Detection and quantification of epithelial progenitor cell populations in human healthy and IPF lungs

Transcription

1 Smirnov et l. Respirtory Reserch (2016) 17:83 DOI /s x RESEARCH Open Access Detection nd quntifiction of epithelil progenitor cell popultions in humn helthy nd IPF lungs N. F. Smirnov 1, A. C. Schmerger 1, S. Nyknti 1, R. Htz 2,3,5, J. Behr 3,4,5 nd O. Eickelerg 1,5,6* Astrct Bckground: In the humn lung, epithelil progenitor cells in the irwys give rise to the differentited pseudostrtified irwy epithelium. In mice, emerging evidence confers progenitor function to cytokertin 5 ( + ) or cytokertin 14 ( + )-positive sl cells of the irwy epithelium. Little is known, however, out the distriution of progenitor supopultions in the humn lung, prticulrly out errnt epithelil differentition in lung disese, such s idiopthic pulmonry firosis (IPF). Methods: Here, we used multi-color immunofluorescence nlysis to detect nd quntify the distriution of irwy epithelil progenitor supopultions in humn lungs otined from helthy donors or IPF ptients. Results: In lungs from oth, helthy donors nd IPF ptients, we detected + -, - + nd + + popultions in the proximl irwys. + cells, however, were sent in the distl irwys of helthy lungs. In IPF, we detected drmtic increse in the mount of + cells nd the emergence of frequent + + epithelil popultion, in prticulr in distl irwys nd lveolr regions. While the - progenitor popultion exhiited signs of proper epithelil differentition, s evidenced y co-stining with pro-spc, quporin 5, CC10, or MUC5B, the + cell popultion did not co-stin with ronchil/lveolr differentition mrkers in IPF. Conclusions: We provide, for the first time, quntittive profile of the distriution of epithelil progenitor popultions in humn lungs. We show compelling evidence for dysregultion nd errnt differentition of these popultions in IPF. Keywords: Epithelium, Progenitor cells, Bsl cells, Supopultions, Humn explnts Bckground Lung function, rrier integrity, nd epithelil homeostsis re mintined, in lrge prts, y its inner lining, which is constituted y lung epithelil cells. Different types of lung epithelil cells hve een identified nd chrcterized in the pst, including cilited, golet, or sl cells in proximl irwys, nd cilited, clu (Clr), neuroendocrine, or sl cells in distl irwys. The pseudostrtified irwy epithelil lyer continues into flt epithelil lyer composed of lveolr type 1 (AT1) or * Correspondence: oliver.eickelerg@helmholtz-muenchen.de 1 Comprehensive Pneumology Center, Institute of Lung Biology nd Disese, University Hospitl, Ludwig-Mximilins-University nd Helmholtz Zentrum München, Mx-Lesche-Pltz 31, Munich 81377, Germny 5 Germn Center of Lung Reserch (DZL), Hnnover, Germny Full list of uthor informtion is ville t the end of the rticle type 2 (AT2) cells in the lveolr regions. Lung injury induced y injurious gents (e.g. cigrette smoke, prticulte mtter, viruses) primrily ffects the integrity of this epithelil comprtment, either y inducing rrier dysfunction or epithelil progenitor cell perturtion [1 4]. Recently, lung epithelil progenitor cells hve ttrcted significnt interest, s they serve s stem cells for differentited lung epithelil cell types, such s secretory, cilited, or AT1 cells in mice [3]. Bsl cells re reltively undifferentited epithelil cells, which re locted ttched to the sl lmin of the strtified nd pseudostrtified irwy epithelium [5]. Bsl cells prticipte in nchoring of the irwy epithelium to the sement memrne nd thus seprte the underlying strom from the epithelil tues. Airwy sl cells lso 2016 The Author(s). Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Smirnov et l. Respirtory Reserch (2016) 17:83 Pge 2 of 11 mintin the homeostsis of the norml epithelil lyer y giving rise to the differentited irwy epithelil cells during postntl growth nd in the dult under stedy stte, s previously shown y us nd others using vriety of in vivo nd in vitro ssys [6 8]. Airwy sl cell dysfunction contriutes to numer of pthologies, s disruption in the lnce etween sl cell prolifertion nd differentition leds to epithelil hyperplsi/hypoplsi [5] or norml differentition ptterns [4, 9], e.g. in lung cncer. Hyperplstic sl cell lesions re lso oserved in chronic ostructive pulmonry disese (COPD) [5, 10] nd cystic firosis (CF) [11], wheres the epithelil injury nd loss in ronchiolitis oliterns syndrome (BOS) my indicte stem cell exhustion or loss in epithelil progenitor popultions [5]. In mice, recent studies using numer of reporter nd fte-mpped mouse strins hve demonstrted tht lung sl cells contriute to regenertion of the lung in response to injury, sed on their cpcity to proliferte nd differentite into mture cell types [12, 13]. Evidence of the regenertive cpcity of sl cells in humn results from the oservtion tht sl cells isolted from different regions of the humn lung proliferte nd differentite to mture cilited, golet, or clu (Clr) cells in ir-liquid interfce (ALI) cultures or ronchosphere ssys ex vivo [14]. In vivo, injury/repir models hve demonstrted tht disruption of