ILC3-derived acetylcholine promotes protease-driven allergic lung pathology

Initiation of allergic airway pathology often depends on the protease activity inhaled allergen. Previous studies have shown that IL-17–driven neutrophil and eosinophil responses can promote pathology1Kim J. Chang Y. Bae B. Sohn K.H. Cho S.H. Chung D.H. et al.Innate immune crosstalk in asthmatic airways: innate lymphoid cells coordinate polarization lung macrophages.J Allergy Clin Immunol. 2019; 143: 1769-1782.e11Abstract Full Text PDF PubMed Scopus (33) Google Scholar are associated with an initial epithelial release IL-23, which is accepted as important promoting TH17 group 3 cell (ILC3) responses. ILC3s a developmentally phenotypically diverse (ILC) subset includes natural killer (NK) receptor (NCR)-positive NCR-negative populations (NCR+ NCR– ILC3s) well CCR6+ tissue inducer cells. However, all defined by expression transcript variant 2 RORC, encoding retinoid-related orphan ?t (RoR?t) production IL-17 IL-22.1Kim Scholar,2Ardain A. Domingo-Gonzalez R. Das S. Kazer S.W. Howard N.C. Singh al.Group mediate early protective immunity against tuberculosis.Nature. 570: 528-532Crossref (95) Work carried out murine models has resulted emerging critical regulators infectious2Ardain noninfectious1Kim pulmonary diseases despite representing only minor population mice. In contrast, major ILC human lung.3De Grove K.C. Provoost Verhamme F.M. Bracke K.R. Joos G.F. Maes T. al.Characterization quantification subsets lung.PLoS One. 2016; 11e0145961Crossref (96) Importantly, ILC3-associated preclinical phenotypes reflective observations humans.2Ardain Scholar,4Jonckheere A.C. Bullens D.M.A. Seys S.F. Innate asthma: pathophysiological insights from to asthma phenotypes.Curr Opin 19: 53-60Crossref (21) our understanding molecular machinery enabling exact their influence incomplete. this study, we demonstrated expansion occurs response papain these IL-17–associated pathology. Critically, induction papain-driven strongly ILC3 synthesis acetylcholine (ACh). We previously identified ACh responsiveness being for CD4+ T-cell–driven adaptive Nippostrongylus brasiliensis infection.5Darby M. Schnoeller C. Vira Culley F.J. Bobat Logan E. al.The M3 muscarinic required optimal helminth bacterial infection.PLoS Pathog. 2015; 11e1004636Crossref (30) Here, extended insight demonstrating lineage-negative (Lin–) CD127+ lymphocytes expressing RoR?t instrumental inflammation. This identifies new paradigm how contribute promotion distinct traditional ACh-driven neuromuscular interactions causing resistance. association protease-induced following acute challenge wild-type (WT) C57BL/6 Papain increased concentrations IL-13, IL-17A, IL-22, IL-23 comparison saline-challenged mice (Fig 1, A). IL-17– IL-23–promoted suggests RoR?t-driven RoR?t–green fluorescent protein (GFP) reporter significant numbers (Lin–CD45+CD127+ICOS–RoR?t-GFP+), ILCs (ILC2s) (Lin–CD45+CD127+ICOS+RoR?t-GFP–), CD3+CD4+ RoR?t-GFP+ T relative saline-treated controls B see Fig E1 article's Online Repository at www.jacionline.org). Restimulation intracellular cytokine capture papain-challenged detected levels IL-5 IL-13 but not when compared C). CD45+CD3–Lin– cells, addition raised level was Moreover, anti-CD3 depletion did protect fact promoted (see E2 These findings support independent RoR?t+ T-cell production. To test requirement any (and B-cell) contribution IL-23/IL-17–promoted pathology, challenged RAG2–/– presence or absence IL-23–neutralizing mAb (anti–IL-23) D). Decreased inflammation IL-23–depleted revealed histologic analysis reduced detection Evans blue (EB) leakage into bronchoalveolar lavage fluid (BALF) D) isotype-treated BALF infiltration populations, IL-17A (but IL-13) were lower abrogation papain-induced anti–IL-23–treated supports key contributing driving protease-mediated