ILC3-derived acetylcholine promotes protease-driven allergic lung pathology. Darby, M., Roberts, L. B, Mackowiak, C., Chetty, A., Tinelli, S., Schnoeller, C., Quesniaux, V., Berrard, S., Togbe, D., Selkirk, M. E, Ryffel, B., & Horsnell, W. G C Journal of Allergy and Clinical Immunology, 147(4):P1513–1516.E4, Elsevier, jan, 2021. ![ILC3-derived acetylcholine promotes protease-driven allergic lung pathology [link]](https://bibbase.org/img/filetypes/link.svg "link")
Paper doi abstract bibtex ILC3-derived acetylcholine promotes protease-driven allergic lung pathology To the Editor: Initiation of allergic airway pathology often depends on the protease activity of the inhaled allergen. Previous studies have shown that IL-17-driven neutrophil and eosinophil responses can promote allergic pathology 1 and are associated with an initial epithelial release of IL-23, which is accepted as important in promoting T H 17 and group 3 innate lymphoid cell (ILC3) responses. ILC3s are a developmentally and phenotypically diverse innate lymphoid cell (ILC) subset that includes natural killer (NK) cell receptor (NCR)-positive and NCR-negative populations (NCR 1 ILC3s and NCR-ILC3s) as well as CCR6 1 lymphoid tissue inducer cells. However, all ILC3s are defined by expression of transcript variant 2 of RORC, encoding retinoid-related orphan receptor gt (RoRgt) and production of IL-17 and IL-22. 1,2 Work carried out in murine models has resulted in ILC3s emerging as critical regulators of infectious 2 and noninfectious 1 pulmonary diseases despite ILC3s representing only a minor lung immune cell population in mice. In contrast, ILC3s are the major ILC population in the human lung. 3 Importantly, ILC3-associated preclin-ical phenotypes are reflective of observations in humans. 2,4 However, our understanding of the molecular machinery enabling ILC3s to exact their influence on lung immunity is incomplete. In this study, we have demonstrated that expansion of lung ILC3s occurs in response to the protease papain and that these cells promote an IL-17-associated lung pathology. Critically, we have shown that induction of papain-driven pathology is strongly associated with ILC3 synthesis of acetylcholine (ACh). We have previously identified ACh responsiveness by immune cells in the lung as being important for CD4 1 T-cell-driven adaptive immunity to Nippostrongylus brasiliensis infection. 5 Here, we have extended this insight by demonstrating that ACh from lineage-negative (Lin-) CD127 1 lymphocytes expressing RoRgt is instrumental for promoting protease induction of allergic inflammation. This identifies a new paradigm for how ILC3s and ACh contribute to early promotion of allergic pathology that is distinct from our traditional understanding of ACh-driven neuromuscular interactions causing allergic pulmonary airway resistance. We have identified association of ILC3s with protease-induced lung pathology following acute papain challenge of wild-type (WT) C57BL/6 mice. Papain challenge increased lung concentrations of IL-13, IL-17A, IL-22, and IL-23 in comparison with the concentrations in saline-challenged mice (Fig 1, A). IL-17-and IL-23-promoted pathology suggests a RoRgt-driven inflammation. Papain challenge of RoRgt-green fluorescent protein (GFP) reporter mice demonstrated a significant expansion of the numbers of ILC3s (Lin-CD45 1 CD127 1 ICOS-RoRgt-GFP 1), group 2 ILCs (ILC2s) (Lin-CD45 1 CD127 1 ICOS 1 RoRgt-GFP-), and CD3 1 CD4 1 RoRgt-GFP 1 T cells relative to the numbers in saline-treated controls (Fig 1, B and see Fig E1 in this article's On-line Repository at www.jacionline.org). Restimulation and intra-cellular cytokine capture of lung CD4 1 T cells from papain-challenged mice detected increased levels of IL-5 and IL-13 but not IL-17 when compared with the levels in saline-treated controls (Fig 1, C). However, in CD45 1 CD3-Lin-cells, in addition to increased levels of IL-5 and IL-13, a raised IL-17 level was detected in papain-challenged mice when compared with the levels in saline-treated controls (Fig 1, C). Moreover, anti-CD3 depletion