RESEARCH ARTICLE

Influenza Virus Infects Epithelial Stem/ Progenitor Cells of the Distal Lung: Impact on Fgfr2b-Driven Epithelial Repair Jennifer Quantius1, Carole Schmoldt1, Ana I. Vazquez-Armendariz1, Christin Becker1, Elie El Agha2, Jochen Wilhelm2,3, Rory E. Morty2, István Vadász1,2, Konstantin Mayer1,2, Stefan Gattenloehner3, Ludger Fink4, Mikhail Matrosovich5, Xiaokun Li6, Werner Seeger1,2,7, Juergen Lohmeyer1,2, Saverio Bellusci2,8, Susanne Herold1,2*

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OPEN ACCESS Citation: Quantius J, Schmoldt C, VazquezArmendariz AI, Becker C, El Agha E, Wilhelm J, et al. (2016) Influenza Virus Infects Epithelial Stem/ Progenitor Cells of the Distal Lung: Impact on Fgfr2bDriven Epithelial Repair. PLoS Pathog 12(6): e1005544. doi:10.1371/journal.ppat.1005544 Editor: Paul G. Thomas, St. Jude Children's Research Hospital, UNITED STATES Received: December 28, 2015 Accepted: March 11, 2016 Published: June 20, 2016 Copyright: © 2016 Quantius et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by: German Research Foundation (SFB1021 C05, SFB-TR84 B2, grants LO271/4-1, BE 443/1-1, EXC147) http://www. sfb-tr84.de/ https://www.uni-marburg.de/sfb1021 http://www.eccps.de/; German Federal Ministry of Research and Education (FluReasearchNet grant 01 KI 1006M); Universities Giessen and Marburg Hospital (UKGM Reseach Support); State Government of Hesse LOEWE Program (UGMLC) https://www.uni-giessen.de/cms/forschung/

1 Department of Internal Medicine II, Universities Giessen & Marburg Lung Center (UGMLC), member of the German Center for Lung Research (DZL), Giessen, Germany, 2 Excellence Cluster Cardio-Pulmonary System (ECCPS), Universities Giessen & Marburg Lung Center (UGMLC), member of the German Center for Lung Research (DZL), Giessen, Germany, 3 Department of Pathology, Justus-Liebig-University Giessen, Giessen, Germany, 4 Institute of Pathology and Cytology, Wetzlar, Germany, member of the German Center for Lung Research (DZL), Giessen, Germany, 5 Institute of Virology, Philipps University, Marburg, Germany, 6 College of Pharmacy, Wenzhou Medical University, Wenzhou, Zhejiang, China, 7 Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany, 8 College of life and Environmental sciences and College of Chemistry and Materials Engineering, Wenzhou University, Wenzhou University town, Zhejiang, China * [email protected]

Abstract Influenza Virus (IV) pneumonia is associated with severe damage of the lung epithelium and respiratory failure. Apart from efficient host defense, structural repair of the injured epithelium is crucial for survival of severe pneumonia. The molecular mechanisms underlying stem/progenitor cell mediated regenerative responses are not well characterized. In particular, the impact of IV infection on lung stem cells and their regenerative responses remains elusive. Our study demonstrates that a highly pathogenic IV infects various cell populations in the murine lung, but displays a strong tropism to an epithelial cell subset with high proliferative capacity, defined by the signature EpCamhighCD24lowintegrin(α6)high. This cell fraction expressed the stem cell antigen-1, highly enriched lung stem/progenitor cells previously characterized by the signature integrin(β4)+CD200+, and upregulated the p63/krt5 regeneration program after IV-induced injury. Using 3-dimensional organoid cultures derived from these epithelial stem/progenitor cells (EpiSPC), and in vivo infection models including transgenic mice, we reveal that their expansion, barrier renewal and outcome after IV-induced injury critically depended on Fgfr2b signaling. Importantly, IV infected EpiSPC exhibited severely impaired renewal capacity due to IV-induced blockade of β-catenin-dependent Fgfr2b signaling, evidenced by loss of alveolar tissue repair capacity after intrapulmonary EpiSPC transplantation in vivo. Intratracheal application of exogenous Fgf10, however, resulted in increased engagement of non-infected EpiSPC for tissue regeneration, demonstrated by improved proliferative potential, restoration of alveolar barrier function and increased survival following IV pneumonia. Together, these data suggest that tropism of IV to distal lung stem cell niches represents an important factor of pathogenicity and highlight

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einrichtungen/loewe/ugmlc; and German Center for Lung Research (DZL) http://www.dzl.de/index.php/de/ . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

impaired Fgfr2b signaling as underlying mechanism. Furthermore, increase of alveolar Fgf10 levels may represent a putative therapy to overcome regeneration failure after IVinduced lung injury.

