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MrgprA3 neurons drive cutaneous immunity against helminths through selective control of myeloid-derived IL-33

Nature Immunology (2024)Cite this article

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Abstract

Skin uses interdependent cellular networks for barrier integrity and host immunity, but most underlying mechanisms remain obscure. Herein, we demonstrate that the human parasitic helminth Schistosoma mansoni inhibited pruritus evoked by itch-sensing afferents bearing the Mas-related G-protein-coupled receptor A3 (MrgprA3) in mice. MrgprA3 neurons controlled interleukin (IL)-17+ γδ T cell expansion, epidermal hyperplasia and host resistance against S. mansoni through shaping cytokine expression in cutaneous antigen-presenting cells. MrgprA3 neuron activation downregulated IL-33 but induced IL-1β and tumor necrosis factor in macrophages and type 2 conventional dendritic cells partially through the neuropeptide calcitonin gene-related peptide. Macrophages exposed to MrgprA3-derived secretions or bearing cell-intrinsic IL-33 deletion showed increased chromatin accessibility at multiple inflammatory cytokine loci, promoting IL-17/IL-23-dependent changes to the epidermis and anti-helminth resistance. This study reveals a previously unrecognized intercellular communication mechanism wherein itch-inducing MrgprA3 neurons initiate host immunity against skin-invasive parasites by directing cytokine expression patterns in myeloid antigen-presenting cell subsets.

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Fig. 1: S. mansoni antigens block the activation of itch-inducing MrgprA3 neurons.
Fig. 2: Activation of MrgprA3 neurons provides cutaneous immunity against S. mansoni.
Fig. 3: MrgprA3 neurons promote skin immunity against S. mansoni through myeloid APCs and γδ T cells.
Fig. 4: MrgprA3 neurons promote host resistance to S. mansoni by stimulating pro-inflammatory cytokines in macrophages dependent on RAMP1 signaling.
Fig. 5: MrgprA3 neurons selectively control IL-33 expression in skin myeloid APCs.
Fig. 6: Myeloid APC-derived IL-33 maintains skin homeostasis by limiting IL-17-driven excessive keratinization.
Fig. 7: IL-33 intrinsically regulates pro-inflammatory cytokine expression in cutaneous myeloid APCs.
Fig. 8: Genetic loss of IL-33 or stimulation with soluble factors from MrgprA3 neurons control chromatin accessibility at pro-inflammatory cytokine gene loci in macrophages.

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Data availability

All reagents generated or used in this study are available on request from the corresponding author with a completed Material Transfer Agreement. Information on reagents used in this study is available in Supplementary Table 1. Code pertinent to the data for this paper is available upon request. All the data supporting the findings of the article are available within the main text or Supplementary Information. The published article includes datasets generated during this study. Original bulk RNA-seq, single-cell RNA-seq and ATAC-seq data have been deposited in the Gene Expression Omnibus under accession GSE218834. Source data are provided with this paper.

Code availability

All newly generated code for data analysis regarding this paper is currently deposited on https://github.com/Cailu086Lin/scRNASeq_ATACSeq_IL33_Project/.

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Acknowledgements

We thank P. Bryce, for initially providing the IL-33fl/fl-eGFP mouse strain. We thank V. Kuchroo and K. Caron for providing tissues to generate BMDMs and BMDCs from RAMP1 KO mice. Foxp3YFP-Cre mice were a generous gift from C. Hunter. We thank the laboratories of E. Behrens and T. Kambayashi for supplying bone marrow from Il1rl1−/− mice. Langerin Cre mice were a kind gift from B. Igyártó. We kindly thank A. Blanco and R. C. Petroni for assistance performing the ATAC-seq analysis on BMDMs. We thank the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute for providing B. glabrata (NMRI) exposed to S. mansoni (NMRI) through NIH-NIAID contract HHSN272201700014I. This work was supported by the NIH (grant nos. T32 AI007532-24, R01 AI164715-01, U01 AI163062-01 and R01 AI123173-05 to D.R.H). J.M.I.-R. is supported by the Life Sciences Research Foundation and by the Skin Biology and Disease Research Core Pilot and Feasibility Grant of the University of Pennsylvania (NIAMS P30-AR069589). T.M. and P.H. are supported by Charles University institutional funding (Cooperatio Biology, UNCE24/SCI/011, SVV 260687) and Czech Science Foundation (GA24-11031S). The data for this paper were generated in the Penn Cytomics and Cell Sorting Shared Resource Laboratory at the University of Pennsylvania and research is partially supported by the Abramson Cancer Center NCI grant (P30 016520). The research identifier number is RRID: SCR_022376. Some figures were created in BioRender.com.

