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Basophils prime group 2 innate lymphoid cells for neuropeptide-mediated inhibition

Abstract

Type 2 cytokine responses promote parasitic immunity and initiate tissue repair; however, they can also result in immunopathologies when not properly restricted. Although basophilia is recognized as a common feature of type 2 inflammation, the roles basophils play in regulating these responses are unknown. Here, we demonstrate that helminth-induced group 2 innate lymphoid cell (ILC2) responses are exaggerated in the absence of basophils, resulting in increased inflammation and diminished lung function. Additionally, we show that ILC2s from basophil-depleted mice express reduced amounts of the receptor for the neuropeptide neuromedin B (NMB). Critically, NMB stimulation inhibited ILC2 responses from control but not basophil-depleted mice, and basophils were sufficient to directly enhance NMB receptor expression on ILC2s. These studies suggest that basophils prime ILC2s to respond to neuron-derived signals necessary to maintain tissue integrity. Further, these data provide mechanistic insight into the functions of basophils and identify NMB as a potent inhibitor of type 2 inflammation.

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Fig. 1: Basophils regulate helminth-induced inflammation.
Fig. 2: Basophils negatively regulate ILC2 responses.
Fig. 3: Basophils regulate ILC2 responses independently of T cells.
Fig. 4: Basophils promote the expression of NMBR on ILC2s.
Fig. 5: NMB suppresses helminth-induced type 2 cytokine responses.
Fig. 6: NMBR expression is required to limit helminth-induced inflammation.
Fig. 7: NMB inhibits ILC2-mediated anthelmintic immunity.
Fig. 8: Basophils prime ILC2s for negative regulation by NMB.

Data availability

Bulk RNA-seq and single-cell RNA-seq data are deposited in the Gene Expression Omnibus under accession code GSE150793. Source data are provided with this paper.

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Acknowledgements

We thank members of the Center for Immunity and Inflammation for discussions and critical reading, the New Jersey Medical School (NJMS) Flow Cytometry and Immunology Core Laboratory for technical assistance and the NJMS Histology Core and the NJMS Genomics Research Lab for bioinformatics assistance. We thank D. Voehringer for kindly providing the Mcpt8Cre mice and D. Sant’Angelo for providing the OP9-DL1 cell line. This work was supported by the National Institutes of Health (grant nos. RO1 AI123224 and RO1 AI131634 to M.C.S., T32AI125185 to C.B.S. and 3R01AI131634-02W1 to C.M.H.). J.M.I.R. is supported by the Mexican Council of Science & Technology (CVU 536876).

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J.M.I.R., J.J.P., N.V.P., C.M.H., C.B.S., A.D.L. and M.C.S. designed and performed the research. A.M.B. contributed to experimental design and data analysis, conceptualization and manuscript editing. J.M.I.R. and M.C.S. analyzed the experimental data and wrote the paper.

Corresponding author

Correspondence to Mark C. Siracusa.

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Mark C. Siracusa is the founder and president of Nemagen Discoveries.

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Peer review information Jamie D. K. Wilson was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Basophils limit helminth-induced pulmonary inflammation.

a, Supernatant levels of IL-4, IL-5 and IL-13 from re-stimulated mesenteric lymph nodes (mLNs) isolated from control or basophil-depleted mice. Mucus production was evaluated in control and basophil-depleted mice on day 7 post-Nb infection by (b), periodic acid shiff (PAS) staining and (c), Muc5ac expression in the lungs by real-time PCR. d, Lung pathology was evaluated by H&E-stained sections with individual images digitally tiled together to provide a larger overview. P values were determined by two-tailed Student’s t-tests. a-d, Representative of at least 3 separate experiments with at least 5 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. (b), Illustrate data pooled from 2 separate experiments.

Source data

Extended Data Fig. 2 Basophil depletion results in elevated ILC2 responses.

a, Lung neutrophils and (b), eosinophils were quantified by flow cytometry on day 7 post-Nb infection in control or baso-dep mice. c, Representative flow cytometric gating strategy to evaluate neutrophils and eosinophils. d, ILC2s in the lung were quantified on day 7 post-Nb infection in control or baso-dep mice. Intracellular cytokine staining for (e, f), IL-5 and IL-13 was performed on lineage negative, CD90+, CD127+ ILC2s in lung on day 7 post-Nb infection and cytokine positive cells were quantified. g, Representative flow cytometric gating strategy to evaluate ILC2 populations. P values were determined by two-tailed Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. a-g, Representative of at least 3 separate experiments with at least 5 mice per group.

Source data

Extended Data Fig. 3 Constitutive ablation of basophils is associated with increased ILC2 activation.

a, b, IL-5+ and IL-13+ ILC2s, as well as (c), eosinophils in the BAL and (d-f), lungs were quantified in control and Mcpt8Cre-4get mice that constitutively lack basophils, 7 days post-Nb infection. P values were determined by two-tailed Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. a-f, Representative of at least 3 separate experiments with at least 5 mice per group.

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Extended Data Fig. 4 Basophils are sufficient to limit helminth-induced ILC2 responses.

a, ILC2 numbers, (b), ILC2 production of IL-5, and (c), IL-13, as well as (d) eosinophil numbers were quantified in the lung on day 7 post-Nb infection in control mice, baso-dep mice, or baso-dep mice that received adoptive transfers of basophils. e, H&E staining of lung sections on day 7 post-Nb infection with individual images digitally tiled together to provide a larger overview. Mucus production in the lung was evaluated by (f), PAS staining of lung sections and (g), Muc5ac expression. P values were determined by two-tailed Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. a-g, Representative of at least 3 separate experiments with at least 4 mice per group.