further characterize input WT Rorc–/– E). show baseline differences C57Bl/6 terms composition E3 www.jacionline.org), sections decreased EB versus Total also both agreement others6Lim A.I. Li Lopez-Lastra Stadhouders Paul F. Casrouge al.Systemic precursors provide substrate differentiation.Cell. 2017; 168: 1086-1100.e10Abstract (299) had expanded ILC2s (Lin–CD45+CD127+ICOS+) expected, such NCR+ (Lin–CD45+CD127+ICOS–NKp46+) acutely opposed mice; small number most likely be non-NK, non–RoR?t-expressing 1 (ILC1s). body work unappreciated, T-cell–independent role IL-23–responsive onset An additional striking feature results protection cholinergic-promoted resistance during Rorc Lymphocytes responders to, sources of, neurotransmitters. For example, ILC2 neurotransmitter neuromedin U inducing type immunity,7Cardoso V. Chesne Ribeiro H. Garcia-Cassani Carvalho Bouchery al.Neuronal regulation via U.Nature. 549: 277-281Crossref (305) host responses.8Roberts L.B. The non-neuronal cholinergic signalling response. Imperial College London, UK2017Google ACh-producing control chronic viral infection,9Cox M.A. Duncan G.S. Lin G.H.Y. Steinberg B.E. Yu L.X. Brenner D. al.Choline acetyltransferase-expressing infection.Science. 363: 639-644Crossref (45) infections.5Darby spleen, effectors anti-inflammatory pathway through ACh, downregulates stimulation regulating neutrophilia sepsis models. whether themselves may cell–derived source capable been investigated. identify produce inflammation, ChAT(BAC)-eGFP papain. choline acetyltransferase (ChAT) trend toward Lin–CD45+CD127+ICOS+ ILC2s), increase ChAT-expressing ILC3-enriched (Lin–CD45+CD127+ICOS–) population, no effect NK (CD3–DX5+) lungs 2, Therefore, RoR?t-expressing challenge. contributes generated RoR?tCreChATloxp lack ability generate deletion ChAT vascular ChATloxp B), along methacholine-induced neutrophils eosinophils B). Detection cytokines homogenates IL-22 levels, disruption sufficient recapitulate seen 1). Quantification equivalent between phenotype Alternaria alternata extract–driven As papain, A extract resistance, BALF, summary, challenge, found ILC3s. induce elevated irrespective RoR?t+CD4+ numbers. maintained abrogated function disrupted. factor Reduction led us investigate physiologically relevant tested generating Remarkably, ILC3-biased protected extent component Indeed, data playing central initiation IL-17–promoted inflammatory cascade. place adding would like acknowledge Elodie Culerier her technical assistance Marc Le Bert (INEM UMR7355 Experimental Molecular Immunology Neurogenetics, CNRS University Orleans) expert advice mouse genetics. All used study 6- 12-week-old background strains C57BL/6, RAG2–/–, ChAT(BAC)-eGFP,E1Tallini Y.N. Shui Greene K.S. Deng K.Y. Doran Fisher P.J. al.BAC transgenic express enhanced green peripheral neurons.Physiol Genomics. 2006; 27: 391-397Crossref (129) RoR?t-eGFP,E2Eberl G. Marmon Sunshine M.J. Rennert PD, Choi Y, Littman DR. essential nuclear RORgamma(t) generation fetal cells.Nat 2004; 5: 64-73Crossref (778) RoR?t-KO (Rorc–/–),E3Sun Z. Unutmaz Zou Y.R. Pierani Brenner-Morton al.Requirement RORgamma thymocyte survival organ development.Science. 2000; 288: 2369-2373Crossref (588) Scholar, E4Eberl D.R. Thymic origin intestinal alphabeta fate mapping RORgammat+ cells.Science. 305: 248-251Crossref (402) RoR?tCre,E3Sun ChATloxp.E5Misgeld Burgess R.W. Lewis R.M. Cunningham J.M. Lichtman J.W. Sanes J.R. Roles synapse formation: development junctions lacking acetyltransferase.Neuron. 