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Authors and Affiliations

  1. Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Juan M. Inclan-Rico, Camila M. Napuri, Li-Yin Hung, Annabel A. Ferguson, Xiaohong Liu, Christopher F. Pastore, Adriana Stephenson, Ulrich M. Femoe, Fungai Musaigwa, Heather L. Rossi, Bruce D. Freedman & De’Broski R. Herbert

  2. Monell Chemical Senses Center, Philadelphia, PA, USA

    Cailu Lin & Danielle R. Reed

  3. Department of Neuroscience, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Qinxue Wu & Wenqin Luo

  4. Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic

    Tomáš Macháček & Petr Horák

  5. Department of Biological Sciences, Zuckerman Mind, Brain, Behavior Institute, Columbia University, New York, NY, USA

    Ishmail Abdus-Saboor

  6. Institute for Regenerative Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Wenqin Luo

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  1. Juan M. Inclan-Rico
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Contributions

Conceptualization: J.M.I.-R. and D.R.H. Methodology: J.M.I.-R., C.N., L.-Y.H., C.F.P., A.F., X.L., A.S., U.F., F.M., Q.W., H.L.R., T.M. and P.H. Investigation: J.M.I.-R., C.N., C.L., A.F., F.M., H.L.R., B.F., D.R., D.R.H., T.M. and P.H. Visualization: J.M.I.-R., D.R.H., C.L. and D.R. Funding acquisition: J.M.I.-R. and D.R.H. Supervision: W.L., I.A.-S. and D.R.H. Writing—original draft: J.M.I.-R. and D.R.H. Writing—review and editing: J.M.I.-R., H.L.R. and D.R.H.

Corresponding author

Correspondence to De’Broski R. Herbert.

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Peer review information

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

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Extended data

Extended Data Fig. 1 The avian schistosome Trichobilharzia szidati directly activates DRG neurons and enhance itch evoked by MrgprA3 neurons.

(a), Mean ± s.e.m (n = 9-10) of total number of scratch bouts and accumulated scratch time of wildtype mice videorecorded for 30mins after subcutaneous injection with vehicle (PBS) or 50μg T. szidati cercarial antigen (TCA). (b), Mean ± s.e.m (n = 9) of total number of scratch bouts and accumulated scratch time of wildtype mice injected with vehicle or 50μg TCA 1hr prior to injection with 100μg CQ. (c-e), Calcium traces of DRG neurons sequentially treated with vehicle (PBS), 25μg/mL SCA, or 25μg/mL TCA for 5 minutes followed by 5-minute stimulation with 1 μM capsaicin and stimulation with 70mM KCl for 1 minute. Proportions of responsive cells per treatment are indicated below each temporal segment for all cells imaged in one experiment. (f), AUC values of intracellular calcium levels of WT DRG neurons during treatment with vehicle, 25μg/mL SCA, or 25μg/mL TCA for 5 minutes, each symbol represents one cell. (g), Capsaicin-induced AUC values of intracellular calcium levels of DRG neurons exposed to vehicle, 25μg/mL SCA, or 25μg/mL TCA prior to treatment with 1 μM capsaicin for 5 minutes. (h), Mean ± s.e.m (n = 5) CGRP supernatant levels of wildtype DRG neurons treated with vehicle or 25μg/mL TCA for 15 minutes prior to stimulation with media or 10μM CQ for 1 hour. (i), Experimental approach for intradermal ear injection with vehicle (PBS) or MrgprA3 agonist, chloroquine (CQ). Created with BioRender.com. (j), Percentage of non-penetrating cercariae and D6 lung larval burden from mice treated with vehicle or CQ prior to skin exposure to S. mansoni. (k,l), Individual channels of IFA staining of MrgprA3+ and βIII-tubulin+ nerve afferents in unmanipulated dorsal skin and dorsal root ganglia (DRG) sections of MrgprA3Cre+/−:Rosa26tdTomato mice. (m) Individual channels of MHC-II+ cells and MrgprA3+ nerve afferents in unmanipulated skin sections of MrgprA3Cre+/−:Rosa26tdTomato mice. P values were determined by two-tailed Student’s t-tests or Two-way ANOVA with post hoc correction. *P<0.05, **P<0.01, ***P<0.001. Representative of 2-3 independent experiments, each with ≥4 biological replicates or 10 technical replicates for calcium influx experiments.

