et al. Neuropilin-1 mediates lung tissue-specific control of ILC2 function in type 2 immunity. Nat Immunol 23, 237–250 (2022). https://doi.org/10.1038/s41590-021-01097-8

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