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STAT3–BDNF–TrkB signalling promotes alveolar epithelial regeneration after lung injury

Nature Cell Biology volume 22, pages 1197–1210 (2020)Cite this article

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Abstract

Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3–BDNF–TrkB axis in orchestrating alveolar epithelial regeneration.

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Fig. 1: The response to injury is marked by significant changes in AT2 chromatin architecture.
Fig. 2: STAT3 regulates key pathways in alveolar epithelial regeneration.
Fig. 3: Deletion of Stat3 in AT2 cells worsens outcomes following sterile and infectious lung injuries.
Fig. 4: Identification of BDNF as important for alveolar epithelial regeneration.
Fig. 5: Loss of AT2-specific BDNF worsens outcomes following sterile and infectious lung injuries.
Fig. 6: BDNF–TrkB signalling promotes alveolar epithelial regeneration.
Fig. 7: The STAT3–BDNF–TrkB axis modulates Fgf7 expression by mesenchymal niche cells.
Fig. 8: The TrkB agonist 7,8-DHF improves outcomes following sterile and infectious lung injury.

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Data availability

ATAC-seq and scRNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE132535. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We acknowledge S. M. Albelda, J. S. Brenner and A. E. Vaughan for help with preparing this manuscript, as well as the Pathology, Flow Cytometry, Nucleic Acid and PCR cores and the Center for Applied Genomics at the Children’s Hospital of Philadelphia Research Institute. This work was supported by the Parker B. Fellowship Program (A.J.P.) and multiple grants from the National Institutes of Health (K08 HL136698 (A.J.P.), R01 AI121321 (M.D.W.), R01 AI099479 (G.S.W.) and R01 DK114054 (G.S.W.)). We also thank the patients and families who donated lung tissue to support this research.

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Authors and Affiliations

  1. Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Andrew J. Paris, Jarod A. Zepp, Jeremy B. Katzen, Maria C. Basil, Ping Wang, Michael F. Beers & Edward E. Morrisey

  2. Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Katharina E. Hayer

  3. Division of Hematology, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Joseph H. Oved

  4. Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Daphne C. Avgousti

  5. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Sushila A. Toulmin, Laurence C. Eisenlohr & Matthew D. Weitzman

  6. Penn-CHOP Lung Biology Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Jarod A. Zepp, Jeremy B. Katzen, Maria C. Basil, Madison M. Kremp, David B. Frank, Michael F. Beers, Edward E. Morrisey & G. Scott Worthen

  7. Division of Pulmonary Biology, Perinatal Institute, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    William J. Zacharias

  8. Department of Pediatrics, Children’s National, Washington, DC, USA

    April R. Slamowitz

  9. Division of Cardiology, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Sowmya Jayachandran, Aravind Sivakumar & David B. Frank

  10. Division of Neonatology, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Ning Dai & G. Scott Worthen

  11. Division of Protective Immunity, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Laurence C. Eisenlohr & Matthew D. Weitzman

  12. Division of Cardiovascular Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Edward Cantu III

  13. Penn Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Edward E. Morrisey

  14. Penn Institute for Regenerative Medicine, Perelman School of Medicine, Philadelphia, PA, USA

    Edward E. Morrisey

  15. Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Edward E. Morrisey

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  1. Andrew J. Paris
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  21. Edward E. Morrisey
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  22. G. Scott Worthen
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Contributions

A.J.P. conceived of and co-performed the ATAC-seq experiment, performed all of the acid-induced lung injury experiments, analysed the histological data and co-wrote the manuscript. K.E.H. analysed the ATAC-seq and scRNA-seq data. J.H.O. assisted with the data analysis and co-wrote the manuscript. D.C.A. co-performed the ATAC-seq experiment. S.A.T. performed the influenza injury experiments. J.A.Z. performed the scRNA-seq of mesenchymal cells and assisted with the murine alveolar organoid experiments. W.J.Z. assisted with the murine and human alveolar organoid experiments. J.B.K. performed the survival analysis and assisted with isolating the human AT2 cells. M.C.B. identified the human histological samples. M.C.B. and M.M.K. isolated the human AT2 cells. A.R.S. assisted with performing the murine and human organoid experiments. S.J., A.S. and D.B.F. analysed the mean linear intercept and alveolar wall thickness of the murine lung specimens. N.D. and P.W. bred the mice, genotyped the mice, administered the tamoxifen and isolated the murine AT2 cells and fibroblasts. P.W. cultured the MRC5 cells and set up the murine and human organoid experiments. E.C. procured the human lung tissue. G.S.W. blindly scored the histological specimens. L.C.E., M.F.B. and E.E.M. assisted with the experimental design and with preparing the manuscript. M.D.W. and G.S.W. designed, conceived of and supervised the experiments and co-wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to G. Scott Worthen.

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The authors have applied for a provisional patent in the United States related to this work. A.J.P. and G.S.W. are listed as co-inventors on this application.

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Extended data

Extended Data Fig. 1 Sorting Strategies for Isolating AT2 cells.

Lungs from C57BL/6 mice were digested into a single-cell suspension. We collected EpCAM positive cells (APC) that were PDPN, CD45, CD34, CD31 and SCA1 negative (all PE).