the sl cell lyer is ssocited with n uncontrolled prolifertion of the underlying strom, resulting in n ccumultion of firolsts nd immune cells tht susequently oliterte the irwys [15]. Emerging evidence shows tht sl cells re composed of multiple heterogeneous supopultions, under physiologicl s well s pthologicl conditions. As n exmple, mouse trchel sl cells chrcteristiclly express cytokertin 5 (), while only limited suset expresses cytokertin 14 (). Interestingly, is upregulted in mouse lung sl cells in response to nphtlene-injury [16]. As such, ongoing evidence highlights role for + + sl cells in post-injury regenertion of the mouse lung [6, 12 14]. Detils out definitive sl cell supopultions, however, remin to e elucidted, in prticulr in the humn lung. In this context, sl cell susets expressing distinct kertin (KRT) isoforms hve een descried [17] nd recent evidence suggests ltertions in KRT undnce nd expression in lung disese with fetures of diffuse lveolr dmge [18, 19]. Incresed nd expression hs lso een reported in the lveolr regions in idiopthic pulmonry firosis (IPF) [19]. Yet, the distinct quntittive nd sptil undnce of + nd + cells to IPF is unknown. To this end, we sought to investigte nd quntify the distriution of + nd + cell popultions in humn lungs, otined from helthy donors or IPF ptients. We provide here, for the first time, quntittive nlysis of the distriution of + nd + singlend doule-positive cell popultions in the helthy humn lung. Importntly, we descrie drmtic chnges in the distriution nd morphology of these cells in IPF. Finlly, we seek to chrcterize their differentition potentil y fluorescent co-stining of these popultions with well-ccepted epithelil differentition mrkers, such s cetylted tuulin, Mucin 5B, or Clr Cell 10 kd Protein (CC10) in IPF. Methods Humn lung mteril Resected humn lung tissue nd explnt mteril ws otined from the iorchive t the Comprehensive Pneumology Center (CPC). Biopsies were otined from 6 helthy donors nd 5 IPF ptients (UIP pttern, men ge: 57,6 ± 3,25, 3 mles, 2 femles). All prticipnts gve written informed consent nd the study ws pproved y the locl ethics committee of Ludwig-Mximilins University of Munich, Germny (333-10). For stining, humn lung tissue ws fixed in 4 % PFA prior to prffin emedding. The 4 μm-sections were prepred with microtome (Hyrx M 55, Zeiss) nd mounted on Superfrost slides. Isoltion of primry humn ronchil epithelil cells Bsl cells were isolted from ronchil tissue (>2 mm) resected from the peripherl tumor region of otherwise norml helthy lungs. For this, the tissue ws longitudinlly cut, wshed 3 times in MEM, supplemented with L-glutmine (2 mm) nd pen/strep (100 U/ml, 100 μg/ml), nd digested with Pronse E (1 mg/ml) in MEM with L-glutmine nd pen/strep for 20 h t 4 C under constnt gittion. The next dy, the epithelium ws scrped off using sclpel, cells further seprted with n 18G nd 25G needle nd collected y centrifugtion t 300 g for 5 min. Isolted cells were suspended in BEGM medium (Lonz; Wokinghm, UK), seeded onto rt-til collgen type I (Sigm-Aldrich; St. Louis, MO) coted dishes, nd incuted t 37 C in humidified incutor with 95 % ir nd 5 % CO 2. Cells which hve reched 80 % confluence were detched using the Clonetics RegentPck for suculturing (Lonz) nd cells/cm 2 were seeded onto collgen type I coted coverslips. Two dys lter, cells were wshed with HBSS, fixed with 4 % PFA for 15 min nd stored in HBSS t 4 C until nlysis.

3 Smirnov et l. Respirtory Reserch (2016) 17:83 Pge 3 of 11 Dul immunofluorescence nlysis All tissue stinings were performed on 4 μm-sections of lung tissue. Sections were clered of prffin y incution t 60 C (1 h) nd susequent xylene wshes. Sections were then rehydrted using 100 % ethnol, followed y distilled wter. Antigen retrievl ws performed in 0.01 M Citric Acid (ph 6.0) in declocking chmer for 30 s t 125 C. After wshing in distilled wter, slides were locked for 1 h t room temperture using 5 % BSA in PBS (locking solution, BS). Primry ntiodies were diluted in BS nd pplied to sections overnight, t 4 C. The primry ntiodies nd their dilutions used to identify the cell types indicted in Tle 1 were s follows: α-cytokertin 5 (oth 1:250, clones EP1601Y or 2C2, respectively or , Acm), α-cytokertin 14 (1:200, clone LL002, 7800, Acm), α- (oth 1:25, clone 4A4, sc-8431, Snt-Cruz; or clone EPR5701, , Acm), α-quporin 5 (1:100, clone C-19, sc-9891, Snt-Cruz), α-pro-surfctnt Protein C (1:100, polyclonl, AB3786, EMD Millipore), nti-cc10 (1:100, clone H-75, sc , Snt-Cruz), α-cetylted tuulin (1:250, clone AcetylK40, , Acm), α-mucin 5B (1:100, clone H-300, sc-20119, Snt-Cruz). Developmentl pthwy mrkers were used s follows: α-sonic Hedgehog (1:100, clone , 50515, Acm), α-gli1 (1:50, polyclonl, 49314, Acm), α-sox9 (1:50, polyclonl, AF3075, R&D Systems), α-hes1 (1:50, clone H-140, sc-25392, Snt-Cruz). Alex488 (green) nd Alex586 (red)-leled secondry ntiodies (Life Technologies) were used t 1:250 dilution in BS for 30 min. Nuclei were counterstined with nd the slides mounted in fluorescent mounting medium (Dko, Hmurg, Germny). For stinings of isolted primry humn ronchil epithelil cells, cells on coverslips were permeilized with DPBS/0.1 % Triton X-100 for 5 min nd locked with BS for 1 h t room temperture. Stining for Cytokertin 5 nd 14 ws performed s descried ove. Tle 1 Mrkers used in the study nd commonly ssocited cell types Mrker Cell type Kertin 5 () Bsl cells (irwys) Kertin 14 () Bsl cells (regions of squmous metplsi) Bsl cells Acetylted tuulin (ctub) Cilited cells CC10 Clr cells Mucin 5B (MUC5B) Golet cells ProSPC Alveolr type II cells Aquporin 5 (AQP5) Alveolr type I cells Imge cquisition nd nlysis All tissue imges were cquired using the confocl LSM710 Microscope (Crl Zeiss; Oerkochen, Germny), coupled to the Zen2009 softwre, nd exmined y three different oservers. The quntifiction of +, + nd + cells ws performed y counting cells in which the cytoplsm (, ) or the nucleus () exhiited positive signl. The numers of the indicted cells were normlized to the length of the corresponding region of the sement memrne, in cse of conducting or distl irwys, or expressed s cell/mm 2 of nlyzed tissue. Imges of primry humn ronchil epithelil cells were otined using the Axiovert II (Crl Zeiss) nd processed using the AxioVision 4.9 softwre (Crl Zeiss). Results Identifiction nd quntifiction of sl cell popultions in the conducting helthy irwys Humn helthy irwys were clssified into conducting nd distl irwys, ccording to the presence (conducting) or sence (distl) of crtilge nd sumucosl glnds in the vicinity, s previously descried [5, 20]. Prominent stining for ws oserved ll long the sement memrne of the conducting irwy epithelium (Fig. 1). All + sl cells/mm sement memrne were +, while 14 % of ll sl cells were + - (Fig. 3). This frction loclized luminl to the sl + + popultion in the strtified ronchil epithelium (Fig. 2, left pnel). A costining for nd reveled the existence of + + supopultion tht ws restricted to the sl lyer of the helthy conducting irwys (Figs. 1 nd 2, right pnel). This popultion ws heterogeneously distriuted, s some regions of the conducting irwys were densely populted with + + sl cells, while other regions hd few + cells (Fig. 1). All + cells long the sement memrne were positive for (Fig. 2, left pnel nd Fig. 3). + + cells represented 40 % of ll + cells, suggesting tht the remining + cells were or (Fig. 3). To exclude stining is of complex tissues, nd to document these popultions y n independent nlysis, we isolted primry humn ronchil epithelil sl cells nd sujected these to immunofluorescence stining. We oserved oth + - nd + + sl cell popultions in primry isolted humn sl cells, in similr proportions tht we oserved for lung tissue outlined ove (Fig. 1c nd dt not shown).

4 Smirnov et l. Respirtory Reserch (2016) 17:83 Pge 4 of 11 conducting irwys distl irwys c primry hbec Fig. 1 Proximl-to-distl distriution of + nd + sl cells supopultions in the helthy humn lung., Sections from helthy humn lungs (6 donors) were co-stined for Kertin (KRT) 5 (green), (red) nd counterstined with (lue). Two representtive pictures, with corresponding high mgnifiction pnels, re shown for the conducting irwys ( left nd right pnels) nd the distl irwys ( left nd right pnels). Arrow: + cell with chrcteristic luminl epithelil cell morphology. Full rs = 500 μm. Scttered rs = 100 μm. c Isolted primry humn ronchil epithelil cells (hbec) were co-stined for KRT 5 (green), (red) nd counterstined with (lue). The pnels show: Kertin5- (left), Kertin14- (middle) nd merged (right) imges. Full rs = 500 μm. Scttered rs = 100 μm Only nd sl cells populte helthy humn distl irwys In comprison to the lrge conducting irwys, the proportion of + + sl cells decresed drmticlly (30 cells/mm sl memrne) in the distl irwys of the helthy lung (Fig. 3). Here, 40 % of + cells did not express, demonstrting the existence of - + sl cell popultion. Importntly, we never oserved stining in norml distl irwys, suggesting the complete sence of this popultion in the helthy lung (Fig. 1). Distriution nd nture of sl cell supopultions in conducting irwys nd the distl IPF lung Similr to the helthy humn lung, (112 cells/mm sement memrne) nd rre popultion were present in conducting irwys of IPF ptients (Fig. 4). Similrly, 41 % of the totl + + popultion expressed (Fig. 6). In the distl IPF lung, however, the profile of nd -expressing cells ws drmticlly ltered compred with helthy lungs (Fig. 4). While + cells were only present in the sl lyers of helthy distl irwys, this cell popultion ws more widely distriuted in the IPF interstitium. This + progenitor popultion ws present in firotic res, mostly s pods (Fig. 6), nd dominnt in ronchiolized res (85 % of ll the + cells found in the distl IPF lung). + cells exhiited cuoidl or elongted morphology in IPF irwys, while they were pyrmidl in helthy distl irwys (Fig. 4). + were frequently orgnized into metplstic epithelil-like structures (Fig. 4, two upper pnels), single epithelil lyers with distinct cell morphology (Fig. 4, lower left pnel), or grouped in clusters or pods (Fig. 4, lower right pnel). While lmost ll + cells were + (92 %), some lso were -, mostly in inner res of centrl pod regions (Fig. 5c), suggesting