of T cells did not protect against and in fact promoted papain-driven pathology (see Fig E2 in this article's Online Repository at www.jacionline.org). These findings support IL-17-driven pathology as being independent of RoRgt 1 T H 17 T-cell IL-17 production. To test a requirement for any T-cell (and B-cell) contribution to IL-23/IL-17-promoted pathology, we challenged RAG2-/-mice with papain in the presence or absence of an IL-23-neutralizing mAb (anti-IL-23) (Fig 1, D). Decreased lung inflammation in IL-23-depleted RAG2-/-mice was revealed by histologic analysis as well as by reduced detection of Evans blue (EB) leakage into bronchoalveolar lavage fluid (BALF) (Fig 1, D) when compared with that in isotype-treated RAG2-/-mice. BALF immune cell infiltration was reduced in all immune cell populations, and tissue levels of IL-17A (but not IL-13) were lower in the IL-23-depleted RAG2-/-mice (Fig 1, D). This abrogation of papain-induced allergic inflammation in anti-IL-23-treated RAG2-/-mice supports ILC3s as being a key contributing lymphoid cell population driving protease-mediated lung inflammation. To further characterize the input of ILC3s to allergic airway pathology, we compared pulmonary responses to papain in WTand Rorc-/-mice (Fig 1, E). Rorc-/-mice did not show significant baseline differences from C57Bl/6 mice in terms of BALF cell composition (see Fig E3 in this article's Online Repository at www.jacionline.org), but histologic analysis of lung sections revealed decreased inflammation and detection of EB leakage into the BALF in Rorc-/-mice challenged with papain versus in WT mice (Fig 1, E). Total BALF immune cell infiltration was also reduced for all immune cell populations (Fig 1, E). Moreover, detection of both IL-13 and IL-17A, as well as IL-22, was reduced in Rorc-/-mice (Fig 1, E). In agreement with findings by others 6 Rorc-/-mice had expanded numbers of ILC2s (Lin-CD45 1 CD127 1 ICOS 1) when compared with the numbers in WT mice (Fig 1, E). However, as expected, detection of ILC3 subsets such as NCR 1 ILC3s (Lin-CD45 1 CD127 1 ICOS-NKp46 1) in Rorc-/-mice was acutely reduced as opposed to in WT mice; the small number of cells detected were most likely to be non-NK, non-RoRgt-expressing group 1 ILCs (ILC1s). This body of work identifies a previously unappreciated, T-cell-independent role for IL-23-responsive ILC3s in contributing to the onset of papain-driven lung pathology. An additional striking feature of these results was protection from cholinergic-promoted airway resistance during papain challenge in the absence of Rorc expression (Fig 1, E). Lympho-cytes are important responders to, and sources of, neurotransmit-ters. For example, the ILC2 response to the neurotransmitter neuromedin U is critical for inducing type 2 immunity, 7 and production of ACh by immune cells following type 2 immune challenge can promote host type 2 immune responses. 8 Moreover, ACh-producing T cells can contribute to control of chronic viral infection, 9 and CD4 1 T-cell responses to ACh via the M3 musca-rinic receptor are required for optimal adaptive immunity to
@article{Darby2021,
abstract = {ILC3-derived acetylcholine promotes protease-driven allergic lung pathology To the Editor: Initiation of allergic airway pathology often depends on the protease activity of the inhaled allergen. Previous studies have shown that IL-17-driven neutrophil and eosinophil responses can promote allergic pathology 1 and are associated with an initial epithelial release of IL-23, which is accepted as important in promoting T H 17 and group 3 innate lymphoid cell (ILC3) responses. ILC3s are a developmentally and phenotypically diverse innate lymphoid cell (ILC) subset that includes natural killer (NK) cell receptor (NCR)-positive and NCR-negative populations (NCR 1 ILC3s and NCR-ILC3s) as well as CCR6 1 lymphoid tissue inducer cells. However, all ILC3s are defined by expression of transcript variant 2 of RORC, encoding retinoid-related orphan receptor gt (RoRgt) and production of IL-17 and IL-22. 