Source data

Extended Data Fig. 2 MrgprA3 neurons promote cutaneous inflammation and host immunity against S. mansoni.

(a), Representative counterplots and quantification of skin macrophages by flow cytometry of wildtype (WT) mice treated with vehicle or chloroquine (CQ). Mean ± s.e.m (n = 4). (b), Ear thickness measured daily during photostimulation of ChR2 control or MrgprA3-ChR2 mice. Mean ± s.e.m (n = 3-4). (c,d), Proportions and absolute cell numbers of skin IL-17+ αβ T cells, IL-17+ ILCs, and Foxp3+ Tregs were quantified by flow cytometry of control or MrgprA3-ChR2 mice 5 days post-optogenetic stimulation. Mean ± s.e.m (n = 6-7). (e), Ear thickness was measured daily during vehicle or CQ treatment of WT mice. Mean ± s.e.m (n = 4). (f), FFPE ear skin sections from mice treated with vehicle or CQ were analyzed by H&E staining and (g), Epidermal thickness was quantified. Mean ± s.e.m (n = 4-5). (h), Representative counterplots and quantification of skin IL-17+ γδ T cells by flow cytometry of WT mice treated with vehicle or CQ. Mean ± s.e.m (n = 4). (i), Ear thickness was measured every 3-4 days of control or MrgprA3-DTR mice treated with diphtheria toxin (DT) administered every 3 days for 3 weeks. Mean ± s.e.m (n = 4-5). (j-l), One day after S. mansoni challenge, neutrophil (CD11b+ Ly6G+ Ly6Cint) ear skin responses were evaluated in ChR2 control or MrgprA3-ChR2 mice; vehicle or CQ-treated mice; and DTR control or MrgprA3-DTR mice. Mean ± s.e.m (n = 4-5). P values were determined by two-tailed Student’s t-tests or Two-way ANOVA with post hoc correction. *P<0.05, **P<0.01, ***P<0.001. Representative of 2-3 independent experiments, each with ≥4 biological replicates.

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Extended Data Fig. 3 Optogenetic activation of MrgprA3 neurons induces TNF+ skin macrophages.

(a-c), Mean ± s.e.m (n = 6-7). Evaluation of skin eosinophil (CD11b+ Siglec-F+), neutrophil (CD11b+ Ly6G+ Ly6Cint), and γδ T cell (CD90+ CD127+ TCRδ+) populations in vehicle- or clodronate treated control or MrgprA3-ChR2 mice following 5 days of optogenetic stimulation. (d), Wildtype (WT) or TCRδKO mice were s.c. injected in the ear with vehicle (PBS) or 0.2mg of chloroquine (CQ) every 2 days for 5 days prior to exposure to 150–200 S. mansoni cercariae. The proportion of parasites remaining in the inoculum was quantified 30 minutes after skin exposure. (e), UMAP plots illustrating expression of signature genes in defined clusters of cells generated by single-cell RNA-sequencing of skin-resident live CD11c+ MHC-II+ populations sort-purified from naïve mice. (f), Bone marrow-derived macrophages (BMDMs) were exposed to 10nM rCGRP for 24 hrs and expression of pro-IL-1β and TNF was evaluated by flow cytometry. Mean ± s.e.m (n = 4). (g,h), Representative counterplots and quantification of skin TNF+ macrophages following optogenetic activation of MrgprA3 neurons. Mean ± s.e.m (n = 6-7). P values were determined by two-tailed Student’s t-tests or One-way ANOVA with post hoc correction. *P<0.05, **P<0.01, ***P<0.001. A-D, F-H, Representative of 2-3 independent experiments, each with ≥4 biological replicates.