5 Smirnov et l. Respirtory Reserch (2016) 17:83 Pge 5 of 11 conducting irwys distl irwys Fig. 2 Coexpression of with nd in the helthy humn lung. Sections from helthy humn lungs (6 donors) were co-stined either for (green) nd (red) ( left; left), or (red) nd(green) ( right; right) nd counterstined with (lue). One representtive picture, with the corresponding high mgnifiction pnel, is shown for ech stining, for the conducting () nd distl () helthy irwys. Arrow: - + cell. Full rs = 500 μm. Scttered rs = 100 μm ongoing differentition processes similr to the + luminl cells in the conducting irwys. In striking contrst to the findings oserved in helthy lungs, + + popultion emerged in the distl IPF lung, nd ccounted for significnt prt of the totl + popultion (24 % in the ronchiolized res, nd 30 % in the firotic res) (Fig. 6). These cells were frequently detected in res of sl cell metplsi nd mostly grouped together in clusters, suggesting clonl expnsion. memrne cells/mm sl *** *** *** ### ### ### Conducting irwys Distl irwys Fig. 3 Quntifiction of totl sl cells nd + nd + sl cell supopultions in the helthy humn lung. Cells were counted ccording to their positivity for, or immunostining in conducting irwys (1 donor, 2 smples, n = 7 ssessed fields/smple) nd in distl irwys (5 donors, 1-3 smples/donor, n = 34 ssessed ronchioles). Dt re expressed s men ± SEM. A 2-Wy ANOVA with Bonferroni post-test reveled significnt interction ccording to the type of irwy (Conducting versus distl, p < ); ll 6 quntified popultions were significntly different etween conducting nd distl irwys (***p < 0.001). Ech dtset (conducting nd distl) ws then compred seprtely with One-Wy ANOVA test: ***p <0.001versus Conducting +, ### p < versus Distl + + cells, ut not + cells, coexpress epithelil cell differentition mrkers in the distl IPF lung In order to ssess the differentition potentil of + - nd + +, costinings of + or + cells with vriety of lveolr or ronchiolr differentition mrkers were performed (Fig. 7). Clusters of + cells in the distl IPF lung exhiited coexpression of quporin (AQP) 5 or Pro-Surfctnt Protein C (SPC), representing AT1 or AT2 mrkers, respectively (Fig. 7, ). While ll + cells exhiited co-expression of such differentition mrkers, + cells, in striking contrst, did not co-express ny of the known differentition mrkers. In ddition, + cells did not even loclize in proximity of AQP5 + or ProSPC + cells. Thus, our results suggest the existence of + - popultion in the IPF distl lung, which potentilly exhiits differentition cpcity into proper lveolr epithelil cell phenotype (AT1 or AT2 cells), wheres + + cell did not exhiit this phenotype. We lso chrcterized the hyperplstic ronchiolr lesions in IPF. Lyers composed of + sl cells in the distl IPF pods presented in close proximity of AcTu + -cilited cells (Fig. 7c, left pnel) or CC10 + -clu (Clr) cells (Fig. 7d, upper left pnel). Interestingly, the presence of + cells within the sl lyer ws ssocited with n sence of differentited cilited or Clr cells in the luminl lyer, similr to our oservtion of AQP5 stining (Fig. 7c, upper right pnel nd Fig. 7d, upper right pnel). Importntly, CC10 ws found to e coexpressed in + nd + cells, which

6 Smirnov et l. Respirtory Reserch (2016) 17:83 Pge 6 of 11 conducting irwys distl irwys Fig. 4 + nd + sl cell supopultions in the IPF lung. Sections from IPF humn lungs (5 ptients) were co-stined for (green), (red) nd counterstined with (lue). Two representtive pictures, with corresponding high mgnifiction pnels, re shown for the conducting irwys. 4 representtive pictures, with corresponding high mgnifiction pnels, illustrte the distriution ptterns (metplstic, simple nd pod-like) of + nd + sl cell supopultions in the distl IPF lung. Full rs = 500 μm. Scttered rs = 100 μm lcked the morphology of differentited Clr cells (Fig. 7d). In some hyperplstic epithelil structures,, ut not, costined with Mucin (MUC) 5B in luminl lyers (Fig. 7e, left pnel). + cells, on the other hnd, were confined to the sl lyer in which secreted MUC5B filled ronchiolr structure (Fig. 7e, right pnel). To further ssess whether these sl cell progenitors exhiited evidence of rectivtion of developmentl signling pthwys in IPF, we co-stined + -sl cells with Sonic Hedgehog, GLI1, SOX9, or HES1 (Fig. 8). Sonic Hedgehog filed to costin in + cells (Fig. 8), while its trget trnscription fctor (GLI1) loclized to the nucleus of most of the + cells (Fig. 8). SOX9 nd HES1 stined the nuclei of some + cells (Fig. 8c, d), indicting the presence of + SOX9 +, + SOX9 -, + HES1 + nd + HES1 - supopultions mongst the IPF sl cell progenitors. Discussion We show, for the first time, proximl-to-distl quntifiction of ronchil sl cell sutypes in the humn lung, chrcterized y vrious comintions of,, nd expression. While severl comintions of +/- +/- +/- sl cells populte the humn irwys in distinct loctions, the totl sence of popultion in the helthy distl lung is