RESEARCH ARTICLE SUMMARY◥

IMMUNOLOGY

Pulmonary neuroendocrine cellsamplify allergic asthma responsesPengfei Sui, Darin L. Wiesner, Jinhao Xu, Yan Zhang, Jinwoo Lee, Steven Van Dyken,Amber Lashua, Chuyue Yu, Bruce S. Klein, Richard M. Locksley,Gail Deutsch, Xin Sun*

INTRODUCTION: The lung, with its vast sur-face area, senses and responds to signals ininhaled air. Aberrant interactions between thelung and the environment underlie many dis-eases, including asthma. In vitro data showthat pulmonary neuroendocrine cells (PNECs),a rare airway epithelial cell population, can actas chemosensors. Once stimulated in culture,they release dense core vesicles rich in neuro-peptides, amines, andneurotransmitters. Thesebioactive molecules are capable of elicitingimmune and physiological responses. A recentin vivo study by our group revealed that theproper development of PNECs into self-clusteringunits called neuroepithelial bodies is essentialfor restricting the number of immune cells in

the naïve lung. However, whether PNECs canfunction in vivo to translate exogenous airwaysignals such as allergens into the cascade ofdownstream responses is unknown.

RATIONALE: To test thehypothesis that PNECsact as sensors in the lung, we generatedmousemutants that lack PNECs by inactivating Ascl1in the airway epithelium—i.e., mutants thatweredepleted of PNECs starting at development. Weexposed these mutants to either ovalbumin orhouse dustmites, following regimes of existingasthma models. We determined whether themutants showed different asthmatic responsesthan controls. We elucidated the underlyingmechanisms by identifyingmolecular effectors

and cellular targets of PNECs. To complementthe functional tests in mice, we investigatedwhether human asthmapatients showed path-ological changes in their PNECs.

RESULTS: Although normal at baseline,Ascl1-mutant mice exhibited severely reducedgoblet cell hyperplasia and immune cellnumbers compared with controls after aller-gen challenge. In investigating possible mol-ecular effectors, we found that several PNEC

productswere decreased inmutants relative tocontrolsafter allergen challenge,including calcitonin gene-related peptide (CGRP)and g-aminobutyric acid(GABA). In exploring pos-

sible cellular targets, we found that innatelymphoid group 2 cells (ILC2s) were enrichedat airway branch points, similar to PNECs.The PNEC product CGRP stimulated ILC2production of interleukin-5 in culture. Con-versely, inactivation of the CGRP receptorgeneCalcrl in ILC2s led to dampened immuneresponses to allergens. In contrast to CGRP,GABA did not increase ILC2 cytokine secre-tion. Rather, inactivation of GABA biogenesisled to defective goblet cell hyperplasia af-ter allergen challenge, suggesting that GABAis required for this response in the airwayepithelium. The instillation of a mixture ofCGRP and GABA in Ascl1 mutants restoredboth immune cell increases and goblet cellhyperplasia after allergen challenge, indi-cating that these products are the primarymolecular effectors of PNECs in vivo. Con-sistent with these results frommice, we foundincreased PNEC numbers and cluster sizes inhuman asthma patients, whichmay underliethe heightened response to allergens in theseindividuals.

CONCLUSION: Our results demonstrate thatPNECs, despite being a rare population of cellsin the airway, are critical for amplifying theairway allergen signal into mucosal type 2responses. Specifically, PNECs act throughtheir product GABA to stimulate airway epi-thelial mucus production. In parallel, PNECsact through another product, CGRP, to stim-ulate ILC2 production of cytokines, which inturn recruit downstream immune cells. PNECsand ILC2s formneuroimmunologicalmodulesat the airway branch points, which are also thesites where airway particles are enriched. Ourfindings indicate that the PNEC-ILC2 axisfunctions to sense inhaled inputs, such as al-lergens, and amplify them into lung outputs,such as the allergic asthma response. ▪

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Sui et al., Science 360, 1086 (2018) 8 June 2018 1 of 1

The list of author affiliations is available in the full article online.*Corresponding author. Email: xinsun@ucsd.eduCite this article as P. Sui et al., Science 360, eaan8546(2018). DOI: 10.1126/science.aan8546

PNECs are preferentially localized at branch points. A mouse airway stained by antibodyagainst CGRP, to label PNECs (magenta) and antibody against SCGB1A1 to label club cells(green) (200× magnification). PNECs often cluster into neuroepithelial bodies and arepreferentially localized at branch points.

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RESEARCH ARTICLE◥

IMMUNOLOGY

Pulmonary neuroendocrine cellsamplify allergic asthma responsesPengfei Sui1,2, Darin L. Wiesner3, Jinhao Xu1,2, Yan Zhang1,2, Jinwoo Lee4,Steven Van Dyken4, Amber Lashua2, Chuyue Yu5, Bruce S. Klein3,Richard M. Locksley4, Gail Deutsch6, Xin Sun1,2*

Pulmonary neuroendocrine cells (PNECs) are rare airway epithelial cells whose function ispoorly understood. Here we show that Ascl1-mutant mice that have no PNECs exhibitseverely blunted mucosal type 2 response in models of allergic asthma. PNECs reside inclose proximity to group 2 innate lymphoid cells (ILC2s) near airway branch points. PNECsact through calcitonin gene-related peptide (CGRP) to stimulate ILC2s and elicitdownstream immune responses. In addition, PNECs act through the neurotransmitterg-aminobutyric acid (GABA) to induce goblet cell hyperplasia.The instillation of a mixture ofCGRPandGABA in Ascl1-mutant airways restores both immune and goblet cell responses. Inaccordance, lungs from human asthmatics show increased PNECs.These findingsdemonstrate that the PNEC-ILC2 neuroimmunological modules function at airway branchpoints to amplify allergic asthma responses.

Areport by the National Center for HealthStatistics of the U.S. Centers for DiseaseControl and Prevention estimates that24.6 million people in the United Stateshave asthma (1). Although much attention

has been placed on immune cells as the sourceof symptoms, in recent years, the concept thatnonhematopoietic tissues are a critical source ofcytokines has gained support (2, 3). For exam-ple, lung epithelial cells such as alveolar type 2cells produce interleukin-33 (IL-33), which inturn activates immune cells, including resi-dential group 2 innate lymphoid cells (ILC2s)(4, 5). ILC2s secrete type 2 cytokines, includingIL-5 and IL-13, which promote smooth musclecontraction, eosinophil infiltration, and gobletcell hyperplasia—key features of an asthmaticresponse (6, 7).PNECs are a distinctive and poorly under-

stood cell population in the lung. They are rare,endoderm-derived epithelial cells that constitute~1% of the airway cell population (8, 9). They arethe earliest specified lung epithelial cells, ex-pressing Ascl1, their defining marker, startingat embryonic day 12.5. In rodents, a majority ofPNECs form clusters, which are called neuro-epithelial bodies (NEBs). NEBs are highly in-nervated by both afferent and efferent neurons

(10). PNECs contain dense core vesicles filledwithneuropeptides, amines, and neurotransmitters.In vitro, PNECs can be stimulated by oxygen,mechanical stretch, and chemical stimuli andrelease their vesicular content (11). Excess PNECshave been reported in lungswith various diseases,including chronic obstructive pulmonary dis-ease, sudden infant death syndrome, broncho-pulmonary dysplasia, and small cell lung cancer(12–16). Recently, we showed that proper cluster-ing of PNECs is essential for restricting immunecell number in the naïve neonatal lung (17). How-ever, the precise in vivo role of PNECs in lungpathogenesis remains unknown.

PNECs are required for goblet cellhyperplasia in asthma models

To generate a mouse mutant lacking PNECs, weinactivated Ascl1, which encodes a transcriptionfactor that is essential for their formation, asshown in Ascl1 global null mutants (18, 19). Tobypass the perinatal lethality of the global nulls,we inactivated Ascl1 in precursors of PNECs inthe airway epithelium by using Shhcre (a cre in-sertion into the sonic hedgehog gene) at the onsetof lung development (hereafter, Ascl1CKO forconditional knockout) (fig. S1) (20). This earlyinactivation led to a complete absence of PNECs,as indicated by the lack of calcitonin gene-relatedpeptide (CGRP)– or synaptophysin-positivecells in the airway epithelium (Fig. 1). Synapto-physin also labels the nerves that normally in-nervate PNECs. In the mutant, these nervesremained present subjacent to the airway, eventhough they no longer intercalated into theepithelium (Fig. 1, C, D, C', and D'). In contrastto the absent PNECs, the intrinsic neurons,which are also dependent on Ascl1, remained,as indicated by TUJ1 (tubulin b3) expression—

presumably because Shhcre is not active in thesecells to delete Ascl1 (fig. S2).Ascl1CKO mutants were viable at birth, which

is counter to the postulation that PNECs arerequired for the transition from the intrauterineenvironment to breathing air. The mutant lungsshowed normal branching morphogenesis andalveolar morphology (fig. S3). Using immuno-fluorescent staining and quantitative reversetranscription polymerase chain reaction (qRT-PCR) of key cell type markers, we found no ap-parent defects in airway club and ciliated cellsor in type I and II alveolar cells. Like in the air-ways of control mice, no goblet cells were de-tected in the naïve mutant airways. These dataindicate that, despite PNECs being the first dif-ferentiated cell type in the airway, their absencehas no gross effects on the development of othercell types in the lung at baseline.To test whether disruption of PNECs affects

lung responses to environmental cues, we usedan established model to induce allergic asthma-like responses by sensitizing early postnatal miceto ovalbumin (OVA) injected in combination withalum. Mice were then challenged with OVA in-halation (Fig. 2A) (21). As predicted, control miceshowed robust goblet cell hyperplasia along theairways (Fig. 2B). All MUC5AC-positive gobletcells were also positive for SCGB1A1, a club cellmarker, suggesting that club cells were partiallyconverted intomucus-producing cells (fig. S4). Incontrast, Ascl1CKO mice showed substantiallyreduced goblet cell hyperplasia, which registeredat about 10% of the control response on the basisof a histological mucin index (Fig. 2, C to F). Thisobservation was corroborated by dramaticallyreduced expression of Muc5ac and goblet cell–promoting factors including Spdef and Foxa3(Fig. 2G). Markers for other airway epithelialcell types—club cells and ciliated cells—remainednormal in Ascl1CKO mice after OVA challenge(fig. S4).To determinewhether PNECs are also required

for allergen-induced goblet cell hyperplasia inthe adult lung, we exposed mutant and controlmice to house dust mites (HDMs), a commonallergen that also causes asthma in humans(22). By periodic acid–Schiff (PAS) staining andquantification ofMuc5ac transcripts, we foundthat goblet cell hyperplasia response was re-duced in the Ascl1CKOmutants compared withcontrols in theHDMmodel (fig. S5). Thus, PNECsamplify airway goblet cell hyperplasia in theasthma models tested.

PNECs are required for type 2 immuneresponses in asthma models

A cardinal feature of asthma is eosinophilic in-filtration and type 2 T helper (TH2) cell primingin the lung (7). To further explore the role ofPNECs in this aspect of the asthmatic response,we compared immune cell frequencies in neo-natal OVA and alum–treated Ascl1CKO and con-trol mice. The Ascl1CKO mice exhibited lowernumbers of ILC2s, eosinophils, and TH2 cellsthan control mice after OVA challenge (Fig. 2, Hto J, and fig. S6). Further, Ascl1CKOmice showed

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Sui et al., Science 360, eaan8546 (2018) 8 June 2018 1 of 10

1Department of Pediatrics, University of California, SanDiego, San Diego, CA 92093, USA. 2Laboratory of Genetics,University of Wisconsin–Madison, Madison, WI 53706, USA.3Department of Pediatrics, University of Wisconsin–Madison,Madison, WI 53706, USA. 4Department of Medicine, HowardHughes Medical Institute, University of California, SanFrancisco, San Francisco, CA 94143, USA. 5Zhiyuan College,Shanghai JiaoTong University, Shanghai, China. 6Departmentof Laboratories, Seattle Children’s Hospital, University ofWashington, Seattle, WA 98105, USA.*Corresponding author. Email: xinsun@ucsd.edu

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reduced Il5 and Il13 expression (Fig. 2K). Thesedifferences were not due to baseline immune celldifferences, given that phosphate-buffered saline(PBS)–treatedAscl1CKO and controlmice had sim-ilar levels of immune cells (Fig. 2,H to J, and fig. S6).To determine whether PNECs are required for

allergen-induced immune response in the adultlung, we returned to the adult HDM model. Inthis adult model, Ascl1CKO mice showed de-creased immune cell infiltration compared withcontrols (fig. S7). We also depleted PNECs inthe postnatal stage by using a different geneticmodel. We used Shhcre to activate diphtheriatoxin receptor (DTR) expression under the tran-scriptional control of the PNEC product CGRP(encoded by Calca) in a CalcaloxP-GFP-loxP-DTR

strain (GFP, green fluorescent protein) (23). Theintroduction of diphtheria toxin into the lungresulted in a ~70% reduction in PNECs. Whenchallenged with HDMs, these mice also showedan immune response that was dampened, al-though to a lesser extent than in the Ascl1CKOmutant, likely owing to residual PNECs in theDTRmodel (fig. S8). Thus, PNECs amplify allergen-induced type 2 immune responses in the asthmamodels tested.

Absence of PNECs leads to thereduction of key neuropeptides and theneurotransmitter GABA

In a normal lung, type 2 immune responses areled by changes in cytokines such as IL-25, IL-33, and TSLP (thymic stromal lymphopoietin)(24–26). In Ascl1CKO lungs, these cytokines weredetected at similar levels to those in controlsafter each of the OVA challenges (fig. S9). Theseresults suggest that these epithelium-derivedcytokines may not explain why type 2 immuneresponses are different when PNECs are absent.A key feature of PNECs is their production and

secretion of neuropeptides and neurotransmitters(11). To test the hypothesis that PNECs controltype 2 responses through someof thesemolecules,we assayed the expression of genes encodingseveral neuropeptides that have been implicatedin inflammatory diseases. These included Vip(encoding vasoactive intestinal peptide), Calca(CGRP), Chga (chromogranin A), andNpy (neuro-peptide Y) (11, 27–29). In control lungs, Calca,Chga,Npy, andVip levels were greater after OVAchallenge than after administration of PBS,with Calca showing the largest fold increase.In Ascl1CKO lungs, Calca, Chga, and Npy, butnot Vip, were significantly lower than in lungs ofcontrol mice after OVA challenge (Fig. 3A). Therelative fold changes in Calca and Chga werelarger than that in Npy, possibly owing to theconcentrated expression of Calca and Chga,but not Npy, in PNECs (11, 27).To further investigate the source of the neuro-

peptide changes, we focused on CGRP as anexample. CGRP is expressed in the PNECs andin lung sensory nerves with cell bodies locatedin the vagal ganglia. By qRT-PCR, we found thatCalca expression in the vagal ganglia was sim-ilar in wild-type mice treated with OVA andPBS controls under the regime that we used

(fig. S10A). Calca expression was also similar inthe vagal ganglia of OVA-treated Ascl1CKO miceand genotype controls (fig. S10B). Within thelung epithelium, we found by immunofluorescentstaining that CGRP expression remained re-stricted to PNECs after challenge (fig. S11). PNECproliferation did not increase (fig. S11). Rather,the CGRP signal was detected at a notably high-er intensity, suggesting that this PNEC-specificincrease contributes to the overall increase ofCGRP levels in the lung (fig. S11).In addition to neuropeptides, we also assayed

for the levels of g-aminobutyric acid (GABA), aPNEC-produced neurotransmitter that can stim-ulate goblet cell hyperplasia (30, 31). GABA levelswere reduced in Ascl1CKOmutants, possibly ow-ing to loss of GAD1, a rate-limiting enzyme inGABA biosynthesis, and VGAT, the vesicularGABA transporter, which are both specificallyexpressed in PNECs in the lung (Fig. 3B) (31).Together, these differences in neuropeptideand neurotransmitter levels suggest that theymay be responsible for the reduced immunecell infiltration and goblet cell hyperplasia inthe Ascl1CKO mutant.

PNECs reside in proximity to ILC2s andcan stimulate ILC2 cytokine production

To identify possible direct targets of PNEC sig-naling, we made use of a distinct feature of thesecells: their preferential localization to airwaybranch points (8, 9). We searched for immunecells residing in close proximity to these junc-tions. ILC2s are a population of tissue-resident

immune cells that have been shown to play acentral role in type 2 immune responses (32, 33).We investigated the localization of ILC2s byusing a mouse reporter line with tdTomatoknocked into the Il5 locus (28). In naïve mice, inaddition to confirming the previous finding thatILC2s are localized near the basement membranesubjacent to the airway epithelium (11, 27–29),we found that amajority of ILC2s (178 of 217 totalcells counted, or 82%) reside within 70 mm ofairway branch points , also called nodal points(Fig. 3, C to F) (9). By labeling PNECs with anti-body against CGRP (anti-CGRP antibody) in theIl5-tdTomato background, we found that ILC2swere preferentially localized within 70 mm ofPNECs (132 of 217 total cells counted, or 61%)(Fig. 3G). This proximity suggests ILC2s as acandidate direct target of PNEC signaling.To test whether ILC2s can perceive PNEC sig-

nals, we first addressed whether they express theappropriate receptors. Existing RNA-sequencingdata indicate that ILC2s express the CGRP co-receptor genes Calcrl and Ramp1 and the GABAreceptor geneGabrr1, but not the CHGA receptorgene (34). We confirmed the expression of CGRPand GABA receptor genes by qPCR assay inprimary ILC2s sorted from lungs of naïve wild-type mice (fig. S12).To test whether ILC2s could be stimulated by

PNEC signals, we cultured primary lung ILC2sfromnaïve lungs and addressed their response toCGRP or GABA. ILC2s respond to cytokines suchas IL-33 (35). Consistent with this, after in vivoOVA challenge, there was an increase in IL-33

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Fig. 1. Inactivation of Ascl1 by Shhcre in lung epithelium prevents PNEC formation, whereasnerves remain subjacent to airways. (A to D and A' to D') At embryonic day 18.5 (E18.5),CGRP staining outlines PNECs in E-cadherin–positive epithelium. Synaptophysin staining outlinesPNECs and nerves, including those that innervate PNECs. Arrows indicate PNECs in the controlairway. Boxed areas in the upper panels are magnified in the corresponding lower panels. NoCGRP or synaptophysin staining is evident in the epithelium in Ascl1CKO mice, indicating acomplete absence of PNECs. In the mutant, although the nerves no longer innervate epithelial cells[compare (C') and (D')], the nerve tracks are still apparent nearby. Data are representative ofsections from n = 3 mice of each genotype.

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receptor gene St2, but no change in the moregeneral IL-1 family signal mediator gene Il1/Rap, in sorted primary ILC2s (fig. S13). CGRPincreased IL-5 production by ILC2s when cul-tured in the presence of IL-33. However, GABAhad a negligible effect (Fig. 3H). In addition,

CGRP enhanced IL-5 production in the presenceof both IL-25 and IL-33 (Fig. 3I) (35). CGRPstimulation did not occur in the absence of IL-25and IL-33 (Fig. 3J). To complement data fromIL-5 enzyme-linked immunosorbent assays(ELISAs), we also assayed an array of candidate

cytokines using the LEGENDplex TH2 system(Biolegend). We confirmed that IL-5 and IL-6were increased when ILC2s were cultured inthe presence of CGRP (fig. S14).Using CFSE (carboxyfluorescein succinimidyl

ester) staining, we observed little change in ILC2

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Student’s t test. Arrows in (E) indicate residual PAS staining. (G) Gobletcell–related gene expression as assayed by qRT-PCR of P24 whole lungsafter OVA challenge (n = 3 for each). Student’s t test. (H to J) Flowcytometry analysis of immune cells from whole lungs, after eitherPBS (control) or OVA challenge as labeled. Mann-Whitney U test (n = 4 forPBS groups and 7 for OVA groups). (K) Cytokine gene expression asassayed by qRT-PCR of P24 whole lungs (n = 3 each). Student’s t test.N.S. (not significant), P ≥ 0.05; *P < 0.05; **P < 0.01. Data arerepresentative of three experiments. Error bars represent means ± SEM.

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division, indicating that CGRP promotes ILC2cytokine production but not proliferation (fig.S15). IL-33 did not appear to alter CGRP receptorgene expression level in ILC2s, suggesting thatCGRP and IL-33 may work in parallel to stim-ulate ILC2s (table S1). We also tested CGRP andGABA function on sorted TH2 cells but observedno change in IL-5 production (fig. S16). Further-more, the addition of CGRP to sorted eosinophilsled to only a slight increase in leukotriene C4production, a minor change compared with theeffect of CGRP on ILC2s at the same concentra-tion (fig. S17). Thus, CGRP can directly act onILC2s to promote their maturation and produc-tion of cytokines such as IL-5. Activated ILC2s, inturn, have been shown to recruit eosinophils andtrigger a cascade of TH2 responses (32, 33).To further validate the PNEC-CGRP-ILC2 axis

in vivo, we deleted Calcrl in ILC2 cells by usingIl5cre.When treatedwithHDMs,mutants showedreduced immune cell infiltration comparedwith controls, consistentwith thenotion thatCGRPsignaling through ILC2s is required for a full TH2immune response (Fig. 3, K to M, and fig. S18).

Inactivation of GABA synthesis ortransport disrupts goblet cell hyperplasiawithout affecting immune responses

Increased expression of GABA receptors in lungepithelial cells from asthmatic patients has beenreported, and intranasal administration of GABAinhibitor has been shown to suppress OVA-induced goblet cell hyperplasia in mice (30).Thus, even though GABA had no effect on ILC2stimulation, we tested the hypothesis that GABAsignaling from PNECs is required for goblet cellhyperplasia in vivo. We disrupted GABA signal-ing by inactivating Gad1, which encodes a GABAsynthesis protein, or Vgat, which encodes GABAtransporter. Because PNECs are the only cells inlung that express these genes, inactivation byShhcre in these animals (hereafter, Gad1CKO orVgatCKOmice) led to the specific disruption ofGABA production from PNECs (31). Airway epi-thelial cell development remained normal in thesemutants (fig. S19). However, when treated withOVA as neonates, both mutants showed a strik-ing deficit in goblet cell hyperplasia (Fig. 4, Ato D), confirming recent findings (36). To testwhether GABA is also required in adults for thegoblet cell hyperplasia response, we subjectedadult Gad1CKO mice to the adult OVA modeland found dampened goblet cell production ofmucus (fig. S20). To further validate that dis-rupting GABA production in the lung was re-sponsible for reduced goblet cell hyperplasia,we inactivated Gad1 in the lung epithelium byusing Nkx2-1creERT2 [generating Nkx2-1creERT2

Gad1fl/fl (Nkx2-1creER;Gad1) mutant]. Nkx2-1creER;Gad1mutants also showed a deficit ingoblet cell hyperplasia (fig. S21). These resultssuggest that GABA production from the PNECsis essential for the goblet cell hyperplasia response.Next, we addressed whether the goblet cell

phenotype in the GABA-signaling mutants wasassociated with a disruption of immune cellinfiltration and cytokine production in vivo.

We found that the Gad1CKO mice had similarlevels of ILC2s, TH2 cells, and eosinophils com-pared with controls upon OVA challenge, unlikein the case of theAscl1CKOmutant (Fig. 4, E to G,and fig. S22). Il5 and Il13 levels were not sig-

nificantly different. Thus, consistent with in vitrodata (Fig. 3H), GABA signaling from PNECs isessential for goblet cell hyperplasia, but not type2 immune cell recruitment, in the OVA asthmamodel.

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Fig. 3. PNECs reside in proximity to ILC2s and stimulate their cytokine production throughCGRP. (A) Neuropeptide gene expression as assayed by qRT-PCR in P24 whole lungs after PBS or OVAchallenge (n = 3 each). Student’s t test. (B) GABA level as assayed by ELISA in P24 whole lungs afterOVA challenge (n = 3 each). Student’s t test. (C to E) From naïve mice, representative images of PNEC(green, anti-CGRP antibody, indicated by arrows) and ILC2 (magenta, Il5-tdTomato reporter, indicated byarrowheads) localization near branch points in longitudinal [(C) and (D)] or transverse (E) vibratomesections of airways.The boxed area in (C) is magnified in (D). Anti-CGRP antibody also labels sensorynerve (E). (F) Of the 217 total ILC2s analyzed, 178 (82%) were localized within 70 mm of airway branch(nodal) points. (G) Of the 217 total ILC2s analyzed, 132 (61%) of them were localized within 70 mm ofPNECs. (H to J) Effects of CGRP or GABA on IL-5 production by sorted primary ILC2s in culture, in thepresence of cytokines indicated at the top left.CGRP, but not GABA,can increase IL-5 secretion as assayedby ELISA (n = 3 each). Student’s t test. (K to M) Data from flow cytometry analysis of immune cellsfromwhole lungs of HDM-treated adult control andmutantmice as labeled.Mann-WhitneyU test (n=7 foreach group). N.S., P ≥ 0.05; *P < 0.05; **P < 0.01. Error bars represent means ± SEM.

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Instillation of CGRP and GABA in theAscl1CKOmutant reverses defective gobletcell hyperplasia and immune cell infiltrationTo address whether CGRP and GABA are suf-ficient to reverse the defective asthma-like re-sponse in the absence of PNECs, we introduceda mixture of CGRP and GABA via intratrachealadministration immediately after each OVA chal-lenge to mimic the in vivo role of PNECs. On thebasis of previously published doses, we used a40-ml mixture of 100 nM CGRP and 10 mM GABA(37, 38). We found that the exogenous adminis-tration of CGRP and GABA was sufficient torestore asthma-like phenotypes in Ascl1CKOmice,including goblet cell hyperplasia, immune cellinfiltration, and type 2 cytokine expression (Fig. 5and fig. S23). Other airway cell types were notperturbed (fig. S24). As a control, we also ad-

ministered the same mixture to wild-type micethat were sensitized but not challenged withOVA. GABA and CGRP did not induce gobletcell hyperplasia in these mice, indicating thatthese PNEC products collaborate with immunecells to mount goblet cell hyperplasia triggeredby allergen challenge (fig. S25). Together, thesefindings offer an in vivo demonstration thatCGRP and GABA are key bioactive products fromPNECs that can drive lung allergic responses inthe absence of these cells.

PNECs are increased in the airways ofhuman asthma patients

Although PNECpathology has been documentedin a number of lung diseases, whether they changein human asthma has not been directly addressed.To investigate this, we stained lung sections from

asthmatics and age-matched controls by usinganti-bombesin antibody, which is commonlyused to outline all human PNECs. We also usedanti-CGRP antibody, which, like inmice, outlinesthe subset of CGRP-producing PNECs (Fig. 6, Ato D, and table S1 for patient characteristics,treatment regime, and primary autopsy findings).We quantified total PNECs, PNECs in the prox-imal bronchiole and distal respiratory bron-chiole, and PNEC cluster size, and we foundthat all of these were increased in asthmaticsamples compared with controls (Fig. 6, E to L).The increase in the CGRP-positive subset oftotal PNECs was more significant than that inthe total bombesin-positive PNECs (P = 0.0052and 0.0485, respectively). These findings in hu-mans are consistent with our findings in miceand suggest that an increase in PNECs, particu-larly CGRP-expressing PNECs, may contribute toallergic asthma.

Discussion

The precise in vivo function of PNECs has been along-standing question in lung biology. Here weshow that PNECs are critical for mounting type 2immune responses in mouse models of asthma.They act through secreted neuropeptides andneurotransmitters, including CGRP and GABA(Fig. 7A). PNECs signal directly to ILC2s, andtogether they form a neuroimmunological mod-ule to sense and respond to environmental stimulithat enter the airway (Fig. 7B).The finding that the collaboration between

PNECs and ILCs occurs preferentially at airwaybranch points is intriguing because computa-tional modeling of particle dynamics shows thatover time, particles that enter the airway con-centrate at branch points (39). Thus, cells thatreside at these sites are at a prime location tosample diverse inputs, including chemosignals(Fig. 7B). This preferential positioning of PNECsis established in utero, poising these cells to re-spond to the environment at first breath. In linewith growing evidence supporting the impact ofearly life exposures on the immune system (40),we propose that in the early postnatal environ-ment, with every stimulation, the PNEC-ILC2neuroimmunological modules mature into sig-naling centers that are critical in airway defenseagainst pathogens and tissue damage.Our results show that PNECs act through dis-

tinct secreted products to control different aspectsof the responses to allergens (Fig. 7A). AlthoughGABA synthesis and secretion are essential forgoblet cell hyperplasia (36), GABA regulatesneither cytokine levels nor immune cell infiltra-tion. In contrast, CGRP can stimulate residentialILC2s at close range, inducing them to releaseIL-5, which can in turn recruit eosinophils andelicit a cascade of immune responses (32, 33). Inthe absence of PNECs and consequently of theCGRP from these cells, the ILC2 response isseverely dampened even though IL-33 is stillpresent. Consistent with this finding, globalinactivation of either Calcrl or Ramp1—genesencoding co-receptors for CGRP—results in dim-inished lung inflammation upon OVA challenge

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(29). Furthermore, our data show that Il5cre-mediated inactivation of Calcrl also results in adeficit in the immune response. These resultsdemonstrate that signals from PNECs are im-portant triggers of ILC2 activation in the twoallergic asthma models that we used.It remains unclear how PNECs are activated

after allergen challenge. Our data suggest thatthe increase in CGRP production in the lung isnot due to an increase in PNEC cells, but ratherto an increase in the levels of CGRP per PNEC.Recent data indicate that innervation of PNECsis required for the increased production of GABA,suggesting that stimulating efferent neuronsthat innervate PNECs may be a mechanism thatcan activate PNECs (36). It is also possible thatallergen-induced upstream immune cells and sig-nals may activate PNECs.

In addition to PNECs, neurons are also asource of neuropeptides in the lung. Althoughgenetic ablation of TRPV1-expressing sensoryneurons did not disrupt anOVA-induced immuneresponse, ablation of a larger pool of NAV1.8nociceptor–expressing sensory neurons bluntedeosinophil and T cell responses (41, 42). Severalrecent studies also showed that neuron-derivedneuromedin can directly interact with ILC2s andregulate immune responses in both the intestineand lung (43–45). Another study showed thatTRPV1 neurons, acting through CGRP, regulateprotective immunity against lethal Staphylococcusaureus pneumonia (46). With the increasingawareness that neuropeptides are important reg-ulators of lung immune responses, our find-ings here establish PNECs as a crucial source ofsignals.

PNECs are the first specialized cell type thatforms in the lung epithelium, suggesting thatearly establishment of this sensory populationis of prime importance to building a functionalorgan that is responsive to environmental inputs.Our finding that PNECs are increased in asth-matic patient lungs raises the possibility that thisincrease may contribute to disease. Given theevolutionary conservation of these cells, it is un-likely that PNECs act as the lung “appendix,” asite of pathogenesis with no apparent role in ho-meostasis. Rather, PNECs are likely essential forlung defense, in addition to conferring responsesto allergens as demonstrated here. Elucidatingthe full capacity of PNECs is important to under-standing how blocking their function, or the func-tion of their secreted products, can be used safelyand effectively to treat allergic lung diseases

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Fig. 5. In vivo restoration of OVA responses inAscl1CKO mutants with intratracheal instillation ofCGRP and GABA. (A to C) PAS staining after OVAchallenge. Arrows from insets point to the areas thatare magnified. (D to F) Immune cell numbers fromwhole lungs after OVA challenge (n = 13 for control,6 for Ascl1CKO, and 11 for Ascl1CKO + CGRP + GABA).Mann-Whitney U test. (G) Gene expression asassayed by qRT-PCR in whole lungs after OVAchallenge. Student’s t test (n = 3 for each group). N.S.,P ≥ 0.05; *P < 0.05; **P < 0.01. Error bars representmeans ± SEM.

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such as asthma. Cells similar to PNECs are foundin other tissues—for example, enteric endocrinecells in the intestine. These neuroendocrine cellsmay prove to play critical roles in translatingorgan-specific environmental inputs into appro-priate tissue responses.

Materials and methodsAnimalsAll mice were housed and all experimental pro-cedures were carried out in American Associa-tion for Accreditation of Laboratory Animal Careaccredited facilities and labs at either the Uni-

versity of Wisconsin–Madison or University ofCalifornia, San Diego. This study followed theGuide for the Care andUse of Laboratory Animals.Shhcre,Ascl1 fl,Vgat fl,Gad1fl,Calcrl fl, Il5tdTomato-cre

Nkx2-1creERT2, and Calcalox-Gfp-lox-Dta mice have allbeen described previously (20, 23, 28, 47–49).

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Fig. 6. The number of PNECs is higher in asthmatic lungs. (A to D)Representative images of control and asthma autopsy lung sections stainedwith anti-bombesin antibody or anti-CGRP antibody to delineate PNECsexpressing eachmarker. Arrows indicate labeled PNECs. (E to J) Quantificationof the percentage of bombesin- and CGRP-positive epithelial area [total, inthe proximal bronchiole (PB), and in the distal respiratory bronchiole (RB)].Asthmatic samples have higher percentages in all cases. Bombesin: total,

P = 0.0485; PB, P = 0.0274; and RB, P = 0.0037. CGRP: total, P = 0.0052;PB, P = 0.0076; and RB, P = 0.0274. (K and L) Quantification of themean largebombesin- or CGRP-positive cluster size. Asthmatic samples have largercluster sizes in both cases. Bombesin, P = 0.010; CGRP, P = 0.0075.Mann-Whitney U test (n = 7 controls and 7 asthmatics). Ages, 16 months to13 years (table S2 shows age-match and other clinical data). *P < 0.05;**P < 0.01. Error bars represent means ± SEM.

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Il5tdTomato-cre, Gad1 fl, Calcrl fl, and Ascl1 fl micewere on the B6 background. Shhcre, Nkx2-1creERT2, Calcafl-Gfp-fl-Dta, and Vgat fl mice wereback-crossed to the B6 background for at leastthree generations starting from a mixed back-ground. Littermates were used as controls tominimize potential genetic background effects.

Allergen-induced asthma model

For the early postnatal OVA asthmamodel, pupswere sensitized by intraperitoneal injections of10 mg of ovalbumin in 10 ml of PBS (OVA, A5503;Sigma) with 10 ml Imject alum (ThermoScientific)on postnatal day (P) 5 and P10, followed by three15min challenges with 3% aerosolized OVA solu-tion on P18, P19, and P20. For controls in this pro-tocol, all pups were injected on P5 and P10 withOVAandalum, and challengedonP18, P19, andP20with PBS. Mice were sacrificed on P24 for analysis.For the adult OVA asthmamodel, bothmutant

and controlmicewere sensitizedby intraperitonealinjections of 50 mg of ovalbumin (OVA, A5503;Sigma) with 10 ml Imject alum (ThermoScientific)at 6 weeks of age on day 0 and day 7, followed byfive 15 min challenges with 3% aerosolized OVAsolution on days 15, 16, 17, 18, and 19. Mice weresacrificed on day 23 for analysis.For the adult HDM model, 50 mg of HDM

extract (Dermatophagoides pteronyssinus, GreerLabs) was introduced intranasally at 4 weeks ofage on days 0, 7, 17, and 21. For controls in thisprotocol, PBS was administered intranasally onthe same schedule instead of HDM. Mice weresacrificed on day 24 for analysis.In Shhcre;Calcalox-Gfp-lox-DTR mutant and cor-

responding controls, diphtheria toxin (List labs)at 10 ng/g body weight was administered intra-nasally at P18 and P22. Treated mice were restedfor at least 4 days before HDM treatment.In pregnant females used to generate Nkx2-

1creERT2;Gad1 fl/fl mutant and corresponding con-trols, tamoxifen was injected at embryonic days(E) 11 and 12 to induce cre for the inactivationof Gad1.

Histology, mean linear intercept, andhistological mucus index analysis

Mice were euthanized using CO2. Lungs wereinflated with 4% PFA (paraformaldehyde) at35 cmH2O airway pressure, and then fixed over-night. Lungswere thenprepared forparaffin (8mm),cryo (10 mm), or vibratome (100 mm) sectioning.Goblet cells were stained using a periodic acid–Schiff (PAS) staining kit (Sigma). The histologicalmucus index (the percentage of PAS-positivecells in the bronchial epithelium)was determinedby using Image-J software (NIH). To quantifymean linear intercept (MLI), 20× H & E imageswere used. For each genotype, three mice, threesections per mouse and three independent fieldsper section were analyzed. Samples were com-pared using Student’s t test, by MLI ± SEM withstatistical significance set at P < 0.05.

Immunostaining

The following primary antibodies were used atthe indicated final concentrations for immuno-

fluorescenceor immunohistological staining: rabbitanti-Synaptophysin polyclonal antibody [5 mg/ml](Fisher), rabbit anti-bombesin polyclonal anti-body [5 mg/ml] (ImmunoStar), rabbit anti-Calcitoningene-related peptide/CGRP polyclonal antibody[5 mg/ml] (LifeSpanBioSciences), rabbit anti-CGRPpolyclonal antibody [2 mg/ml] (Sigma), rat anti-E-Cadherinpolyclonal antibody [5mg/ml] (Abcam),rabbit anti-SCGB1A1 polyclonal antibody [5 mg/ml](Seven Hills Bioreagents), rabbit anti-SPC poly-clonal antibody [5 mg/ml] (SevenHills Bioreagents),mouse anti-MUC5ACmonoclonal antibody [5 mg/ml] (MRQ-9, Sigma), and syrian hamster anti-T1alpha (PDPN) polyclonal antibody [5 mg/ml](Developmental Studies Hybridoma Bank). Thefollowing secondary antibodies were used: Cy3-conjugated goat anti-rabbit IgG [2 mg/ml], FITC-conjugated goat anti-rabbit IgG [2 mg/ml],FITC-conjugated goat anti-rabbit IgG [2 mg/ml],goat anti-rat FITC [2 mg/ml] (all from JacksonImmunoresearch),HRP-conjugatedgoat anti-rabbitIgG [2 mg/ml] (Vector Laboratories), and ImmPACTDAB peroxidase (HRP) substrate following manu-facturer’s protocol (Vector Laboratories). Imageswere acquired either by ZEISS LSM 880 withAiryscan (Fig. 3, C andD, or by ZEISSAxio Imager2 (the rest of mouse images), or Nikon DSRi1digital camera mounted on a Nikon Eclipse 80imicroscope (human sample images).

EdU analysis of cell proliferation

For EdU analysis of cell proliferation, ~300 ml of400 mM EdU solution (ThermoScientific) was

intraperitoneally injected. Mice were sacrificed1 hour after EdU injection. Lungs were fixed in4% PFA overnight and prepared for cryo section-ing (20 mm). EdUwas detected using the Click-iTEdU Kit with Alexa Fluor 488 (Invitrogen).

Tissue processing for cell sorting andflow cytometry

After euthanasia and before tissue harvest, micewere transcardially perfused with 5 ml of coldPBS. Whole lungs were digested by gentle shak-ing in 5 ml of HBSS with 0.1 Wünsch units (WU)ml of Liberase (Roche) and 25mgDNase I (Roche)for 60 min at 37°C, and then mechanically dis-sociated using GentleMACS C tubes (MiltenyiBiotec) followed by straining through a 70-mmfilter. The cells were then re-suspended in 40%Percoll, underlined with 66% Percoll and cen-trifuged. Hematopoietic cells were isolated fromthe interphase for sorting and analysis.

Cell sorting and flow cytometry

The single-cell suspensions from above werepelleted, aliquoted at ~1 × 106 cells per tube,and stained with Fc blocking antibody (5 mg/ml,BD) and Live/Dead Fixable Dead Cell Stain Kit(Invitrogen) at room temperature for 10 min.The cells were washed and then incubated withsurface marker antibody cocktail for 45 min at4°C. For sorting ILC2s and TH2 cells, the fol-lowing antibodies were used (all antibodies arefrom Biolegend, used at 2 mg/ml, unless other-wise specified): BV785-conjugated anti-CD90.2

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Fig. 7. The PNEC-ILC2 axis functions at airway branch points to amplify the allergic asthmaresponse. (A) A model for PNEC function in asthmatic responses. PNECs act through CGRP tostimulate ILC2s and through GABA to stimulate goblet cell hyperplasia. (B) A diagram illustratingPNEC-ILC2 interactions in neuroimmunological modules (boxed areas) at airway branch points,where particles carrying allergens or other antigens congregate.

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(30/H12), AF700-conjugated anti-B220 (RA36B2),AF700-conjugated anti-CD11b (M1/70), AF700-conjugated anti-CD11c (N418), AF700-conjugatedanti-Nk1.1 (PK136), BV510-conjugated anti-CD4(GK1.5), PE-Cy7-conjugated anti-TCR beta (H57-597), FITC-conjugated anti-TCR delta (GL3),and PE-conjugated anti-ST2 (DIH9). For sortingeosinophils, the following antibodies were used:PE-conjugated anti-CD64 (X54-5/7.1), PE-Cy7-conjugated anti-CD11c (N418), and BB515 anti-Siglec F (2 mg/ml, E50-2440, BD). Samples wereanalyzed on an LSR II (BD Biosciences) with fourlasers (405nm, 488nm, 561 nm, and635nm).Datawere analyzed with FlowJo software (Treestar).

Cell culture

Freshly sorted ILC2s were cultured in RPMIculture media (Sigma) with 10% (vol/vol) heat-inactivated FBS (fromJapanBioSerum), 100U/mlpenicillin/streptomycin, 10 mM HEPES buffersolution, 1× MEM nonessential amino acids,1 mMsodiumpyruvate, 50 mM2-mercaptoethanoland 50 mg/ml gentamycin sulfate. Cells were cul-tured with IL-7 (10 ng/ml) and additional indi-cated cytokines (each at 10 ng/ml) for 12 hours.Then either CGRP (Sigma, 100 nM) or GABA(Sigma, 10 mM) were added. Freshly sorted TH2cells were cultured with immobilized anti-CD3antibody (Biolegend, 145-2C11, 10 mg/mL) withIL-33 (R&D Systems, 10 ng/ml) and IL-7 (R&DSystems, 10 ng/ml), and either CGRP (100 nM)or GABA (100mM). Freshly sorted eosinophilswere cultured with IL-5 (R&D Systems, 10 ng/ml),andwith or without CGRP (100 nM). Cytokine pro-duction were determined by using IL-5 ELISA kitfrom eBioscience or the Biolegend LEGENDplexTH2 system. Leukotriene C4 production weremeasure by using the LTC4 ELISA kit (CaymanChemical).

Human asthma study population

After Institutional Review Board approval, thepathology database at Seattle Children’s Hospitalwas queried from 1960–2016 to identify autopsiesin which asthma was documented in the finalreport. An age-matched control was obtainedfrom the same year as each asthma case.

Human sample morphometric analysis

All slides were stained, imaged, and quantifiedtogether. For each subject, immunostaining forCGRP and bombesin was quantified in up to 12random peripheral (non–cartilage-containing) air-ways. Airways were classified as either proximal(comprising membranous and terminal bron-chioles with complete muscular walls) or respi-ratory bronchioles (air passages composed ofpart muscular bronchial wall and part alveoli).Airways were visualized and captured with aNikonDSRi1 digital cameramounted on aNikonEclipse 80i microscope and analyzed using NIS-Elements Advanced Research Software v4.13(Nikon Instruments, Melville, NY). All cases werephotographed and analyzed using the samemag-nification, exposure time, lamp intensity, andcamera gain. No image processing was carriedout prior to intensity analysis. In each airway,

the immunostained area was expressed as a per-centage of the total airway epithelial area (cell %).The largest immune-positive cluster size was doc-umented for each airway and divided by totalairways counted to obtainmean large cluster size.

Statistical methods

As indicated in the figure legends, Student’s t testwas used for parametric data and the Mann-WhitneyU test was used for nonparametric data.All statistical analyses were performed usingPrism 6 (GraphPad).

Primers

All primers are listed in table S3.

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ACKNOWLEDGMENTS

We thank Q. Wu, K. Caron, and S. Han for sharing mouse strains;J. Whitsett for providing antibody; V. Nizet for the use of a flowcytometer; D. Broide for assistance with the HDM model; L. Nantiefor help with imaging; S. Gong and Y. Wang for consultation on

the delivery of peptides; and H. Hoffman, G. Haddad, andM. Kronenberg for advice. We thank the University of WisconsinCarbone Cancer Center Flow Cytometry Laboratory for assistancewith the use of flow cytometers. Funding: This work was supportedby NIH grants OT2OD023857, R01HL113870, R01HL119946, andR01HL122406 and by Wisconsin Partnership Program grant 2897(to X.S.). Author contributions: P.S., D.L.W., J.X., Y.Z., J.L.,S.V.D., A.L., C.Y., R.M.L., B.S.K., G.D., and X.S. designed andperformed the research; P.S. and D.L.W. analyzed the data; andP.S. and X.S. wrote the manuscript with input from other authors.Competing interests: The authors declare no competing interests.Data and materials availability: Ascl1fl mice were obtainedfrom F. Guillemot under a material transfer agreement with theFrancis Crick Institute, Mill Hill Laboratory. All data are presentedeither in the body of the paper or in the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/360/6393/eaan8546/suppl/DC1Figs. S1 to S25Tables S1 to S3

28 May 2017; resubmitted 11 February 2018Accepted 21 March 2018Published online 29 March 201810.1126/science.aan8546

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Pulmonary neuroendocrine cells amplify allergic asthma responses

Klein, Richard M. Locksley, Gail Deutsch and Xin SunPengfei Sui, Darin L. Wiesner, Jinhao Xu, Yan Zhang, Jinwoo Lee, Steven Van Dyken, Amber Lashua, Chuyue Yu, Bruce S.

originally published online March 29, 2018DOI: 10.1126/science.aan8546 (6393), eaan8546.360Science 

, this issue p. eaan8546Sciencetarget for the treatment of asthma.Interestingly, human asthma patients were found to have increased PNEC numbers, suggesting a potential therapeutic

-aminobutyric acid).γgene-related peptide). They also induced goblet-cell hyperplasia via the neurotransmitter GABA (cells (ILC2s) around airway branch points. The PNECs enhanced ILC2 activity by secreting CGRP (calcitoninsuppressed type 2 (allergic) immune responses. PNECs were observed in close proximity to group 2 innate lymphoid

found that mice lacking PNECs haveet al.primitive lungs of amphibia and reptiles, suggesting a critical function. Sui often in contact with sensory nerve fibers. They have a wide phylogenic distribution and are found even in the relatively

Pulmonary neuroendocrine cells (PNECs) are a rare cell type located in airway and alveolar epithelia and areFinding a role for PNECs in asthma

ARTICLE TOOLS http://science.sciencemag.org/content/360/6393/eaan8546

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