ARTICLES Local lung hypoxia determines epithelial fate decisions during alveolar regeneration Ying Xi1,6, Thomas Kim1, Alexis N. Brumwell1, Ian H. Driver2, Ying Wei1, Victor Tan1, Julia R. Jackson1, Jianming Xu3, Dong-Kee Lee3, Jeffrey E. Gotts1, Michael A. Matthay1, John M. Shannon4, Harold A. Chapman1,7,8 and Andrew E. Vaughan1,5,6,7 After influenza infection, lineage-negative epithelial progenitors (LNEPs) exhibit a binary response to reconstitute epithelial barriers: activating a Notch-dependent 1Np63/cytokeratin 5 (Krt5) remodelling program or differentiating into alveolar type II cells (AEC2s). Here we show that local lung hypoxia, through hypoxia-inducible factor (HIF1α), drives Notch signalling and Krt5pos basal-like cell expansion. Single-cell transcriptional profiling of human AEC2s from fibrotic lungs revealed a hypoxic subpopulation with activated Notch, suppressed surfactant protein C (SPC), and transdifferentiation toward a Krt5pos basal-like state. Activated murine Krt5pos LNEPs and diseased human AEC2s upregulate strikingly similar core pathways underlying migration and squamous metaplasia. While robust, HIF1α-driven metaplasia is ultimately inferior to AEC2 reconstitution in restoring normal lung function. HIF1α deletion or enhanced Wnt/β-catenin activity in Sox2pos LNEPs blocks Notch and Krt5 activation, instead promoting rapid AEC2 differentiation and migration and improving the quality of alveolar repair. The adult lung is a largely quiescent tissue but responds effectively In this study we explored the basis for activation and expansion to injury by activating stem/progenitor populations and promoting of lung epithelial stem/progenitor cells observed in mice infected proliferation of surviving, mature lineages. Depending on the type with H1N1 (PR8) influenza. Mice and humans infected with H1N1 and severity of cellular injury, different cell types are involved influenza develop large areas virtually devoid of alveolar epithelial in repair1–5. A remarkable expansion of potentially regenerative cells, requiring both expansion and migration of surviving AEC2s 1Np63pos/Krt5pos cells was observed in mouse lungs infected by and more undifferentiated progenitors to restore alveolar epithelial murine-adapted H1N1 (PR8) influenza A virus6. Lineage-tracing barriers2,9,10. Studies here interrogate the underlying mechanisms experiments showed that >80% of the expanded Krt5pos cells arise and sources of epithelial progenitors that determine the alternative from LNEP populations in distal airways and probably alveoli2. These pathways of alveolar repair. LNEPs proliferate and migrate dramatically to damaged sites to reconstitute the alveolar epithelium and promote recovery. Specific RESULTS ablation of the newly expanded Krt5pos cells by diphtheria toxin Influenza-activated Krt5pos cells are derived from p63pos resulted in prolonged hypoxemia, confirming involvement of these lineage-negative progenitors cells in the repair process7. Notch signalling is critical for activating Our previous studies indicated that LNEPs are a Sox2pos this 1Np63/Krt5 remodelling program; however, persistent Notch heterogeneous population comprised of both cells expressing signalling prevents alveolar epithelial differentiation8, leading to cystic the stem cell transcription factor 1Np63 and 1Np63neg cells, structures indicative of incomplete regeneration. Similar Krt5pos although p63 expression was restricted to a minor LNEP fraction2,11. honeycombing cysts also exist in the lungs from patients with Transplantation studies suggested the possibility that these subtypes pulmonary fibrosis, suggesting that common mechanisms apply to are biased toward either basal-like or AEC2 differentiation, injury responses in both mice and humans2. respectively2. To clarify this, we infected tamoxifen-treated 1Department of Medicine, Cardiovascular Research Institute, UCSF, San Francisco, California 94143, USA. 2Department of Anatomy, UCSF, San Francisco, California 94143, USA. 3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA. 4Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, USA. 5Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. 6Present addresses: Department of Immunology, Genentech Inc., South San Francisco, California 94080, USA (Y.X.); Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA (A.E.V.). 