1,2 Work carried out in murine models has resulted in ILC3s emerging as critical regulators of infectious 2 and noninfectious 1 pulmonary diseases despite ILC3s representing only a minor lung immune cell population in mice. In contrast, ILC3s are the major ILC population in the human lung. 3 Importantly, ILC3-associated preclin-ical phenotypes are reflective of observations in humans. 2,4 However, our understanding of the molecular machinery enabling ILC3s to exact their influence on lung immunity is incomplete. In this study, we have demonstrated that expansion of lung ILC3s occurs in response to the protease papain and that these cells promote an IL-17-associated lung pathology. Critically, we have shown that induction of papain-driven pathology is strongly associated with ILC3 synthesis of acetylcholine (ACh). We have previously identified ACh responsiveness by immune cells in the lung as being important for CD4 1 T-cell-driven adaptive immunity to Nippostrongylus brasiliensis infection. 5 Here, we have extended this insight by demonstrating that ACh from lineage-negative (Lin-) CD127 1 lymphocytes expressing RoRgt is instrumental for promoting protease induction of allergic inflammation. This identifies a new paradigm for how ILC3s and ACh contribute to early promotion of allergic pathology that is distinct from our traditional understanding of ACh-driven neuromuscular interactions causing allergic pulmonary airway resistance. We have identified association of ILC3s with protease-induced lung pathology following acute papain challenge of wild-type (WT) C57BL/6 mice. Papain challenge increased lung concentrations of IL-13, IL-17A, IL-22, and IL-23 in comparison with the concentrations in saline-challenged mice (Fig 1, A). IL-17-and IL-23-promoted pathology suggests a RoRgt-driven inflammation. Papain challenge of RoRgt-green fluorescent protein (GFP) reporter mice demonstrated a significant expansion of the numbers of ILC3s (Lin-CD45 1 CD127 1 ICOS-RoRgt-GFP 1), group 2 ILCs (ILC2s) (Lin-CD45 1 CD127 1 ICOS 1 RoRgt-GFP-), and CD3 1 CD4 1 RoRgt-GFP 1 T cells relative to the numbers in saline-treated controls (Fig 1, B and see Fig E1 in this article's On-line Repository at www.jacionline.org). Restimulation and intra-cellular cytokine capture of lung CD4 1 T cells from papain-challenged mice detected increased levels of IL-5 and IL-13 but not IL-17 when compared with the levels in saline-treated controls (Fig 1, C). However, in CD45 1 CD3-Lin-cells, in addition to increased levels of IL-5 and IL-13, a raised IL-17 level was detected in papain-challenged mice when compared with the levels in saline-treated controls (Fig 1, C). Moreover, anti-CD3 depletion of T cells did not protect against and in fact promoted papain-driven pathology (see Fig E2 in this article's Online Repository at www.jacionline.org). These findings support IL-17-driven pathology as being independent of RoRgt 1 T H 17 T-cell IL-17 production. To test a requirement for any T-cell (and B-cell) contribution to IL-23/IL-17-promoted pathology, we challenged RAG2-/-mice with papain in the presence or absence of an IL-23-neutralizing mAb (anti-IL-23) (Fig 1, D). Decreased lung inflammation in IL-23-depleted RAG2-/-mice was revealed by histologic analysis as well as by reduced detection of Evans blue (EB) leakage into bronchoalveolar lavage fluid (BALF) (Fig 1, D) when compared with that in isotype-treated RAG2-/-mice. BALF immune cell infiltration was reduced in all immune cell populations, and tissue levels of IL-17A (but not IL-13) were lower in the IL-23-depleted RAG2-/-mice (Fig 1, D). This abrogation of papain-induced allergic inflammation in anti-IL-23-treated RAG2-/-mice supports ILC3s as being a key contributing lymphoid cell population driving protease-mediated lung inflammation. To further characterize the input of ILC3s to allergic airway pathology, we compared pulmonary responses to papain in WTand Rorc-/-mice (Fig 1, E). Rorc-/-mice did not show significant baseline differences from C57Bl/6 mice in terms of BALF cell composition (see Fig E3 in this article's Online Repository at