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Extended Data Fig. 4 MrgprA3+ neurons promote cutaneous inflammation and host immunity against S. mansoni through CGRP-RAMP1 signaling.

(a), Representative counterplots of CD45.1 vs. CD45.2 expression in blood-derived Live CD45+ hematopoietic cells evaluated in WT or RAMP1KO bone marrow (BM) chimeras. (b,c), TNF+ macrophages and IL-17+ γδ T cells quantified 5 days after injection with vehicle or CQ of WT or RAMP1KO BM chimeras. Mean ± s.e.m (n = 5-6). (d), WT or RAMP1KO reconstituted mice were treated with CQ and exposed to 500 S. mansoni cercariae exposed in the ear followed by quantification of non-penetrating cercariae. (e), Experimental approach for CGRP inhibition by intradermal administration of CGRP antagonist, CGRP8-37, prior to optogenetic ear stimulation of MrgprA3-ChR2 mice. Created with BioRender.com. (f), Ear thickness of control, vehicle-treated MrgprA3-ChR2, or MrgprA3-ChR2 treated with CGRP inhibitor prior to post-optogenetic stimulation for 5 days. Mean ± s.e.m (n = 3-4). (g), Percentage of non-penetrating S. mansoni cercariae from control or MrgprA3-ChR2 mice were treated with vehicle or CGRP inhibitor prior to optogenetic stimulation followed by parasite exposure. (h), IL-17+ γδ T cells, (i), macrophages, and (j), TNF+ macrophages from control, vehicle-treated MrgprA3-ChR2, and CGRP inhibitor-treated MrgprA3-ChR2 mice were quantified by flow cytometry 5 days post-optogenetic stimulation. Mean ± s.e.m (n = 4-6). (k), Individual channels of Immunofluorescence (IFA) staining for Keratin 5 (Krt5), MHC-II, βIII tubulin and IL-33-GFP in tissue sections from naïve skin of IL-33fl/fl IRES-eGFP mice. (l), Quantification of the distance between IL-33-GFP+, MHC-II+ or IL-33-GFP+ MHC-II+ cells with respect to βIII tubulin+ neurons. Mean ± s.e.m (n = 27,42,120). (m), Individual channels of MHC-II, MrgprA3, and IL-33 in naïve skin sections of MrgprA3Cre+/−:Rosa26tdTomato mice. (n), Quantification of the distance between IL-33+, MHC-II+ or IL-33+ MHC-II+ cells with respect to MrgprA3+ neurons. Mean ± s.e.m (n = 18,32,50). P values were determined by two-tailed Student’s t-tests or One-way ANOVA with post hoc correction. *P<0.05, **P<0.01, ***P<0.001. Representative of 2-3 independent experiments, each with ≥3 biological replicates.

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Extended Data Fig. 5 Dermal myeloid APCs express IL-33 that is downmodulated by MrgprA3 neurons or CGRP.

(a), Flow cytometric gating strategy to analyze skin-resident hematopoietic and non-hematopoietic cell subsets from naïve IL-33fl/fl IRES-eGFP mice. (b), Representative flow cytometric plots of IL-33 protein and IL-33-GFP transcript expression within specific subsets of skin-resident cells from IL-33fl/fl GFP mice as well as staining controls. (c), Tissue and cell IL-33 protein levels quantified in CMV-IL-33KO mice. Mean ± s.e.m (n = 3). (d), Immunofluorescence (IFA) staining for Keratin 5 (Krt5), MHC-II, and IL-33 protein in tissue sections from naïve skin of CMV-IL-33KO mice. (e), Representative flow cytometric plots of IL-33 protein and IL-33-GFP transcript expression within specific subsets of skin-resident cells (f), Quantification of percentages and absolute numbers of IL-33-GFP+, IL-33-protein+, and IL-33-GFP+ IL-33protein+ cutaneous cells. Mean ± s.e.m (n = 4). (g), IL-33+ macrophages and cDC2s from control, vehicle-treated MrgprA3-ChR2, and CGRP inhibitor-treated MrgprA3-ChR2 mice quantified by flow cytometry 5 days post-optogenetic stimulation. Mean ± s.e.m (n = 3,4). (h), IL-33 protein expression in bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) evaluated 24hrs. after treatment with 10nM rCGRP. Mean ± s.e.m (n = 5). P values were determined by two-tailed Student’s t-test or One-way ANOVA with post hoc correction. *P<0.05, **P<0.01, ***P<0.001. Representative of 3 independent experiments, each with ≥4 biological replicates.