remrkle. In IPF, the overll mount nd distriution of the + + cells is drmticlly modified, nd frequent popultion emerges in the distl IPF lung. While - cells co-expressed lveolr nd ronchil epithelil differentition mrkers, + cells exhiit metplstic phenotype nd filed to costin with ny of these mrkers. This strongly suggests tht the cell type identified in the distl IPF lung is functionlly different from the cell popultion. The decrese in sl cell numers long the proximl-distl xis reported herein is consistent with the profile reported 27 yers go y Boers et l. [21]. In modifiction to those oservtions, we used the wellccepted sl cell mrker for cell quntifiction, which gives more precise estimtion of totl sl cell numers compred with morphologicl mesures lone [4]. We sought to chrcterize nd quntify irwy epithelil progenitors in the humn lung, s such dt is

7 Smirnov et l. Respirtory Reserch (2016) 17:83 Pge 7 of 11 conducting irwys distl irwys Fig. 5 Coexpression of with nd in the IPF lung., Sections from IPF humn lungs (5 ptients) were co-stined either for (green) nd (red) ( left; left), or (red) nd (green) ( right; right) nd counterstined with (lue). One representtive picture, with the corresponding high mgnifiction pnel, is shown for ech stining, for the conducting () nd distl () helthy irwys. c Chrcteristic + pod, stined for (green) nd (red). Arrow: + - cell. Full rs = 500 μm. Scttered rs = 100 μm missing up-to-dte, while quntittive investigtions hve een performed for + - nd + + popultions in the mouse trche [22]. Evidence of the expression of, ut not, y humn sl cells cme from colony forming-sl cells, isolted from the proximl or distl humn lung [14]. The expression of in humn sl cells ws oserved in sl lyers of ronchospheres formed y sl cells from humn lungs [6]. In ddition, erly studies lso descried expression in sl cells in conducting helthy humn irwys, lthough without ny evidence of expression [17]. Thus, our study now provides evidence tht 1) nd popultions coexist in similr proportions in the conducting irwys, 2) cells re sent from distl humn helthy lungs, 3) some + cells re -,whichsuggestssequentilloss of sl cells mrkers during stedy stte cell renewl in the conducting irwys; nd 4) rre popultion of - + exists in the distl humn lung, previously oserved lso y Vughn et l. [12]. This - + popultion of the distl irwys could either express KRT17 [17] or KRT15, which is descried s mrker for ll sl cells in the mouse trche [22]. IPF is progressive, irreversile, nd ftl lung disese, in which the lung epithelium is replced y scrring tissue rich in collgens nd extrcellulr mtrix, honeycoming, nd myofirolst-rich regions. Aville therpeutic pproches slow down progression of the disese, ut regenertive medicine pproches would represent promising novel option for the tretment of IPF [23]. As such, one potentil option is to trget sl cells, endogenous lung epithelil cells likely to hve regenertive potentil. While the ccumultion of + cells hs een previously reported in IPF [24], distinct supopultions were undefined. Similrly, n ccumultion of + cells [12], s well s cells [20] hs een oserved, ut neither quntified nor chrcterized. Here, we show tht, in striking contrst to norml lungs, distl IPF lungs present 1) n incresed undnce of + cells, 2) the emergence of frequent + + supopultion, 3) morphologicl chnges in these sl cells, nd 4) the existence of chrcteristic ptterns of + - nd + + structures. Moreover, the quntittive nlysis of these

8 Smirnov et l. Respirtory Reserch (2016) 17:83 Pge 8 of 11 memrne cells/mm sl cells/mm Krt14 + ### *** *** *** *** *** *** ** ** ** *** *** *** *** *** *** Bronchioliztion Firotic Non firotic Fig. 6 Quntifiction of totl sl cells nd + nd + sl cell supopultions in the IPF lung., Cells were counted ccording to their positivity for, or immunostining in () conducting irwys (1 donor, 2 smples, n = 7 ssessed fields/smple) nd () in distl regions (5 donors, 1-3 smples/donor, n=34 ssessed fields in ronchiolized regions (striped rs), n = 24 ssessed fields in firotic regions (lck rs), n=11 ssessed fields in non firotic regions (white rs)). Dt re expressed s men ± SEM. A1-Wy ANOVA reveled significnt interction (p < ). The dt were further nlyzed for significnce with Dunn s post-test (**p < versus + ). A 2-Wy ANOVA with Bonferroni post-test reveled significnt interction ccording to the type of remodeled region (p < ). Ech dtset (ronchioliztion, firotic, non firotic) ws then compred seprtely with 1-Wy ANOVA test, with Dunn s post-test: ***p < versus the neighoring Bronchioliztion column, ### p < etween Bronchioliztion + nd Bronchioliztion + different progenitor supopultions reveled regionl heterogeneity in the distl IPF lung. While in helthylike non firotic, or in firotic res, the sl cell progenitors re rrely found, they re undnt in res of ronchioliztion, suggesting the possiility of n ongoing regenertive process in these specific regions. Current knowledge in this field is limited y the lck of n irreversile niml model more closely mimicking these fetures of IPF, since