www.jacionline.org), but histologic analysis of lung sections revealed decreased inflammation and detection of EB leakage into the BALF in Rorc-/-mice challenged with papain versus in WT mice (Fig 1, E). Total BALF immune cell infiltration was also reduced for all immune cell populations (Fig 1, E). Moreover, detection of both IL-13 and IL-17A, as well as IL-22, was reduced in Rorc-/-mice (Fig 1, E). In agreement with findings by others 6 Rorc-/-mice had expanded numbers of ILC2s (Lin-CD45 1 CD127 1 ICOS 1) when compared with the numbers in WT mice (Fig 1, E). However, as expected, detection of ILC3 subsets such as NCR 1 ILC3s (Lin-CD45 1 CD127 1 ICOS-NKp46 1) in Rorc-/-mice was acutely reduced as opposed to in WT mice; the small number of cells detected were most likely to be non-NK, non-RoRgt-expressing group 1 ILCs (ILC1s). This body of work identifies a previously unappreciated, T-cell-independent role for IL-23-responsive ILC3s in contributing to the onset of papain-driven lung pathology. An additional striking feature of these results was protection from cholinergic-promoted airway resistance during papain challenge in the absence of Rorc expression (Fig 1, E). Lympho-cytes are important responders to, and sources of, neurotransmit-ters. For example, the ILC2 response to the neurotransmitter neuromedin U is critical for inducing type 2 immunity, 7 and production of ACh by immune cells following type 2 immune challenge can promote host type 2 immune responses. 8 Moreover, ACh-producing T cells can contribute to control of chronic viral infection, 9 and CD4 1 T-cell responses to ACh via the M3 musca-rinic receptor are required for optimal adaptive immunity to},
author = {Darby, Matthew and Roberts, Luke B and Mackowiak, Claire and Chetty, Alisha and Tinelli, Sasha and Schnoeller, Corinna and Quesniaux, Valerie and Berrard, Sylvie and Togbe, Dieudonn{\'{e}}e and Selkirk, Murray E and Ryffel, Bernhard and Horsnell, William G C},
doi = {10.1016/j.jaci.2020.10.038},
file = {:C$\backslash$:/Users/01462563/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Darby et al. - 2021 - ILC3-derived acetylcholine promotes protease-driven allergic lung pathology.pdf:pdf},
issn = {00916749},
journal = {Journal of Allergy and Clinical Immunology},
keywords = {OA,fund{\_}ack,letter},
mendeley-tags = {OA,fund{\_}ack,letter},
month = {jan},
number = {4},
pages = {P1513--1516.E4},
pmid = {33461747},
publisher = {Elsevier},
title = {{ILC3-derived acetylcholine promotes protease-driven allergic lung pathology}},
url = {https://linkinghub.elsevier.com/retrieve/pii/S0091674920315864},
volume = {147},
year = {2021}
}
Downloads: 0
{"_id":"4SLdF9T8AR4NnbLjC","bibbaseid":"darby-roberts-mackowiak-chetty-tinelli-schnoeller-quesniaux-berrard-etal-ilc3derivedacetylcholinepromotesproteasedrivenallergiclungpathology-2021","authorIDs":[],"author_short":["Darby, M.","Roberts, L. B","Mackowiak, C.","Chetty, A.","Tinelli, S.","Schnoeller, C.","Quesniaux, V.","Berrard, S.","Togbe, D.","Selkirk, M. E","Ryffel, B.","Horsnell, W. G C"],"bibdata":{"bibtype":"article","type":"article","abstract":"ILC3-derived acetylcholine promotes protease-driven allergic lung pathology To the Editor: Initiation of allergic airway pathology often depends on the protease activity of the inhaled allergen. Previous studies have shown that IL-17-driven neutrophil and eosinophil responses can promote allergic pathology 1 and are associated with an initial epithelial release of IL-23, which is accepted as important in promoting T H 17 and group 3 innate lymphoid cell (ILC3) responses. ILC3s are a developmentally and phenotypically diverse innate lymphoid cell (ILC) subset that includes natural killer (NK) cell receptor (NCR)-positive and NCR-negative populations (NCR 1 ILC3s and NCR-ILC3s) as well as CCR6 1 lymphoid tissue inducer cells. However, all ILC3s are defined by expression of transcript variant 2 of RORC, encoding retinoid-related orphan receptor gt (RoRgt) and production of IL-17 and IL-22. 