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Extended Data Fig. 6 IL-33 expression in APCs limits IL-17 cutaneous responses and sustain dermal T regulatory cells and ILC2s.

(a), RNA and protein levels of cytokines from naïve skin explants of control or CD11c-IL-33KO mice. Mean ± s.e.m (n = 4,5). (b-d), Representative plots and quantification of γδ T cells and IL-17+ γδ T cells in skin-draining lymph nodes (sdLNs) from control or CD11c-IL-33KO mice. Mean ± s.e.m (n = 4). Naïve skin from control or CD11-IL-33KO mice evaluated for (e), DETC, (f,g), CD4+ T helper and IL-17+ CD4+ T cells, (h-j), CD8+ cytotoxic T cells and IFNγ+ CD8+ T cells, (k,l), ST2+ GATA3+ T regulatory cells, and (m,n), GATA3+ Lineage- CD90+ CD127+ ILC2 cell populations. Mean ± s.e.m (n = 4,5). P values were determined by two-tailed Student’s t-test. *P<0.05, **P<0.01, ***P<0.001. Representative of 3 independent experiments, each with ≥4 biological replicates.

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Extended Data Fig. 7 Loss of myeloid APC-derived IL-33 alters cutaneous immunity dependent on IL-17 and gd T cells.

(a), IL-17+ dermal γδ T cells were quantified in Langerin Cre-IL-33KO and control mice. Mean ± s.e.m (n = 4). (b), Dermal γδ T cells were evaluated in Foxp3Cre-ST2fl/fl and control mice. Mean ± s.e.m (n = 4,6). (c), Principal component analysis (PCA) of bulk RNA-seq of naïve skin explants from control or CD11c-IL-33KO mice. (d), Volcano plots of bulk RNA-seq analysis of differentially expressed genes from naïve skin explants from control or CD11c-IL-33KO mice. (e), Gene set enrichment analysis (GSEA) plots of pathways overrepresented by genes enriched in naïve skin from CD11c-IL-33KO mice. (f-h), Individual channels of IFA staining for Loricrin, Desmoglein-1 (Desm-1), or Ki-67 in skin sections from control or CD11c-IL-33KO mice. White arrows indicate localized areas of epidermal thickening. (i), IFA staining for Ki-67 in skin sections from control, Ctrl Ig-treated CD11c-IL-33KO, or CD11c-IL-33KO treated with neutralizing antibodies against IL-17 and IL-23. (j,k), Quantification of Ki-67+ and Krt5+ Ki-67+ cells in skin sections of control, CD11c-IL-33KO, or CD11c-IL-33KO treated with neutralizing antibodies against IL-17 and IL-23. Mean ± s.e.m (n = 12–15). (l), Quantification of epidermal thickness assessed by Loricrin- or Desmoglein-1 staining, as in D,E, of CD11c control, CD11c-IL-33KO, or CD11c-IL-33KO interbred with TCRδKO (CD11c-IL-33KO x TCRδKO) mice. Mean ± s.e.m (n = 12). (m,n), Percentage of non-penetrating S. mansoni cercariae quantified in Langerin Cre-IL-33KO or in Foxp3Cre-ST2fl/fl mice and compared with their respective controls. Mean ± s.e.m (n = 5,10). (o,p), CD11c-IL-33KO mice were treated with neutralizing antibodies against IFNγ for 10 days or interbred with TCRδKO mice (CD11c-IL-33KO x TCRδKO) prior to cercarial skin exposure. The proportion of non-penetrating cercariae was compared to CD11c control or CD11c-IL-33KO mice. Mean ± s.e.m (n = 6,8,18). P values were determined by two-tailed Student’s t-test or One-way ANOVA with post hoc correction. *P<0.05, **P<0.01, ***P<0.001. Representative of 2-3 independent experiments, each with ≥4 biological replicates.