leomycin-treted mice do not exhiit sl cell ccumultion in the lungs [12]. To dte, only the trchel epithelium-injury model using nphtlene relily increses the proportion of + cell popultions in the mouse [22]. Interestingly, upon infection with H1N1, + + cells lso ccumulte in the distl prts of mouse lungs, forming clusters or pods [14]. In our study, we detected, mongst others, similr ptterns of + cells in IPF lungs. Moreover, regions of pronounced + cell metplsi in IPF smples were frequently chrcterized y coexpression, suggesting hyperplstic potentil of the + + sl cell popultion. This is consistent with the hyperprolifertive potentil ttriuted to + cells. Indeed, the knockdown of in sl epithelil cells of the skin leds to cell cycle rrest [25]. In mice, reporter lines hve enled to descrie the different origins of the + sl cell popultion tht rises in the mouse lung fter injury. Vughn et l. showed tht the + cells ccumulting fter H1N1 injury re mostly not of +, ut linege-negtive origin. Another study showed tht Clr cells dedifferentite into + + sl cells [26]. Interestingly, we oserved surprising coexpression of or with the Clr cell mrker CC10 in the sl lyers of typicl ronchiolr structures in IPF. Therefore, lthough we cnnot provide direct evidence of the origin of sl cells in IPF, dedifferentition of CC10 + cells cnnot e excluded. The dedifferentition nd trnsdifferentition cpcities of sl cells re strongly dependent on the type of injury [10] nd their proximl-to-distl origin [14]. In the mouse, trchel + cells give rise to clu (Clr) nd cilited cells under stedy stte or epithelil injury conditions [6]. After selective ltion of + KRT6 + + distl irwy stem cells (DASC), mice filed to regenerte AT1 + structures fter H1N1 injury [13]. The differentition potentil of humn sl cells is tightly dependent on their origin nd ssy-specific environment [14]. In IPF, Vughn et l. could not identify + SPC + coexpressing cells. In contrst, in the humn lung, ws frequently coexpressed with ProSPC, in prticulr in hyperplstic regions. Moreover, further costinings etween nd lveolr/ronchil epithelil cell mrkers suggested multidirectionl differentition cpcity of these cells in IPF. The fte of these sl progenitor cells in IPF is likely to e dependent on the dysregultion of numerous growth fctors nd cytokines, which cn ffect the differentition potentil of sl cells [27], ut lso on the djcent ECM [28], ll of which is drmticlly ltered in IPF. The dysregulted signling environment chnges the intrcellulr signling cscdes in the IPF epithelium [29], in prticulr with respect to errnt ctivtion of developmentl pthwys in IPF [30, 31]. During lung development, SHH, the most rodly expressed Hedgehog lignd, controls proper rnching morphogenesis nd ptterns the lung [32]. SHH is upregulted in IPF, where it loclizes to lveolr nd ronchiolr epithelil cells, s previously reported y Bolños et l. [30]. In greement, we oserved SHH stining in the distl IPF lung, ut not in + sl cells. Interestingly, the SHH-induced

9 Smirnov et l. Respirtory Reserch (2016) 17:83 Pge 9 of 11 AQP5 AQP5 ProSPC ProSPC ctub ctub c d CC10 CC10 e MUC5B MUC5B Fig. 7 Costining of nd with lveolr nd ronchil epithelil differentition mrkers in the distl IPF lung. Humn distl IPF lungs (5 ptients) were costined for or nd AQP5 (Alveolr type 1 cell mrker), ProSPC (Alveolr type 2 cell mrker), ctub (cilited cell mrker), CC10 (Clr cell mrker) nd Mucin 5B (secretory cell mrker). One picture with the corresponding high mgnifiction is shown for ech stining. Left: (green) nd AQP5 (red). Right: (red) nd AQP5 (green). Left: (green) nd ProSPC (red). Right: (red) nd ProSPC (green). c Left: (green) nd ctub (red). Right: (red) nd AQP5 (green). d Left: (green) nd CC10 (red). Right: (red) nd CC10 (green). e Left: (green) nd MUC5B (red). Right: (red) nd MUC5B (green). Full rs = 100 μm. Scttered rs = 50 μm trnscription fctor GLI1, which is known for its involvement in stem cell renewl in non-smll cell lung cncer [33], loclized to the nuclei of most + cells in the distl IPF lung, rguing for ctivtion of the SHH pthwy in these cells. HES1, trnscription fctor ctivted y cnonicl Notch signling, induces Clr cell differentition in the developing lung [34]. HES1 is expressed in + cells in res of honeycoming in IPF, suggesting impired differentition potentil into AT2 cells [12]. Our co-immunostinings for nd HES1 confirmed ptchy Notch signling ctivtion in the + supopultion. In ddition, SOX9 prticiptes in ronchil development. Tight control of SOX9 levels is required for proper epithelil cell prolifertion, since SOX9 inhiits epithelil differentition in the developing mouse lung [35]. Upregultion of SOX9 is ssocited with lung cncer [36]. We oserved significnt numers of + cells in the IPF lung with prominent nucler SOX9+ stining. Altogether, these nd other findings thus suggest norml ctivtion of t lest three signling pthwys involved in cell prolifertion nd differentition in IPF sl cells, nd further indicte