1,2 Work carried out in murine models has resulted in ILC3s emerging as critical regulators of infectious 2 and noninfectious 1 pulmonary diseases despite ILC3s representing only a minor lung immune cell population in mice. In contrast, ILC3s are the major ILC population in the human lung. 3 Importantly, ILC3-associated preclin-ical phenotypes are reflective of observations in humans. 2,4 However, our understanding of the molecular machinery enabling ILC3s to exact their influence on lung immunity is incomplete. In this study, we have demonstrated that expansion of lung ILC3s occurs in response to the protease papain and that these cells promote an IL-17-associated lung pathology. Critically, we have shown that induction of papain-driven pathology is strongly associated with ILC3 synthesis of acetylcholine (ACh). We have previously identified ACh responsiveness by immune cells in the lung as being important for CD4 1 T-cell-driven adaptive immunity to Nippostrongylus brasiliensis infection. 5 Here, we have extended this insight by demonstrating that ACh from lineage-negative (Lin-) CD127 1 lymphocytes expressing RoRgt is instrumental for promoting protease induction of allergic inflammation. This identifies a new paradigm for how ILC3s and ACh contribute to early promotion of allergic pathology that is distinct from our traditional understanding of ACh-driven neuromuscular interactions causing allergic pulmonary airway resistance. We have identified association of ILC3s with protease-induced lung pathology following acute papain challenge of wild-type (WT) C57BL/6 mice. Papain challenge increased lung concentrations of IL-13, IL-17A, IL-22, and IL-23 in comparison with the concentrations in saline-challenged mice (Fig 1, A). IL-17-and IL-23-promoted pathology suggests a RoRgt-driven inflammation. Papain challenge of RoRgt-green fluorescent protein (GFP) reporter mice demonstrated a significant expansion of the numbers of ILC3s (Lin-CD45 1 CD127 1 ICOS-RoRgt-GFP 1), group 2 ILCs (ILC2s) (Lin-CD45 1 CD127 1 ICOS 1 RoRgt-GFP-), and CD3 1 CD4 1 RoRgt-GFP 1 T cells relative to the numbers in saline-treated controls (Fig 1, B and see Fig E1 in this article's On-line Repository at www.jacionline.org). Restimulation and intra-cellular cytokine capture of lung CD4 1 T cells from papain-challenged mice detected increased levels of IL-5 and IL-13 but not IL-17 when compared with the levels in saline-treated controls (Fig 1, C). However, in CD45 1 CD3-Lin-cells, in addition to increased levels of IL-5 and IL-13, a raised IL-17 level was detected in papain-challenged mice when compared with the levels in saline-treated controls (Fig 1, C). Moreover, anti-CD3 depletion of T cells did not protect against and in fact promoted papain-driven pathology (see Fig E2 in this article's Online Repository at www.jacionline.org). These findings support IL-17-driven pathology as being independent of RoRgt 1 T H 17 T-cell IL-17 production. To test a requirement for any T-cell (and B-cell) contribution to IL-23/IL-17-promoted pathology, we challenged RAG2-/-mice with papain in the presence or absence of an IL-23-neutralizing mAb (anti-IL-23) (Fig 1, D). Decreased lung inflammation in IL-23-depleted RAG2-/-mice was revealed by histologic analysis as well as by reduced detection of Evans blue (EB) leakage into bronchoalveolar lavage fluid (BALF) (Fig 1, D) when compared with that in isotype-treated RAG2-/-mice. BALF immune cell infiltration was reduced in all immune cell populations, and tissue levels of IL-17A (but not IL-13) were lower in the IL-23-depleted RAG2-/-mice (Fig 1, D). This abrogation of papain-induced allergic inflammation in anti-IL-23-treated RAG2-/-mice supports ILC3s as being a key contributing lymphoid cell population driving protease-mediated lung inflammation. To further characterize the input of ILC3s to allergic airway pathology, we compared pulmonary responses to papain in WTand Rorc-/-mice (Fig 1, E). Rorc-/-mice did not show significant baseline differences from C57Bl/6 mice in terms of BALF cell composition (see Fig E3 in this article's Online Repository at www.jacionline.org), but histologic analysis of lung