Competing Interests: The authors have declared that no competing interests exist.

Author Summary Influenza Virus (IV) pneumonia causes disruption of the alveolar epithelial barrier, leading to edema formation which severely affects gas exchange. Recovery after IV-induced acute lung injury includes not only innate immune responses to clear virus infection, but also tissue renewal processes to restore the alveolar barrier. We demonstrate that highly pathogenic IV particularly infect cells of the epithelial stem/progenitor cell niche, which significantly impairs repair processes within the damaged lung, as demonstrated by transplantation experiments using intrapulmonary delivery of infected versus non-infected stem cells into IV-injured mice. Analyses of the underlying virus-host interactions reveal that IV infection of epithelial stem/progenitor cells results in blockade of a pathway involving Wnt/β-catenin and fibroblast growth factor receptor 2b (Fgfr2b) activation within a regenerative epithelial-mesenchymal cell signaling network. Our data therefore suggest that tropism of IV to the distal lung stem cell niche represents an important factor of pathogenicity. Therapeutic intrapulmonary application of excess Fgf10 to induce Fgfr2b signaling in the non-infected stem cell niche was found to counteract IV-induced repair failure and to restore barrier function, representing a promising treatment to foster epithelial cell regeneration after IV-induced injury.

Introduction Influenza viruses (IV) may cause primary viral pneumonia in humans with rapid progression to lung failure and fatal outcome, and treatment options for this sometimes devastating disease are limited [1, 2]. Histopathology and clinical features of IV-induced lung injury in humans resemble those of other forms of ARDS (acute respiratory distress syndrome) and are characterized by apoptotic and necrotic airway and alveolar epithelial cell death, loss of pulmonary barrier function and severe hypoxemia [1, 3, 4]. IV primarily infect cell subsets of the upper and lower respiratory tract. In the latter, these are particularly ciliated and goblet cells, club cells and alveolar epithelial cells type II (AECII) [5–7]. Injury of lung epithelial cells is induced by both direct viral cytopathogenicity and unbalanced immune responses [8–11]. The initiation of well-coordinated programs of inflammation termination and of regeneration of the injured distal lung epithelium are a prerequisite for the re-establishment of proper gas exchange. Absence or imbalance of these responses may at best result in chronically organizing infiltrates and aberrant or excess remodeling with tissue fibrosis, associated with long-term pulmonary organ dysfunction in ARDS survivors [12, 13], or in fatal outcome at worst. However, the cellular communication patterns and molecular networks underlying regeneration of the distal lung compartment after severe pathogen-associated injury are incompletely understood to date. In particular, the distinct mechanisms of interaction between injury-causing pathogens with components of regenerative signaling pathways within the lung stem cell niche, determining outcome of the repair response, have not been studied in detail. Alveolar re-epithelialization after injury was shown to involve different populations of endogenous, organ-resident stem/progenitor cells, which express lineage markers of distal lung