10 Smirnov et l. Respirtory Reserch (2016) 17:83 Pge 10 of 11 c d Kertin5-HES1 Kertin5-SOX9 Kertin5-GLI1 Kertin5-SHH Fig. 8 Costining of with developmentl pthwy mrkers in the distl IPF lung.,, c, d Sections from IPF humn lungs (5 ptients) were co-stined for (green) nd () SHH (red), () GLI1 (red), (c) SOX9 (red), nd (d) HES1 (red), nd counterstined with (lue). One representtive picture, with the corresponding high mgnifiction pnel, is shown for ech stining. Full rs = 100 μm. Scttered rs = 50 μm tht the differentition potentil of the + popultion in IPF is modified compred with quiescent helthy cells. While + cells hve een reported to coexpress AT2 cell mrkers in DAD [18], we could not detect ny costining of + with differentition mrkers. The function of the + + popultion therefore remins uncler. Under stedy-stte conditions, oth + - nd + + supopultions re mitotic in the mouse trche [22]. Interestingly, the increse in the numer of + cells nd their contriution to the mitotic pool suggests tht this supopultion gives rise to highly mitotic reprtive pool fter nphtlene injury in the mouse [22]. The hypothesis of prolifertive function of + cells is reinforced y the tumorigenic potentil demonstrted for + cells from the skin [25], ut lso in the lung, where tumors spontneously develop in knock-in mice for humn [37]. Yet, some evidence lso suggests differentition potentil for the + + popultion. Indeed, fter nphtlene injury, some + cells hd luminl morphology in the mouse trche, wheres the differentiting cells were mostly -, which indictes quick shift in the expression during differentition. Ltely, new pthwys determining sl cell ftes fter injury hve een emerging in the mouse. Prdo-Sgnt et l. showed tht depending on the intrcellulr pthwys ctivted in the sl cells, they were committed either to cilited or secretory cell linege [38]. These discoveries, comined with n ccurte description of epithelil progenitor supopultions in humn helthy nd disesed lung, re promising pproches for further use of lung epithelil progenitor cells for therpeutic mnipultion nd epithelil regenertion. Conclusions This work provides quntittive nlysis of the regionl distriution of nd sl cell supopultions in the helthy humn lung. Drmtic chnges in the mount nd distriution of these cell popultions re oserved in IPF. In IPF, these popultions express differentited epithelil cell mrkers, showing tht in context of injury/disese, they re likely ttempting to regenerte the epithelium. This work provides sis for further descriptions of the moleculr pthwys tht my control the fte of progenitor sl cells in humn lung disese. Arevitions ctub, cetylted tuulin; AQP5, quporin 5; AT1, lveolr Type 1; AT2, lveolr type 2; CC10, clr cell 10 kd-protein; IPF, idiopthic pulmonry firosis; KRT, kertin; MUC5B, mucin 5B; ProSPC, pro-surfctnt protein C Acknowledgements The uthors thnk Gerld Burgstller for help nd dvice with the immunofluorescence experiments, in prticulr the imging nd nlysis. Authors contriutions NS prticipted to the design of the study, crried out the immunofluorescence stinings, nlyzed the slides, mde the quntifiction nd sttisticl nlysis, nd drfted the mnuscript; AS performed the hbec isoltion nd immunofluorescence, nd helped to drft the mnuscript; RH provided the humn mteril nd helped to drft the mnuscript; JB provided the humn mteril nd helped to drft the mnuscript; SN performed immunofluorescence stinings nd performed quntifiction thereof; OE prticipted to the design of the study, evluted immunofluorescence stinings, nd drfted nd edited the mnuscript. All uthors red nd pproved the finl mnuscript. Competing interests No competing interest needs to e declred.

11 Smirnov et l. Respirtory Reserch (2016) 17:83 Pge 11 of 11 Author detils 1 Comprehensive Pneumology Center, Institute of Lung Biology nd Disese, University Hospitl, Ludwig-Mximilins-University nd Helmholtz Zentrum München, Mx-Lesche-Pltz 31, Munich 81377, Germny. 2 Thorxchirurgisches Zentrum, Klinik für Allgemeine, Viszerl, Trnsplnttions, Gefäß- und Thorxchirurgie, Klinikum Großhdern, Ludwig-Mximilins-Universität, Munich, Germny. 3 Asklepios Fchkliniken München-Guting, Munich, Germny. 4 Medizinische Klinik und Poliklinik V, Klinikum der Ludwig-Mximilins-Universität, Munich, Germny. 5 Germn Center of Lung Reserch (DZL), Hnnover, Germny. 6 Comprehensive Pneumology Center (CPC), Ludwig-Mximilins-University nd Helmholtz Zentrum München, Mx-Lesche-Pltz 31, Munich D-81377, Germny. Received: 8 Ferury 2016 Accepted: 10 July 2016 References 1. Crosy LM, Wters CM. Epithelil repir mechnisms in the lung. Am J Physiol Lung Cell Mol Physiol. 2010;298(6):L Rckley CR, Stripp BR. Building nd mintining the epithelium of the lung. J Clin Invest. 