sections revealed decreased inflammation and detection of EB leakage into the BALF in Rorc-/-mice challenged with papain versus in WT mice (Fig 1, E). Total BALF immune cell infiltration was also reduced for all immune cell populations (Fig 1, E). Moreover, detection of both IL-13 and IL-17A, as well as IL-22, was reduced in Rorc-/-mice (Fig 1, E). In agreement with findings by others 6 Rorc-/-mice had expanded numbers of ILC2s (Lin-CD45 1 CD127 1 ICOS 1) when compared with the numbers in WT mice (Fig 1, E). However, as expected, detection of ILC3 subsets such as NCR 1 ILC3s (Lin-CD45 1 CD127 1 ICOS-NKp46 1) in Rorc-/-mice was acutely reduced as opposed to in WT mice; the small number of cells detected were most likely to be non-NK, non-RoRgt-expressing group 1 ILCs (ILC1s). This body of work identifies a previously unappreciated, T-cell-independent role for IL-23-responsive ILC3s in contributing to the onset of papain-driven lung pathology. An additional striking feature of these results was protection from cholinergic-promoted airway resistance during papain challenge in the absence of Rorc expression (Fig 1, E). Lympho-cytes are important responders to, and sources of, neurotransmit-ters. For example, the ILC2 response to the neurotransmitter neuromedin U is critical for inducing type 2 immunity, 7 and production of ACh by immune cells following type 2 immune challenge can promote host type 2 immune responses. 8 Moreover, ACh-producing T cells can contribute to control of chronic viral infection, 9 and CD4 1 T-cell responses to ACh via the M3 musca-rinic receptor are required for optimal adaptive immunity to","author":[{"propositions":[],"lastnames":["Darby"],"firstnames":["Matthew"],"suffixes":[]},{"propositions":[],"lastnames":["Roberts"],"firstnames":["Luke","B"],"suffixes":[]},{"propositions":[],"lastnames":["Mackowiak"],"firstnames":["Claire"],"suffixes":[]},{"propositions":[],"lastnames":["Chetty"],"firstnames":["Alisha"],"suffixes":[]},{"propositions":[],"lastnames":["Tinelli"],"firstnames":["Sasha"],"suffixes":[]},{"propositions":[],"lastnames":["Schnoeller"],"firstnames":["Corinna"],"suffixes":[]},{"propositions":[],"lastnames":["Quesniaux"],"firstnames":["Valerie"],"suffixes":[]},{"propositions":[],"lastnames":["Berrard"],"firstnames":["Sylvie"],"suffixes":[]},{"propositions":[],"lastnames":["Togbe"],"firstnames":["Dieudonnée"],"suffixes":[]},{"propositions":[],"lastnames":["Selkirk"],"firstnames":["Murray","E"],"suffixes":[]},{"propositions":[],"lastnames":["Ryffel"],"firstnames":["Bernhard"],"suffixes":[]},{"propositions":[],"lastnames":["Horsnell"],"firstnames":["William","G","C"],"suffixes":[]}],"doi":"10.1016/j.jaci.2020.10.038","file":":C$\\$:/Users/01462563/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Darby et al. - 2021 - ILC3-derived acetylcholine promotes protease-driven allergic lung pathology.pdf:pdf","issn":"00916749","journal":"Journal of Allergy and Clinical Immunology","keywords":"OA,fund_ack,letter","mendeley-tags":"OA,fund_ack,letter","month":"jan","number":"4","pages":"P1513–1516.E4","pmid":"33461747","publisher":"Elsevier","title":"ILC3-derived acetylcholine promotes protease-driven allergic lung pathology","url":"https://linkinghub.elsevier.com/retrieve/pii/S0091674920315864","volume":"147","year":"2021","bibtex":"@article{Darby2021,\r\nabstract = {ILC3-derived acetylcholine promotes protease-driven allergic lung pathology To the Editor: Initiation of allergic airway pathology often depends on the protease activity of the inhaled allergen. Previous studies have shown that IL-17-driven neutrophil and eosinophil responses can promote allergic pathology 1 and are associated with an initial epithelial release of IL-23, which is accepted as important in promoting T H 17 and group 3 innate lymphoid cell (ILC3) responses. ILC3s are a developmentally and phenotypically diverse innate lymphoid cell (ILC) subset that includes natural killer (NK) cell receptor (NCR)-positive and NCR-negative populations (NCR 1 ILC3s and NCR-ILC3s) as well as CCR6 1 lymphoid tissue inducer cells. However, all ILC3s are defined by expression of transcript variant 2 of RORC, encoding retinoid-related orphan receptor gt (RoRgt) and production of IL-17 and IL-22. 