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epithelium such as club cell-specific protein (CC10/scgb1a1) or surfactant protein C (SPC/ sftpc), are quiescent under normal conditions and proliferate during repair [14]. More recent reports revealed that intrinsically committed distal airway stem cells (DASC) expressing keratin 5 (krt5) and the transcription factor p63 were found to contribute to de novo generation of both bronchiolar and alveolar tissue after formation of cell “pods” in a murine model of IV infection [15, 16]. Vaughan et al. defined lineage-negative, integrin(β4)+CD200+ epithelial progenitors as the source of p63/krt5+ amplifying cells regenerating airways and alveoli, highlighting integrin(β4)+CD200+ epithelial cells as important progenitors regenerating the distal lung following IV-induced injury [17]. During regeneration processes, the lung stroma likely plays a key role by maintaining the distinct microenvironment of the stem cell niche, involving extracellular matrix, direct cell-cell contacts and autocrine or paracrine mediators. These signals initiate and co-ordinate selfrenewal, fate determination and terminal differentiation of stem/progenitor cells. Different subsets of resident lung stromal/mesenchymal cells have been attributed a role in these processes, including parabronchial smooth muscle cells [18], Sca-1high lung mesenchymal cells [19, 20] or a human vimentin+ lung fibroblast population [21]. Signals involved in these crosstalk events include, among others, the paracrine fibroblast growth factors (Fgfs), which regulate cell survival, proliferation, differentiation, and motility. In particular, Fgf7 and Fgf10 and their common tyrosine kinase receptor Fgfr2b (fibroblast growth factor receptor 2b), are indispensable for distal lung development including branching morphogenesis [19, 22–24]. Fgfr2b signaling is also re-activated in stem cell niches of the adult lung after different forms of injury to regenerate the epithelium [23, 25, 26]. The regulation of ligand and receptor expression of the Fgf7/10-Fgfr2b network in the context of lung repair after infectious injury, however, is not well understood. In the current study, we demonstrate that a highly proliferating EpCamhighCD24lowintegrin (α6β4)highCD200+ distal lung epithelial cell population represents a primary target of pathogenic IV. This population highly enriched cells expressing key characteristics of distal lung epithelial stem/progenitor cells mediating bronchiolar and alveolar repair. Of note, IV tropism to these cells significantly reduced their regeneration capacity by impairment of β-catenin-dependent Fgfr2b signaling. These data for the first time demonstrate that the extent of lung stem/ progenitor cell infection by IV is a hallmark of pathogenicity as it critically impacts on lung regeneration capacity after severe IV injury. Moreover, IV-induced regeneration failure could be counteracted by intratracheal application of excess recombinant Fgf10, suggesting recruitment of the non-infected Fgfr2bhigh stem cell fraction for repair as putative novel treatment strategy to drive organ regeneration in patients with IV-induced ARDS.

Results Influenza viruses target epithelial cell subsets of the distal murine lung to different extent after intratracheal infection It is well established that IV infect different subsets of the airways and alveoli, particularly ciliated and goblet cells, club cells and AECII [5–7]. However, recent advances in the field resulted in the definition of more specialized subsets of lung epithelial cells, some of which display stem/progenitor cell characteristics and contribute to repair of the injured organ [17, 19, 27]. To address which of these epithelial cell compartments were infected by IV, we fractionated distal lung epithelial cells into different subsets, after dissection of large airways and vessels and depletion of leukocytes and endothelial cells, according to surface expression levels of EpCam and integrin α6 [19], and the lineage markers CD24 (differentiated airway epithelial cells) [19], CC10 (club cells), pro-SPC (AEC II) and T1α (AEC I), by flow cytometry. We identified a

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high-frequent EpCamlowα6low fraction (91.3 ± 1.8%) and a low frequent EpCamhighα6high fraction, the latter of which consisted of a CD24low and CD24high population (1.7 ± 0.3% and 6.3 ± 1.8%, respectively, Fig 1A). The majority of the most abundant EpCamlowα6low cells showed a granular cytoplasm typically observed in AEC II, with approximately 95% of the cells expressing the AEC II signature pro-SPChighT1αneg and around 5% expressing an AEC I signature (SPCnegT1α+) (Fig 1B). EpCamhighα6highCD24high cells contained pro-SPCnegCC10neg differentiated small airway epithelial cells (SAEC, 70%), composed of both β-tubulin+ ciliated and mucin5AC+ goblet cells, and pro-SPCnegCC10+ club cells (30%) (Fig 1C). EpCamhighα6highCD24low cells were cells of homogeneous morphology and stained positive for the stem cell antigen Sca-1+ (Fig 1D). To analyse which of these epithelial cell subset were targeted by IV, we infected C57BL/6 mice using 500pfu of IV strains of increasing pathogenicity, i.e. low-pathogenic H3N2 (x-31), pandemic pH1N1 strain (A/Hamburg/04/09), causing mild to moderate lung injury at this dose in mice, and the highly pathogenic mouse-adapted PR/8 strain [28, 29]. Quantification of the infection rates by staining for IV nucleoprotein (NP) revealed that EpCamlowα6high CD24high and EpCamlowα6low cells were infected with a frequency of *11% and *6%, respectively, by d4 pi after PR/8 infection, a time point where PR/8 replication in the lung reaches a peak [28]. Subfractionation into differentiated alveolar and airway epithelial cells revealed that rates of PR/8 infection in AEC I and AEC II ranged at *8% and *4%, whereas club cells and ciliated/goblet cells displayed similar PR/8 infection rates of *10%. Of note, EpCamhigh α6highCD24low cells were infected by IV to high amounts (around 15% of all EpCamhigh α6highCD24low after PR/8 infection), and the proportion of infected epithelial cell subsets at d4 pi correlated with the level of pathogenicity of the IV strain used (Fig 1E and 1F). To further address whether PR/8 revealed increased tropism to EpCamhighα6highCD24low cells, AEC, SAEC and EpCamhighα6highCD24low cells were flow-sorted, seeded into culture plates at equal numbers and infected ex vivo with x-31, PR/8 and pH1N1 at an MOI of 2, respectively. After one replication cycle (6h), excluding de novo infection by progeny virions, the infection rate was determined, reflecting the capacity of each virus strain to infect the respective cell population. Similar to our in vivo results, we observed that PR/8 reveals an enhanced tropism to EpCamhighα6highCD24low cells (S1 Fig).