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Extended Data Fig. 8 IL-33 intrinsically limits cytokine secretion by myeloid cells.

(a,b), UMAP plots and quantification of cell proportions from scRNA-seq cell clusters in control or CD11c-IL-33KO mice. (c), Quantification of macrophages, cDC1s, and cDC2s in naïve skin from control or CD11c-IL-33KO mice by flow cytometry. Mean ± s.e.m (n = 4,5). (d,e), Percentage of cytokine+ and cytokine levels in supernatant from control or CD11c-IL-33KO bone marrow-derived macrophages (BMDMs) treated with increasing concentrations of LPS or 250ng/mL of LPS followed by ATP stimulation for mature IL-1β release. Mean ± s.e.m (n = 4) (f,g), Cytokine-expressing cells and levels in supernatant from control or CD11c-IL-33KO bone marrow-derived dendritic cells (BMDCs) treated with increasing concentrations of LPS or 250ng/mL of LPS followed by ATP stimulation for mature IL-1β release. Mean ± s.e.m (n = 4). (h), LPS-induced cytokine-expressing cells from CD11c control, anti-ST2-treated CD11c control, ST2-deficient, CD11c-IL-33KO, or rIL-33-treated CD11c-IL-33KO BMDMs. Mean ± s.e.m (n = 4,5) (i,j), Representative counterplots and quantification of plasmid+ control or CMV-IL-33KO BMDMs 2 days post-transfection with empty or IL-33-containing plasmid. (k), IL-33 supernatant levels from control or CMV-IL-33KO BMDMs transfected with empty- or IL-33-containing plasmid. Mean ± s.e.m (n = 5). (l), Total macrophages were quantified in control or CMV-IL-33KO BMDMs that were transfected with empty or IL-33-containing plasmid. Mean ± s.e.m (n = 4,5) P values were determined by two-tailed Student’s t-tests or One-way ANOVA with post hoc correction. *P<0.05, **P<0.01. ***P<0.001. C. Representative of 2-3 independent experiments, each with ≥4 biological replicates.

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Extended Data Fig. 9 Ablation of IL-33 or MrgprA3 neuron stimulation alter chromatin accessibility in macrophages.

(a), Heatmap showing distance of accessible regions to transcriptional start sites (TSS) of control or CD11c-IL-33KO bone-marrow derived macrophages (BMDMs). (b), Genomic features of open chromatin regions in control or CD11c-IL-33KO BMDMs. (c), Heatmaps of accessible chromatin regions in control or CD11c-IL-33KO BMDMs. (d), Heatmaps of accessible chromatin regions in wildtype BMDMs treated with MrgprA3 (A3) neuron supernatant or rCGRP for 24 hours. (e,f), Genome browser views of distinct accessible regions (boxed) in II1b and Arg1 loci of control or CD11c-IL-33KO BMDMs or fibroblasts. (g,h), Genome browser views of distinct accessible regions (boxed) in Il1b and Arg1 loci of wildtype BMDMs treated with MrgprA3 (A3) neuron supernatant or rCGRP. A-C, Representative of 2 independent experiments, each with 3 biological replicates.

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Extended Data Fig. 10 Schistosoma mansoni prevents the activation of MrgprA3 neurons that induce anti-helminth immunity by modulating cytokine expression patterns in myeloid APCs.

In sum, our data propose a model wherein the mammalian schistosome S. mansoni has evolved mechanisms to inhibit the activation of MrgprA3 neurons that promote anti-helminth resistance by re-programming of chromatin accessibility and transcriptional identity in cutaneous myeloid APCs through at least in part, by suppressing IL-33 expression, to rapidly unleash IL-17-mediated cutaneous inflammation. Created with BioRender.com.

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Inclan-Rico, J.M., Napuri, C.M., Lin, C. et al. MrgprA3 neurons drive cutaneous immunity against helminths through selective control of myeloid-derived IL-33. Nat Immunol (2024). https://doi.org/10.1038/s41590-024-01982-y

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