1,2 Work carried out in murine models has resulted in ILC3s emerging as critical regulators of infectious 2 and noninfectious 1 pulmonary diseases despite ILC3s representing only a minor lung immune cell population in mice. In contrast, ILC3s are the major ILC population in the human lung. 3 Importantly, ILC3-associated preclin-ical phenotypes are reflective of observations in humans. 2,4 However, our understanding of the molecular machinery enabling ILC3s to exact their influence on lung immunity is incomplete. In this study, we have demonstrated that expansion of lung ILC3s occurs in response to the protease papain and that these cells promote an IL-17-associated lung pathology. Critically, we have shown that induction of papain-driven pathology is strongly associated with ILC3 synthesis of acetylcholine (ACh). We have previously identified ACh responsiveness by immune cells in the lung as being important for CD4 1 T-cell-driven adaptive immunity to Nippostrongylus brasiliensis infection. 5 Here, we have extended this insight by demonstrating that ACh from lineage-negative (Lin-) CD127 1 lymphocytes expressing RoRgt is instrumental for promoting protease induction of allergic inflammation. This identifies a new paradigm for how ILC3s and ACh contribute to early promotion of allergic pathology that is distinct from our traditional understanding of ACh-driven neuromuscular interactions causing allergic pulmonary airway resistance. We have identified association of ILC3s with protease-induced lung pathology following acute papain challenge of wild-type (WT) C57BL/6 mice. Papain challenge increased lung concentrations of IL-13, IL-17A, IL-22, and IL-23 in comparison with the concentrations in saline-challenged mice (Fig 1, A). IL-17-and IL-23-promoted pathology suggests a RoRgt-driven inflammation. Papain challenge of RoRgt-green fluorescent protein (GFP) reporter mice demonstrated a significant expansion of the numbers of ILC3s (Lin-CD45 1 CD127 1 ICOS-RoRgt-GFP 1), group 2 ILCs (ILC2s) (Lin-CD45 1 CD127 1 ICOS 1 RoRgt-GFP-), and CD3 1 CD4 1 RoRgt-GFP 1 T cells relative to the numbers in saline-treated controls (Fig 1, B and see Fig E1 in this article's On-line Repository at www.jacionline.org). Restimulation and intra-cellular cytokine capture of lung CD4 1 T cells from papain-challenged mice detected increased levels of IL-5 and IL-13 but not IL-17 when compared with the levels in saline-treated controls (Fig 1, C). However, in CD45 1 CD3-Lin-cells, in addition to increased levels of IL-5 and IL-13, a raised IL-17 level was detected in papain-challenged mice when compared with the levels in saline-treated controls (Fig 1, C). Moreover, anti-CD3 depletion of T cells did not protect against and in fact promoted papain-driven pathology (see Fig E2 in this article's Online Repository at www.jacionline.org). These findings support IL-17-driven pathology as being independent of RoRgt 1 T H 17 T-cell IL-17 production. To test a requirement for any T-cell (and B-cell) contribution to IL-23/IL-17-promoted pathology, we challenged RAG2-/-mice with papain in the presence or absence of an IL-23-neutralizing mAb (anti-IL-23) (Fig 1, D). Decreased lung inflammation in IL-23-depleted RAG2-/-mice was revealed by histologic analysis as well as by reduced detection of Evans blue (EB) leakage into bronchoalveolar lavage fluid (BALF) (Fig 1, D) when compared with that in isotype-treated RAG2-/-mice. BALF immune cell infiltration was reduced in all immune cell populations, and tissue levels of IL-17A (but not IL-13) were lower in the IL-23-depleted RAG2-/-mice (Fig 1, D). This abrogation of papain-induced allergic inflammation in anti-IL-23-treated RAG2-/-mice supports ILC3s as being a key contributing lymphoid cell population driving protease-mediated lung inflammation. To further characterize the input of ILC3s to allergic airway pathology, we compared pulmonary responses to papain in