Extended Data Fig. 2 STAT3 is activated following sterile and infectious lung injuries.

Cells staining positive for proSFTPC+ and pSTAT3+ are highlighted with a white box while proSFTPC + pSTAT3- cells are highlighted with a yellow box. Areas that were felt to be indeterminate for cell type or pSTAT3 status are highlighted in blue. Scale bars, 50μm.

Extended Data Fig. 3 Loss of Stat3 diminished alveolar organoid formation.

a, PCR shows the expected 802bp product when corn oil is administered to SftpcCreERT2:Stat3LoxP/LoxP mice and the recombined 514bp product when tamoxifen is administered to SftpcCreERT2:Stat3LoxP/LoxP mice. The gels in this figure represent n=2 mice per condition (corn oil and tamoxifen). This experiment was repeated 4 times with similar results. b, AT2 cells from tamoxifen treated SftpcCreERT2-Rosa26tdTomato and SftpcCreERT2-Rosa26tdTomato:Stat3LoxP/LoxP mice were isolated and co-cultured with PDGFRα mesenchymal cells in transwells containing Matrigel for four weeks (n=4 wells per condition). Organoid forming efficiency was significantly decreased in the absence of STAT3 signaling in AT2 cells (p=2.2x10-8). Data is shown as the mean +/- SEM. Statistical significance was determined using a two-tailed Student’s t-test. Scale bars, 2.5mm.

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Extended Data Fig. 4 Loss of Stat3 in AT2 cells does not alter distal lung morphology.

SftpcCreERT2:Stat3LoxP/LoxP mice were given tamoxifen or vehicle and euthanized three months later. Representative H&E stained histological specimens are shown. The Mean linear intercept was unchanged by the loss of Stat3 in these unchallenged mice (n=4 mice per group). Data is shown as the mean +/- SEM. Statistical significance was determined with a two-tailed Student’s t-test. Scale bars, 500μm.

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Extended Data Fig. 5 Single-cell expression of TrkB, Ccnd1, Tm4sf1 and Sftpc in isolated AT2 cells.

Plots show relative expression of TrkB, Ccnd1, Tm4sf1 and Sftpc in AT2 cells isolated isolated from uninjured mice and mice 24h after acid-induced lung injury.

Extended Data Fig. 6 Pathways analysis of isolated AT2 cells reveals multiple subsets of cells with multiple functions.

Pathways analysis of differentially expressed genes in the clusters shown in Fig. 4b.

Extended Data Fig. 7 AT2 cells express Bdnf after acid-induced lung injury.

BdnfCre-Rosa26tdTomato were subjected to acid-induced lung injury (n=4 mice per group). One day after injury the mice were euthanized, and the lungs were probed for Tomato (red) and proSFTPC (green). Autofluorescence delineates tissue structure. Scale bar, 25μm.

Extended Data Fig. 8 Loss of TrkB in mesenchymal cells worsens outcomes following sterile and infectious lung injuries.

Lungs from PdgfraEGFP mice were digested into a single-cell suspension either before or 24h after acid-induced lung injury (n=4/group). We first gated on PDGFRα+ and then quantified the percent of GFP+ cells within that population.

Extended Data Fig. 9 Loss of TrkB in mesenchymal cells worsens outcomes following sterile and infectious lung injuries.

a, H&E staining and ATS lung injury scores of tamoxifen and corn oil exposed PdgfrαCreERT2:TrkBLoxP/LoxP mice 24 hours after acid-induced lung injury (n=4 mice per group). b-d, H&E (b), PDPN (c) and Krt5 (d) staining of tamoxifen and corn oil exposed PdgfrαCreERT2:TrkBLoxP/LoxP mice 21 days after infection with PR8 influenza (n=3 mice per group). Statistical analysis was performed with a two-tailed Student’s t-test or ANOVA, where appropriate. For panels (a) and (d), data is shown as the mean +/- SEM. Statistical significance was determined with a two-tailed Student’s t-test (a) and (d). Scale bars, 100μm.

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Extended Data Fig. 10 The TrkB agonist 7,8-DHF is unable to rescue mice with an AT2-specific Stat3 deletion.

Survival curves for Tamoxifen-exposed SftpcCreERT2:Stat3LoxP/LoxP mice that had been infected with intranasal PR8 influenza (5x10-5 HAU/mouse) and were given intraperitoneal injections of 7,8-DHF or vehicle every other day (n=5 mice per group). The data analyzed using the Log-rank (Mantel-Cox) test and is not statistically significant.

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Supplementary information

Reporting Summary

Supplementary Tables

Table 1. Table of STAT3 binding sites. Table 2. Table of antibody concentrations. Table 3. Table of RT-PCR primer sequences. Table 4. Table of PCR primer sequences.

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Paris, A.J., Hayer, K.E., Oved, J.H. et al. STAT3–BDNF–TrkB signalling promotes alveolar epithelial regeneration after lung injury. Nat Cell Biol 22, 1197–1210 (2020). https://doi.org/10.1038/s41556-020-0569-x

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