Highly infectable EpCamhighα6highCD24lowSca-1pos cells reveal epithelial stem cell characteristics and generate lung-like organoids in an Fgf10-dependent manner Given that the EpCamhighα6highCD24lowSca-1pos cells which revealed the highest rates of infection were previously described as epithelial stem/progenitor population giving rise to airway and alveolar epithelium [19], we aimed to further characterize their phenotype and stemness properties. Further analyses using established stem cell markers [17] revealed that they were integrin β4+CD200+, a signature which has been confined to a distal lung stem cell phenotype known to engage the krt5/p63 regeneration program [17] (Fig 1D). In accordance, these cells were negative for krt5 and p63 in healthy lungs, but highly upregulated krt5 and p63 gene expression after IV-induced injury (Fig 1G), suggesting that they contain epithelial stem/progenitor cells (EpiSPC). To verify these stem/progenitor cell characteristics ex vivo, EpCam+ cell fractions were flowsorted and seeded in organotypic 3D cultures [30]. As opposed to AEC I/II, and SAEC/club cells, EpiSPC developed typical large organoid spheres with cystic or saccular outgrowth in the presence of the growth factors Fgf10 and Hgf (hepatocyte growth factor), a characteristic feature of lung stem/progenitor cells [19] (S2A Fig). A robust clonogenic potential of EpiSPC was

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Fig 1. Characterization of distal lung epithelial cell subpopulations and analysis of their infection rates in vivo. (A) Gating strategy of three epithelial cell subsets in CD31 and CD45 depleted lung homogenates of wt mice according to the expression of EpCam, α6 integrin and CD24. (B) Pappenheim stained cytospins of flow-sorted EpCamlowα6low epithelial cells and flow cytometric subgating of this fraction

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with proSPC and T1α. (C) Characterization of the EpCamhighα6highCD24high subpopulation by flow cytometry reveals a CC10+ and a CC10neg fraction. Pappenheim stained cytospins of flow-sorted EpCamhighα6highCD24highCC10neg epithelial cells (arrows indicate ciliated cells) and immunofluorescence stainings of this cell subset with mucin5ac and β-tubulin after 4d of culture. (D) Flow-sorted and Pappenheim stained cytospins of the EpCamhighα6highCD24low epithelial cell population (left). Further flow cytometric phenotype characterization of the EpCamhighα6highCD24low population revealed that it is Sca-1+ (middle) and localizes to the β4 integrin+ and CD200+ fraction of EpCam+ cells (right, EpCamhighα6highCD24low population depicted in red). (E-F) Wt mice were infected with 500pfu of the indicated influenza virus strains and the fractions of influenza virus infected (nucleoprotein+, NP+) cells of the different EpCam+ subpopulations were determined by FACS at d4 pi. (G) Cytospins of the flow-sorted EpCamhighα6highCD24low population or of tracheal digests (positive control) from uninfected mice were stained for krt5 and p63 (left). Quantification of p63 and krt5 mRNA levels of flow-sorted EpCamhighα6highCD24low at d14 pi from PR/8 infected mice (right). Bar graphs represent fold induction compared to mock-infected controls. Bar graphs represent means ± SD of n = 4 independent experiments; * p