7These authors jointly supervised this work. 8Correspondence should be addressed to H.C. (e-mail: [email protected]) Received 17 March 2017; accepted 21 June 2017; published online 24 July 2017; DOI: 10.1038/ncb3580 904 NATURE CELL BIOLOGY VOLUME 19 | NUMBER 8 | AUGUST 2017 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ARTICLES a b 95.3% 91.5% TMX Infection Euthanization 120 NS 100 –6 week d0 d13 cells 80 pos 60 40 20 traced Krt5 Percentage of p63-cre 0 Lung Trachea p63 trace 100 μm Krt5 ce HIF1αfl/fl HIF1α–/– Saline Krt5 DAPI Hypoxyprobe 500 μm 100 μm Krt5 Influenza f g P = 0.03 P = 0.02 20 Krt5 450 15 d Saline Influenza 300 10 M (K) lung area r HIF1α 100 pos 5 150 β Percentage of -actin Krt5 0 0 37 fl/fl –/– HIF1αfl/fl HIF1α–/– Relative mRNA level HIF1α HIF1α Saline Influenza 110 P = 0.04 fl/fl d21 d28 h HIF1α (n = 14) i P = 0.007 j P = 0.01 –/– l) μ 100 HIF1α (n = 7) 250 pos 100 pos 90 * 200 90 80 150 saturation 2 100 O 70 Percentage of 80 60 50 0 7 9 13 14 15 16 0 of total EpCAM 70 Excess lung water ( fl/fl –/– Percentage of SPC fl/fl –/– Days post influenza HIF1α HIF1α HIF1α HIF1α Figure 1 Epithelial HIF1α deletion blocks alveolar Krt5 activation post obtained by pulse oximetry are greater in HIF1α−=− at the indicated times. H1N1 infection. (a) Krt5pos cells are completely traced (tdTomatopos) after (i) HIF1α−=− mice exhibit less excess lung water after influenza, indicative influenza injury in p63-CreERT2/tdTomato mice. (b) Quantification of lineage of improved barrier function. Data for h and i are mean s.e.m., n D 7 tracing by manual cell counts in tissue sections. Data are mean s.e.m., HIF1α−=−, n D 14 wild-type (2 Shh-Creneg, 12 C57BL6) or n D 13 wild- n D 3 mice. (c) Krt5pos cells (red) invariably appear in hypoxic alveolar type for i (2 Shh-Creneg, 11 C57BL6) from two independent experiments. regions (hypoxyprobe, green) after influenza injury in mice, although some (j) AEC2 recovery/regeneration is improved in HIF1α−=− as judged by Krt5neg hypoxic regions were also observed (n D 7 mice). (d) HIF1α protein intracellular FACS for SPCpos cells as a fraction of total EpCAMpos cells. accumulates in influenza-infected mouse lungs (nD6 influenza, nD3 saline, The percentage of EpCAMpos live cells is unchanged in HIF1α−=− and β-actin as loading control). (e) Alveolar Krt5pos expansion (green) (left) wild-type mice. Data are mean s.e.m., n D 3 HIF1α−=−, n D 4 wild-type is largely blocked by epithelial HIF1α deletion (right). (f) Quantification mice from 2 independent experiments. Analysis is 11 days post-infection of e. Data are mean s.e.m., n D 5 wild-type, n D 4 HIF1α−=− mice unless otherwise indicated. All P values were derived by unpaired two- from two independent experiments. (g) Krt5 mRNA levels are reduced in tailed Student’s t-test, except in h, derived by Mann–Whitney. NS, not HIF1α−=− mice. Data are mean s.e.m., n D 8 wild-type, n D 9 HIF1α−=− significant. Unprocessed original scans of blots are shown in Supplementary mice from 3 independent experiments. (h) Arterial oxygen saturation values Fig. 8. p63-CreERT2 mice and determined that essentially all arising resistance to infection as well as migratory and proliferative Krt5pos cells were lineage labelled (Fig. 1a,b and Supplementary capacity. However, the scarcity of p63pos LNEPs and their infrequent Fig. 1a). Uninjured mice possessed p63-lineage-labelled cells scattered contribution to AEC2 regeneration (Supplementary Fig. 1a) suggest throughout airways, comprising <0.01% of all lung epithelial cells that additional p63neg Sox2pos LNEPs are a likely source of previously (Supplementary Fig. 1b,c). These rare cells must possess remarkable observed AEC2 differentiation following LNEP transplantation post NATURE CELL BIOLOGY VOLUME 19 | NUMBER 8 | AUGUST 2017 905 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ARTICLES influenza injury2. To begin to dissect these possibilities we considered metaplasia entirely. Given the impact of HIF1α on the quality of signals that impact LNEP expansion in vivo. epithelial recovery, we explored the mechanistic link between hypoxia and Krt5pos cell expansion. Epithelial HIF1α is required for Krt5pos cell expansion post influenza injury Hypoxia promotes Notch signalling and Krt5 expression in a To explore mechanisms for p63pos LNEP expansion during influenza HIF1α-dependent manner infection, we examined the role of hypoxia, a likely consequence We validated that submersion cultures are hypoxic, as previously of extensive epithelial necrosis. Krt5pos areas were found exclusively reported12, in comparison with air/liquid interface demonstrated in hypoxic regions identified by immunostaining of pimonidazole by hypoxyprobe staining and elevation of HIF1α targets (Fig. 3a). (hypoxyprobe) (Fig. 1c). HIF1α, a key mediator of cell responses to Hypoxia also promoted Notch target and Krt5 mRNA expression hypoxia, accumulated in the whole lung lysates 11 days post infection