WTand Rorc-/-mice (Fig 1, E). Rorc-/-mice did not show significant baseline differences from C57Bl/6 mice in terms of BALF cell composition (see Fig E3 in this article's Online Repository at www.jacionline.org), but histologic analysis of lung sections revealed decreased inflammation and detection of EB leakage into the BALF in Rorc-/-mice challenged with papain versus in WT mice (Fig 1, E). Total BALF immune cell infiltration was also reduced for all immune cell populations (Fig 1, E). Moreover, detection of both IL-13 and IL-17A, as well as IL-22, was reduced in Rorc-/-mice (Fig 1, E). In agreement with findings by others 6 Rorc-/-mice had expanded numbers of ILC2s (Lin-CD45 1 CD127 1 ICOS 1) when compared with the numbers in WT mice (Fig 1, E). However, as expected, detection of ILC3 subsets such as NCR 1 ILC3s (Lin-CD45 1 CD127 1 ICOS-NKp46 1) in Rorc-/-mice was acutely reduced as opposed to in WT mice; the small number of cells detected were most likely to be non-NK, non-RoRgt-expressing group 1 ILCs (ILC1s). This body of work identifies a previously unappreciated, T-cell-independent role for IL-23-responsive ILC3s in contributing to the onset of papain-driven lung pathology. An additional striking feature of these results was protection from cholinergic-promoted airway resistance during papain challenge in the absence of Rorc expression (Fig 1, E). Lympho-cytes are important responders to, and sources of, neurotransmit-ters. For example, the ILC2 response to the neurotransmitter neuromedin U is critical for inducing type 2 immunity, 7 and production of ACh by immune cells following type 2 immune challenge can promote host type 2 immune responses. 8 Moreover, ACh-producing T cells can contribute to control of chronic viral infection, 9 and CD4 1 T-cell responses to ACh via the M3 musca-rinic receptor are required for optimal adaptive immunity to},\r\nauthor = {Darby, Matthew and Roberts, Luke B and Mackowiak, Claire and Chetty, Alisha and Tinelli, Sasha and Schnoeller, Corinna and Quesniaux, Valerie and Berrard, Sylvie and Togbe, Dieudonn{\\'{e}}e and Selkirk, Murray E and Ryffel, Bernhard and Horsnell, William G C},\r\ndoi = {10.1016/j.jaci.2020.10.038},\r\nfile = {:C$\\backslash$:/Users/01462563/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Darby et al. - 2021 - ILC3-derived acetylcholine promotes protease-driven allergic lung pathology.pdf:pdf},\r\nissn = {00916749},\r\njournal = {Journal of Allergy and Clinical Immunology},\r\nkeywords = {OA,fund{\\_}ack,letter},\r\nmendeley-tags = {OA,fund{\\_}ack,letter},\r\nmonth = {jan},\r\nnumber = {4},\r\npages = {P1513--1516.E4},\r\npmid = {33461747},\r\npublisher = {Elsevier},\r\ntitle = {{ILC3-derived acetylcholine promotes protease-driven allergic lung pathology}},\r\nurl = {https://linkinghub.elsevier.com/retrieve/pii/S0091674920315864},\r\nvolume = {147},\r\nyear = {2021}\r\n}\r\n","author_short":["Darby, M.","Roberts, L. B","Mackowiak, C.","Chetty, A.","Tinelli, S.","Schnoeller, C.","Quesniaux, V.","Berrard, S.","Togbe, D.","Selkirk, M. E","Ryffel, B.","Horsnell, W. G C"],"key":"Darby2021","id":"Darby2021","bibbaseid":"darby-roberts-mackowiak-chetty-tinelli-schnoeller-quesniaux-berrard-etal-ilc3derivedacetylcholinepromotesproteasedrivenallergiclungpathology-2021","role":"author","urls":{"Paper":"https://linkinghub.elsevier.com/retrieve/pii/S0091674920315864"},"keyword":["OA","fund_ack","letter"],"metadata":{"authorlinks":{}},"downloads":0},"bibtype":"article","biburl":"https://drive.google.com/uc?export=download&id=1-JLqZ7RwZ3VC2d6ErLGHAtOeMRS_7GCz","creationDate":"2021-01-29T10:41:07.032Z","downloads":0,"keywords":["oa","fund_ack","letter"],"search_terms":["ilc3","derived","acetylcholine","promotes","protease","driven","allergic","lung","pathology","darby","roberts","mackowiak","chetty","tinelli","schnoeller","quesniaux","berrard","togbe","selkirk","ryffel","horsnell"],"title":"ILC3-derived acetylcholine promotes protease-driven allergic lung pathology","year":2021,"dataSources":["Krmt6gt9ktB2s6ARh","9bX4N36CTXtCXNFMd","wrEvssexmuYudwQw9","6GMFi5DCojy3jHY44"]}