Source data

Extended Data Fig. 5 Basophils regulate ILC2s independently of adaptive lymphocytes.

Nb-infected Rag2-/- mice were treated with isotype control or the basophil-depleting antibody MAR-1 and (a), ILC2 responses and (b), eosinophilia were determined in the BAL and (c,d), lung on day 7 post-infection. (e), H&E staining of lung sections on day 7 post-Nb infection with individual images digitally tiled together to provide a larger overview. (f), Mucus production in the lung was evaluated by Muc5ac expression. P values were determined by two-tailed Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. a-f, Representative of at least 3 separate experiments with at least 2 mice per naive groups and at least 4 mice per infected groups.

Source data

Extended Data Fig. 6 Elevated ILC2 responses are not associated with increased cytokine alarmin expression.

a-c, Expression of cytokine alarmins in the lungs of control and baso-dep mice was determined on day 7 post-Nb infection by real-time PCR. d, e, Numbers of IL-33-GFP+ type 1 and type 2 pneumocytes were evaluated in IL-33-GFP-reporter mice infected with Nb and treated with the basophil-depleting antibody MAR-1. Expression of (f), Il10 and (g), Areg in the lungs of control and baso-dep mice was determined on day 7 post-Nb infection by real-time PCR. Splenic basophils were sort-purified and cultured (O/N) with IL-3 and anti-IgE antibody and supernatant levels of (h), IL-6, (i), amphiregulin (Areg), and (j), IL-10 were evaluated by ELISA. P values were determined by two-tailed Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. a-g, Representative of at least 2 separate experiments with at least 2 mice per naive groups and at least 5 mice per infected groups. h-j, Representative of at least 3 separate experiments with at least 5 individual samples of sort-purified basophils from 5 mice per experimental group.

Source data

Extended Data Fig. 7 Single cell RNAseq analysis of lung-resident ILC2s.

a, Uniform Manifold Approximation and Projection (UMAP) plot illustrating defined clusters of cells generated by single cell RNA-sequencing of lung-resident live ILC2 populations (CD45+Lin-CD90+CD127+) sort-purified from control (and basophil-depleted (baso-dep) mice 5 days post-Nb infection. b, Top 10 marker genes expressed by each cluster of ILC2s. c, Single-cell expression of Il5, Il13, Areg, Arg1, Il1rl1, and Il17rb in ILC cell clusters as defined in A. Horizontal bars represent mean normalized expression. P values were determined by Wilcoxon signed rank sum test. *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 8 Analysis of NMBR expression in the hematopoietic compartment.

a, Heat map illustrating representative genes of interest expressed in control or baso-dep ILC2s. Surface NMBR expression by (b), CD4+ T cells, (c), alveolar macrophages, (d), non-alveolar macrophages, (e), neutrophils, and (f), eosinophils was determined in lung suspensions of naïve and mice infected with Nb 7 days prior. P values were determined by two-tailed Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. (b-f), Representative of at least 3 separate experiments with at least 2 mice per naive groups and at least 4 mice per infected groups.

Source data

Extended Data Fig. 9 NMB-NMBR signaling suppresses helminth-induced ILC2 responses.

a, Schematic illustrating targeting strategy and placement of loxP cassettes upstream and downstream of exon 2 of the Nmbr gene. b-d, Type 2 cytokine expression in the lungs of NMBRfl/fl controls and NMBRfl/fl x Vav-iCre+ mice was determined on day 7 post-Nb infection by real-time PCR. e, IL-5+ and (f), IL-13+ ILC2s, as well as (g), eosinophils were quantified in the lungs of NMBRfl/fl x Vav-iCre+ mice 7 days post-Nb. Nb-infected Rag2-/- mice were treated with PBS or rNMB (i.t.) and (h, i), the percentage of IL-5+ and IL-13+ ILC2s were determined in the BAL and (j), the total number of IL-5+ and IL-13+ ILC2s were determined in the lung on day 7 post-infection. k, eosinophils and (l), neutrophils were determined in the lungs of Rag2-/- mice treated with PBS or rNMB on day 7-post infection. P values were determined by two-tailed Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. b-l, Representative of 3 separate experiments with at least 3 mice per naive groups and at least 5 mice per infected groups.

Source data

Extended Data Fig. 10 Basophils are required for NMBR-mediated inhibition of ILC2s.

Sort-purified ILC2s were cultured (O/N) with vehicle or rNMB in the presence of IL-2 and IL-7 or IL-2, IL-7, and IL-33. a, b, The percentage of IL-5+ and IL-13+ ILC2s were quantified by intracellular staining. c, d, IL-5 and IL-13 levels in the supernatant were quantified by ELISA. Sort-purified ILC2s were cultured (O/N) alone or with activated basophils. e, Cytokine levels in the supernatant were monitored by ELISA and (f, g), cell proliferation was evaluated by CTV dilution 4 days post-culture. h, Heat map illustrating genes differentially expressed at 2.0-fold or higher between control or NMB-treated ILC2s. i, Heat map illustrating genes not differentially expressed in control or NMB-treated ILC2s. P values were determined by two-tailed Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. a-g, Representative of at least 3 separate experiments with at least 5 individual samples of sort-purified ILC2s in each experimental group.

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Inclan-Rico, J.M., Ponessa, J.J., Valero-Pacheco, N. et al. Basophils prime group 2 innate lymphoid cells for neuropeptide-mediated inhibition. Nat Immunol 21, 1181–1193 (2020). https://doi.org/10.1038/s41590-020-0753-y

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