2012;122(8): Kotton DN, Morrisey EE. Lung regenertion: mechnisms, pplictions nd emerging stem cell popultions. Nt Med. 2014;20(8): Schmerger AC, et l. Cigrette smoke lters primry humn ronchil epithelil cell differentition t the ir-liquid interfce. Sci Rep. 2015;5: Rock JR, Rndell SH, Hogn BL. Airwy sl stem cells: perspective on their roles in epithelil homeostsis nd remodeling. Dis Model Mech. 2010;3(9-10): Rock JR, et l. Bsl cells s stem cells of the mouse trche nd humn irwy epithelium. Proc Ntl Acd Sci U S A. 2009;106(31): Schmerger AC, et l. Cigrette smoke-induced disruption of ronchil epithelil tight junctions is prevented y trnsforming growth fctor-et. Am J Respir Cell Mol Biol. 2014;50(6): Schoch KG, et l. A suset of mouse trchel epithelil sl cells genertes lrge colonies in vitro. Am J Physiol Lung Cell Mol Physiol. 2004;286(4):L Studt MR, et l. Airwy Bsl stem/progenitor cells hve diminished cpcity to regenerte irwy epithelium in chronic ostructive pulmonry disese. Am J Respir Crit Cre Med. 2014;190(8): Hogn BL, et l. Repir nd regenertion of the respirtory system: complexity, plsticity, nd mechnisms of lung stem cell function. Cell Stem Cell. 2014;15(2): Voynow JA, et l. Bsl-like cells constitute the proliferting cell popultion in cystic firosis irwys. Am J Respir Crit Cre Med. 2005;172(8): Vughn AE, et l. Linege-negtive progenitors moilize to regenerte lung epithelium fter mjor injury. Nture. 2015;517(7536): Zuo W, et l. (+)Krt5(+) distl irwy stem cells re essentil for lung regenertion. Nture. 2015;517(7536): Kumr PA, et l. Distl irwy stem cells yield lveoli in vitro nd during lung regenertion following H1N1 influenz infection. Cell. 2011;147(3): O Koren EG, Hogn BL, Gunn MD. Loss of sl cells precedes ronchiolitis oliterns-like pthologicl chnges in murine model of chlorine gs inhltion. Am J Respir Cell Mol Biol. 2013;49(5): Hong KU, et l. In vivo differentition potentil of trchel sl cells: evidence for multipotent nd unipotent supopultions. Am J Physiol Lung Cell Mol Physiol. 2004;286(4):L Nkjim M, et l. Immunohistochemicl nd ultrstructurl studies of sl cells, Clr cells nd ronchiolr cuoidl cells in norml humn irwys. Pthol Int. 1998;48(12): Ficil M, et l. Kertin-14 expression in pneumocytes s mrker of lung regenertion/repir during diffuse lveolr dmge. Am J Respir Crit Cre Med. 2014;189(9): Iyong K, et l. Altertions in cytokertin expression y the lveolr lining epithelil cells in lung tissues from ptients with idiopthic pulmonry firosis. J Pthol. 1997;182(2): Seiold MA, et l. The idiopthic pulmonry firosis honeycom cyst contins mucocilry pseudostrtified epithelium. PLoS One. 2013;8(3):e Boers JE, Amergen AW, Thunnissen FB. Numer nd prolifertion of sl nd prsl cells in norml humn irwy epithelium. Am J Respir Crit Cre Med. 1998;157(6 Pt 1): Cole BB, et l. Trchel Bsl cells: fculttive progenitor cell pool. Am J Pthol. 2010;177(1): Lu AN, et l. Stem cells nd regenertive medicine in lung iology nd diseses. Mol Ther. 2012;20(6): Chilosi M, et l. Anorml re-epitheliliztion nd lung remodeling in idiopthic pulmonry firosis: the role of deltn-. L Invest. 2002;82(10): Alm H, et l. Novel function of kertins 5 nd 14 in prolifertion nd differentition of strtified epithelil cells. Mol Biol Cell. 2011;22(21): Tt PR, et l. Dedifferentition of committed epithelil cells into stem cells in vivo. Nture. 2013;503(7475): Tdokoro T, et l. IL-6/STAT3 promotes regenertion of irwy cilited cells from sl stem cells. Proc Ntl Acd Sci U S A. 2014;111(35):E Discher DE, Mooney DJ, Zndstr PW. Growth fctors, mtrices, nd forces comine nd control stem cells. Science. 2009;324(5935): Konigshoff M, et l. Functionl Wnt signling is incresed in idiopthic pulmonry firosis. PLoS One. 2008;3(5):e Bolnos AL, et l. Role of Sonic Hedgehog in idiopthic pulmonry firosis. Am J Physiol Lung Cell Mol Physiol. 2012;303(11):L Plntier L, et l. Ectopic respirtory epithelil cell differentition in ronchiolised distl irspces in idiopthic pulmonry firosis. Thorx. 2011;66(8): Pepicelli CV, Lewis PM, McMhon AP. Sonic hedgehog regultes rnching morphogenesis in the mmmlin lung. Curr Biol. 1998;8(19): Bor-Singhl N, et l. Gli1-medited regultion of Sox2 Fcilittes self-renewl of stem-like cells nd confers resistnce to EGFR inhiitors in non-smll cell lung cncer. Neoplsi. 2015;17(7): Morimoto M, et l. Cnonicl Notch signling in the developing lung is required for determintion of rteril smooth muscle cells nd selection of Clr versus cilited cell fte. J Cell Sci. 2010;123(Pt 2): Rockich BE, et l. Sox9 plys multiple roles in the lung epithelium during rnching morphogenesis. Proc Ntl Acd Sci U S A. 2013;110(47):E Zhou CH, et l. Clinicl significnce of SOX9 in humn non-smll cell lung cncer progression nd overll ptient survivl. J Exp Clin Cncer Res. 2012;31: Dkir EH, Feigenum L, Linnoil RI. Constitutive expression of humn kertin 14 gene in mouse lung induces premlignnt lesions nd squmous differentition. Crcinogenesis. 2008;29(12): Prdo-Sgnt A, et l. Injury induces direct linege segregtion of functionlly distinct irwy sl stem/progenitor cell supopultions. Cell Stem Cell. 2015;16(2): Sumit your next mnuscript to BioMed Centrl nd we will help you t every step: We ccept pre-sumission inquiries Our selector tool helps you to find the most relevnt journl We provide round the clock customer support Convenient online sumission Thorough peer review Inclusion in PuMed nd ll mjor indexing services Mximum visiility for your reserch Sumit your mnuscript t