2002; 36: 635-648Abstract (248) deficient RoR?t. necessary lymph nodes Peyer patches, fail develop mice.E4Eberl expressed many including immature double-positive (CD4+CD8+) ?? thymocytes, ILC3s, comprise RoR?tCre crossed over 5 generations RoR?tCreChATfl faithfully report capacity across hematopoietic types.E1Tallini rate-limiting enzyme crucial significance synthesis, demonstration specific widely considered reflect cholinergic-synthesizing nature cell. anesthetized isoflurane followed intranasal administration 25 ?g (Calbiochem, Darmstadt, Germany) 40 ?L saline solution per once day days 3. On 4 mice, humanely killed CO2 inhalation 24 hours after final administered daily dose endotracheal route under light anaesthesia, before inhalation. Anti–IL-23 antibody isotype (Bio X cell, Lebanon, NH; BR0313 rat IgG1) given hour 200 route. Anti-CD3 (Biolegend, San Diego, Calif; catalog no. BL100208) intraperitoneal injection rate 50 mouse. After killed, collected cardiac perfusion ISOTON II (acid-free balanced electrolyte solution, Beckman Coulter, Krefeld, Germany), sampled analyses. animal experimental protocols complied French ethical experiments regulations Charte Nationale, Code Rural R 214-122, 214-124 European Union Directive 86/609/EEC) approved Ethics Committee Animal Experimentation Campus Orleans, registered (No. 3) National Ethical Reflexion (CLE Orleans 2013-1006). South African accordance Veterinary Council Cape Town Faculty Health Sciences Committee. left lobe fixed 4% buffered formaldehyde paraffin embedded standard conditions. Tissue (3-?m) stained hematoxylin eosin periodic acid–Schiff. score determined semiquantitative assessment scale 0 (with increasing extent). slides blindly examined investigators Leica microscope (Leica, Solms, Germany). Bronchoalveolar performed lavages 500 cannula introduced trachea. samples centrifuged 400 g 10 minutes 4°C, supernatants stored –20°C analysis, pellets recovered prepare Cytospin (Thermo Scientific, Waltham, Mass) glass staining Diff-Quik stain (Merz & Dade AG, Dudingen, Switzerland). Differential counts least 300 Vascular quantified concentration BALF. measured 45 intravenous 0.3% EB; measurement absorbance 460 nm, described elsewhere.E6Michaudel Mackowiak Maillet I. Fauconnier L. Akdis C.A. Sokolowska al.Ozone exposure induces respiratory barrier biphasic injury controlled IL-33.J 2018; 142: 942-958Abstract (63) Extravasation micrograms milliliter (?g EB/mL) supernatant. Lungs digested RPMI 1640 medium containing 100 U/mL penicillin, streptomycin, mg/mL DNase I (Sigma, St Louis, Mo), 125 liberase (Roche, Basel, Switzerland) 37°C rotation. digestion, supplemented 10% FCS added. Cells dissociated passage 70-?m strainer 4°C. Pellets resuspended red blood lysis buffer (Stem Cell Technologies, Vancouver, British Columbia, Canada) incubated ice. Lysis stopped again passed 40-?m strainer. using cocktail, CD45, CD127, ICOS panel. panel ICOS, NKp46 RoR?t, indicated. Cytokine-expressing CD3, CD4, IL-5, IL-17, CD3 DX5. FACS antibodies Biolegend. Homogenized commercial ELISA kits (eBiosciences, Calif) according manufacturer's instructions. invasive dynamic ketamine (100 mg/kg, Merial, Duluth, Ga) xylazine (10 Bayer, Leverkusen, paralyzed D-tubocuranine (0.125%, Sigma), intubated 18-gauge catheter. Respiratory frequency set 140 breaths minute tidal volume 0.2 mL positive end-expiratory pressure H2O. Increasing aerosolized methacholine (9.375, 18.75, 37.5, 75, 150 mg/mL) administered. Resistance recorded plethysmograph (Buxco, United Kingdom). Baseline restored subsequent doses methacholine.E7Madouri Chenuet P. Beuraud Marchiol Rouxel N. al.Protein kinase Ctheta TH2 house dust mite allergen.J 139: 1650-1666Abstract (18) Data analyzed Prism software, version 6 (GraphPad Software, Calif). Either Mann-Whitney t parametric 1-way ANOVA Bonferroni multiple-comparison assess significance. Values means ± SDs.Fig E2CD3-depleted ChAT-GFP do Mice treated intraperitoneally ?g/mouse. cellular assessed BAL, fluid; Eos., eosinophil; Gra., granulocyte; Lymp, lymphocyte; Mono., monocyte.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Rorc–/– composition. flow cytometry. (PPT)