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Gli1+ mesenchymal stromal cells form a pathological niche to promote airway progenitor metaplasia in the fibrotic lung

Nature Cell Biology volume 22, pages 1295–1306 (2020)Cite this article

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Abstract

Aberrant epithelial reprogramming can induce metaplastic differentiation at sites of tissue injury that culminates in transformed barriers composed of scar and metaplastic epithelium. While the plasticity of epithelial stem cells is well characterized, the identity and role of the niche has not been delineated in metaplasia. Here, we show that Gli1+ mesenchymal stromal cells (MSCs), previously shown to contribute to myofibroblasts during scarring, promote metaplastic differentiation of airway progenitors into KRT5+ basal cells. During fibrotic repair, Gli1+ MSCs integrate hedgehog activation signalling to upregulate BMP antagonism in the progenitor niche that promotes metaplasia. Restoring the balance towards BMP activation attenuated metaplastic KRT5+ differentiation while promoting adaptive alveolar differentiation into SFTPC+ epithelium. Finally, fibrotic human lungs demonstrate altered BMP activation in the metaplastic epithelium. These findings show that Gli1+ MSCs integrate hedgehog signalling as a rheostat to control BMP activation in the progenitor niche to determine regenerative outcome in fibrosis.

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Fig. 1: Gli1+ mesenchymal cells promote metaplastic KRT5 differentiation in vivo.
Fig. 2: Gli1+ mesenchymal cells provide a specialized niche for airway progenitor proliferation and metaplastic differentiation in vitro.
Fig. 3: Mesenchymal Hh activation promotes metaplastic KRT5 differentiation while suppressing SFTPC differentiation from airway.
Fig. 4: Single-cell transcriptome analysis of Gli1 Lin+ mesenchyme reveals upregulated BMP antagonism in the fibrotic niche.
Fig. 5: Hh activation upregulates BMP antagonism in the fibrotic lung.
Fig. 6: BMP activation attenuates metaplastic airway progenitor differentiation.
Fig. 7: IPF lungs display altered BMP activation in the epithelium.

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Data availability

The mouse RNA-seq data reported in this paper (Fig. 4) are deposited in the NCBI Gene Expression Omnibus (GEO) under the accession number GSE140032. The human RNA-seq data reported in this paper (Fig. 7) are deposited in the NCBI GEO under the accession number GSE132771. Previously published sequencing data from the Tabula Muris cellular database were referenced here and are publicly available at https://tabula-muris.ds.czbiohub.org/. There are no restrictions on data availability. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

This study does not include any custom code.

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Acknowledgements

We thank K. N. Cheong, A. Brumwell, A. Baldwin and N. Reyes de Mochel for providing technical assistance; C. Gralapp for assistance with model illustration; M. Looney for critical review of manuscript; the Parnassus Flow Cytometry Core for assistance with cell sorting for bulk and single-cell RNA analysis (P30DK063720); members of the Biological Imaging Development Core (P30 DK063720); and E. Wan and the Institute for Human Genetics Core for the processing of single-cell RNA samples and high-throughput sequencing. This work is supported by NIH grants DP2AG056034, K08HL121146 and R01HL142552 to T.P., along with a Tobacco Related Disease Research Program New Investigator Award and a Pulmonary Hypertension Association award to T.P. and a Nina Ireland Program Award to M.M. for human lung collection.

Author information

Authors and Affiliations

  1. Department of Medicine, Division of Pulmonary and Critical Care Medicine, Cardiovascular Research Institute, San Francisco, CA, USA

    Monica Cassandras, Chaoqun Wang, