for providing the opportunity to participate in the 2013 researchexpedition and A. Pasulka and K. Dawson for their contributionsin shipboard sample processing. This work was supported by theU.S. Department of Energy Biological and EnvironmentalResearch program (grants DE-SC0010574 and DE-SC0004940)and funding by the Gordon and Betty Moore Foundation throughgrants GBMF3306 and GBMF3780 (to V.J.O.). S.S. wassupported in part by the Swiss National Science Foundation(grant no. PBEZP2_142903). All data are available in the

supplementary materials. Archaeal 16S rRNA, mcrA genes, andbacterial 16S rRNA genes were deposited with the NationalCenter for Biotechnology Information under accession numbersKU324182 to KU324260, KU324346 to KU324428, and KU324261to KU324345, respectively. S.S., H.Y., and V.J.O. devised thestudy, and S.S., H.Y., G.L.C., and S.M. conducted the experimentsand analyses. S.S. and V.J.O. wrote the manuscript, withcontributions from all authors to data analysis, figure generation,and the final manuscript.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/351/6274/703/suppl/DC1Materials and MethodsFigs. S1 to S7Tables S1 to S5References (31–57)

26 October 2015; accepted 20 January 201610.1126/science.aad7154

LUNG PHYSIOLOGY

Pulmonary neuroendocrine cellsfunction as airway sensors to controllung immune responseKelsey Branchfield,1 Leah Nantie,1 Jamie M. Verheyden,1 Pengfei Sui,1

Mark D. Wienhold,2 Xin Sun1*

The lung is constantly exposed to environmental atmospheric cues. How it senses andresponds to these cues is poorly defined. Here, we show that Roundabout receptor (Robo)genes are expressed in pulmonary neuroendocrine cells (PNECs), a rare, innervatedepithelial population. Robo inactivation in mouse lung results in an inability of PNECs tocluster into sensory organoids and triggers increased neuropeptide production uponexposure to air. Excess neuropeptides lead to an increase in immune infiltrates, which inturn remodel the matrix and irreversibly simplify the alveoli. We demonstrate in vivothat PNECs act as precise airway sensors that elicit immune responses via neuropeptides.These findings suggest that the PNEC and neuropeptide abnormalities documented in awide array of pulmonary diseases may profoundly affect symptoms and progression.

In humans, approximately 5 to 8 liters of airpasses in and out of the lung per minutewhen resting. The air can vary in oxygen andCO2 concentration, may carry allergens, andconfers different extents of mechanical

stretch of the airway and gas-exchange surfaces.These signals are sensed, relayed, and processedinto physiological outputs such as the control ofpulmonary blood pressure, immune responses,and breathing rhythm, but the mechanism isunclear. Pulmonary neuroendocrine cells (PNECs)are found in a wide array of organisms from fishto mammals (1). In the mammalian lung, PNECsare the only innervated airway epithelial cellsand represent less than 1% of the total lung epi-thelial cell population (2). Although in vitro evi-dence has implicated PNECs in oxygen sensing,bronchial and vascular smooth muscle tonus,and immune responses (1, 3), these roles havenot been demonstrated in vivo. A recent studyshowed that genetic ablation of PNECs in theadult did not compromise homeostasis or airwayrepair, leaving in question the in vivo importanceof these cells (4). PNEC pathologies, in particularan increase in PNEC number, have been docu-mented in a large array of lung diseases, in-

cluding asthma, bronchopulmonary dysplasia,cystic fibrosis, chronic obstructive pulmonarydisease, congenital diaphragmatic hernia, neuro-endocrine hyperplasia of infancy, sudden infantdeath syndrome, and pulmonary hypertension(5–8). In each case, it remains unclear whetherthe PNEC increase is a cause for or the conse-quence of symptoms.In mouse lung, most PNECs reside in clusters

of ~3 to 20 cells called neuroepithelial bodies(NEBs) (3, 9). Both solitary and clustered PNECscontain dense core vesicles, filled with bioactiveneuropeptides such as calcitonin gene-relatedpeptide (CGRP) or amines such as serotonin (1).These are released in response to stimuli, suchas changes in oxygen level. Neuropeptides andamines have been implicated in some of thesame processes as PNECs (10–12), raising thepossibility that they may mediate PNEC func-tion. However, a causal link has not been demon-strated in vivo.We initiated the current study to uncover the

mechanisms underlying congenital diaphrag-matic hernia (CDH), a birth defect associatedwith considerable lung dysfunction, includingheightened immune response and pulmonaryhypertension (13). In a genetic mouse model ofCDH, we uncovered a defect of failed PNECclustering. This is followed by a sequence ofevents: an increase in PNEC neuropeptides, anincrease in immune infiltrates, and remodelingof lung structure. These findings offer an in vivo

demonstration of PNEC function. Because changesin PNEC number and associated neuropeptideshave been documented in many lung diseases,our results have wide implications beyond CDH.In humans, mutations in roundabout receptor

(ROBO) genes have been associated with CDH(13, 14). To study the lung defects associated withCDH, we inactivated both Robo1 and Robo2 inendoderm-derived epithelium, including the lung,using Shhcre (hereafter Shhcre;Robo mutant) inmice (15, 16). Although these mutants survive,they exhibit reduced gas-exchange surface areastarting at postnatal day (P) 15 (Fig. 1, A and B,and fig. S1). We performedmicroarray followedby quantitative reverse transcriptase polymerasechain reaction (qRT-PCR) at P7, before reductionof gas-exchange surface. Fifteen of the top 20 dif-ferentially expressed genes have been implicatedin immune responses, and all are significantly in-creased, including Ccl3, Cxcl2, Tnfa, and Saa3(Fig. 1C). Consistent with this signature, we ob-served elevated numbers of immune cells, includ-ing neutrophils, eosinophils, macrophages, andT cells (Fig. 1, D and E, and fig. S2). Furthermore,there is an increase in the proportion of M2 anda decrease in the proportion of M1 macrophages(fig. S3). These findings indicate that Shhcre;Robomutants show heightened immune sensitivity,mimicking a common CDH comorbidity (13).Although Robo is expressed in the alveolar re-

gion of the lung mesenchyme (fig. S4), its ex-pression in the epithelium is restricted to rarecells along the airway (Fig. 1F). Colabeling withCGRP antibody revealed that Robo-expressing epi-thelial cells are PNECs (Fig. 1G). To confirm thatRobo genes are required within PNECs for func-tion, we inactivated Robo using Ascl1creERT2(17), a knock-in cre driver that confers PNEC-specific activity in the lung epithelium (fig. S5).We found that Ascl1creERT2;Robo mutants ex-hibited both alveolar simplification and macro-phage increase, recapitulating the Shhcre;Robophenotypes (fig. S6). These findings together dem-onstrate that Robo is required specifically inPNECs for restricting immune cell number andpreventing alveolar simplification.At embryonic day (E) 13.5, newly specified

PNECs were solitary cells in both control andShhcre;Robo mutant lungs (Fig. 2, A and B). ByE15.5, a majority of PNECs had aggregated intoNEBs in the control. However, PNECs were notclustered in Shhcre;Robomutants (Fig. 2, C andD). This highly penetrant phenotype persistedin postnatal lungs (Fig. 2, E and F, and fig. S7).Total PNEC cell number appears unaffected, assupported by normal expression of Ascl1 andother PNEC markers (fig. S8). Unclustered cells

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1Laboratory of Genetics, Department of Medical Genetics,University of Wisconsin-Madison, Madison, WI 53706, USA.2Department of Pediatrics, School of Medicine and Public Health,University of Wisconsin-Madison, Madison, WI 53706, USA.*Corresponding author. E-mail: xsun@wisc.edu

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in the mutant lose their wedge shape and aremore rounded (figs. S7 and S9). Furthermore, incontrast to controls, where solitary PNECs arenot innervated (9), ~33.3% (31 of 93 cells) ofunclustered PNECs are innervated in themutant(Fig. 2F and fig. S9), suggesting that PNEC inner-vation is not dependent on cluster formation oron Robo function in PNECs.Ascl1creERT2;Robomutants also exhibit PNEC

unclustering (fig. S10). This phenotype mani-fested even when Robo inactivation was inducedpostnatally, which is subsequent to NEB forma-tion. Together, our results establish that Robo isrequired for PNEC assembly and maintenancein NEBs.Robo can function either dependently or in-

dependently of its ligand, Slit (18). Analysis ofSlit mutants show that, whereas PNEC clus-tering is not affected in any of the single mutants,it is reduced in Slit1;3 mutants (fig. S11, A to D).This result suggests that Robo function in thisprocess is likely ligand dependent.Slit and Robo function primarily to mediate

cellular repulsion and rarely attraction (19). Todetermine whether Slit acts as a repulsive orattractive signal for Robo-expressing PNECs, wefirst determined where Slit genes are expressed.Combined Slit1;2-GFP (green fluorescent pro-tein) reporter revealed expression in only about1 to 3 PNECs within large NEBs, raising the pos-sibility that the Slit1/2-expressing cells may bethe nucleating cells of the cluster (fig. S11E). Thisalso indicates PNEC subspecialization within acluster. Slit3 expression is restricted to the vascularsmooth-muscle-cell layer surrounding arteries,which runs alongside the main bronchi wheremost NEBs are found (fig. S11, F and G). Together,the close proximity of Slit-expressing cells toRobo-expressing PNECs raised the possibility that Slitligands may provide an attractive cue for PNECs.To test this, flow cytometry sorted GAD1-GFP+

PNECs were seeded in the top chamber of aBoyden cell migration culture insert. When Slitprotein was added with the cells in the top cham-ber, ~52% fewer (P = 8.5 × 10−4) PNECs migratedto the bottom (fig. S12, J to I). Conversely, whenSlit protein was added to the bottom chamber,18%more (P = 7.5 × 10−5) PNECs migrated to thebottom (fig. S11, L to N). These results suggestthat Slit-Robo drive PNEC clustering into NEBs,likely through cellular attraction.To test a possible link between PNECs and

immune response, we assayed the expression ofneuropeptides produced by PNECs (1). Of thenine neuropeptide genes assayed, five were sig-nificantly up-regulated in Shhcre;Robo mutants(Fig. 3A). Staining with antibody against CGRPrevealed that, although its expression remainsin PNECs in themutant, the staining intensity isincreased and it is no longer restricted to the basalside of these cells (figs. S7 and S9). We also notethat, while unclustering occurred by E15.5, neu-ropeptide up-regulation is only observed afterbirth, presumably upon exposure to air (fig. S12).To determine whether the increase in neuro-

peptides contributes to the immune response,we focused onCGRPbecause its transcript shows

the largest increase among all assayed (Fig. 3A).We countered this increase by breeding amutantallele of Cgrp into the Shhcre;Robo background(20). In Robo controls, loss of Cgrp did not altermacrophage numbers (Fig. 3, B, D, and F). How-ever, in Shhcre;Robo mutants, loss of Cgrp sig-nificantly decreased macrophage numbers in adose-dependent manner (Fig. 3, C, E, and F). Wealso found that loss of Cgrp partially reversedthe alveolar simplification phenotype (fig. S13).We note that neither macrophage increase noralveolar simplification were entirely prevented,suggesting that the increase in other neuropep-tidesmay add to downstreamoutcomes. Together,these results provide in vivo genetic demonstra-tion that neuropeptides mediate PNEC function.As normal alveologenesis initiates at P4 (21),

the late appearance of alveolar simplification atP15 suggests that disruption of alveologenesismay not be a primary cause. Although no change

in cell death was observed by P10, there was aclear reduction of elastin (fig. S14), which is apossible trigger of simplification (22). Immunecells such as macrophages express matrix metal-loproteinases that degrade elastin (23). Further-more, the increase in macrophages is observedbefore simplification (figs. S1 and S2), raising thepossibility of a causal relationship. To test this,we treated Shhcre;Robo and control lungs withclodronate, a hydrophilic drug that depletesmac-rophages (24). Treatment starting at P5, beforethe immune cell increase, effectively restrictedalveolar macrophage numbers to baseline levelin Shhcre;Robo mutants (Fig. 4, A to E). This at-tenuated the decrease in elastin and entirely pre-vented simplification (Fig. 4, F to J, and fig. S15).Together these data offer in vivo demonstrationthat increased immune infiltrates are responsi-ble for alveolar simplification and that both aredownstream consequences of PNEC dysfunction.

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Fig. 1. Robo mutants exhibit alveolar simplification and heightened immune response. (A andB) Hematoxylin and eosin (H&E) staining of alveolar region at P22. (C) qRT-PCR of P7 lungs. (D and E)IsoB4 labeling of macrophages at P22. (F and G) X-Gal–stained heterozygous Robo1lacZ/+;Robo2lacZ/+

lungs. (F) P0, arrows indicate expression in PNECs. (G) E15.5, with anti-CGRP immunostaining (red). *P <0.05; **P < 0.001.

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Fig. 2. Robo1;2 are required for PNEC clustering. Immunostained vibratomelung slices. (A and B) ASCL1 immunostaining labels nascent PNECs. (C to F)Synaptophysin immunostaining labels differentiated PNECs and their associ-ated nerves. Arrowheads indicate solitary PNECs, arrows indicate clusteredPNECs in NEBs, and asterisks indicate airway lumen. Scale bars, 30 mm.

Fig. 3. Increase in neuropeptide level contributesto increased macrophage infiltration. (A) qRT-PCR of PNEC peptide transcript levels in P5 lungs.*P < 0.05, **P < 0.001. (B to E) IsoB4 labeling ofmacrophages at P22. Scale bars, 40 mm. (F) Quan-tification presented as the relative percentage ofmacrophage to total cell ratio normalized to Shhcre;Robo+/−;Cgrp+/+. **P < 0.01; ***P < 0.0001; n.s., notsignificantly different (P ≥ 0.05).

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In this study, we present in vivo genetic evi-dence demonstrating that PNECs, despite theirrarity, have profound impact on postnatal lungfunction. Although the PNEC defect is already evi-dent at E15.5 in the Shhcre;Robomutant, the phy-siological outcomes, beginningwith up-regulationof neuropeptides, initiates after birth. This sug-gests that the effect of PNEC is dependent on lungexposure to air. Thus, our findings delineate amode of signal transduction in which PNECs aresensitive rheostats on the airway wall that trans-late environmental cues non–cell-autonomouslyinto immune responses.Our results establish Robo1,2 as a set of genes

that control PNEC clustering into NEBs. It alsopresents Slit and Robo as players in selective cellsorting in the epithelium of a mammalian organ.Inactivation of Robo after NEB formation alsoled to unclustering, suggesting that the clustersare actively maintained. Although Slit-Robo arelargely known to mediate cellular repulsion, ourdata indicate that they drive PNEC clusteringthrough cellular attraction. Robo inactivation ledto altered innervation and loss of basal-biased lo-calization of neuropeptides. These changes mayunderlie the altered downstream impact of PNECs.An increase in PNEC number has been docu-

mented in a large array of lung-associated dis-eases, ranging from rare disorders such as CDH tocommon conditions such as asthma (5–8).Wenote

that the Robo mutant PNEC phenotype is dis-tinct from increased PNECnumber. However, bothare associated with increased neuropeptides,which we show to be potent effectors of PNECfunction. Our findings thereby predict that ratherthan being a passive readout of the disease, thedocumentedPNECpathologies andneuropeptideincreasesmay serve as active contributors to symp-toms in a large array of respiratory diseases.

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ACKNOWLEDGMENTS

Supporting data and methods are presented in the supplementarymaterials. We thank X. Ai, T, Gomez, and E. Chapman fordiscussion; N. Hernandez-Santos for immune analysis; L. Ma,M. Tessier-Lavigne, J. Johnson, L. Wadiche, M. Zylka, and MutantMouse Regional Resource Center for mouse strains; and A. Lashuafor technical support. This work was supported by AmericanHeart Association predoctoral fellowship 14PRE20490146 and NIHpredoctoral training grant T32 GM007133 (to K.B.), NIAID postdoctoralfellowship 5T32AI007635 (to L.N.), and NHLBI RO1 HL113870,HL097134, HL122406, University of Wisconsin Romnes FacultyFellowship, and Wisconsin Partnership Program grant 2897 (to X.S.).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/351/6274/707/suppl/DC1Materials and MethodsFigs. S1 to 15References (25–28)

5 September 2015; accepted 16 December 2015Published online 7 January 201610.1126/science.aad7969

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Fig. 4. Macrophage reduction by clodronatetreatment attenuates alveolar simplification.(A to D) IsoB4 labeling of macrophages at P22.Scale bar, 50 mm. (E) Macrophage quantificationas the relative percentage of macrophage to totalcell ratio normalized to control mice with liposomecontrol treatment. (F to I) H&E staining of alveolarregion at P22. Scale bar, 100 mm. (J) Quantifica-tion of mean linear intercept (MLI). ***P < 0.0001;n.s., not significantly different (P ≥ 0.05).

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Pulmonary neuroendocrine cells function as airway sensors to control lung immune responseKelsey Branchfield, Leah Nantie, Jamie M. Verheyden, Pengfei Sui, Mark D. Wienhold and Xin Sun

originally published online January 7, 2016DOI: 10.1126/science.aad7969 (6274), 707-710.351Science

, this issue p. 707; see also p. 662Sciencesensitive and effective rheostats on the airway wall that receive, interpret, and respond to environmental stimuli.production of neuropeptides, which in turn trigger a heightened immune response. Thus PNECs, despite their rarity, are

genes in mouse PNECs prevents normal PNEC clustering and causes an increase in theRoundaboutInactivating neuroendocrine cells (PNECs) sense and respond to airborne cues (see the Perspective by Whitsett and Morrisey).

show that rare airway cells called pulmonaryet al.physiological outputs, including the immune response. Branchfield Liters of air pass through the lung every minute. Signals in the atmospheric environment are processed into

Neuroendocrine cells as air sensors

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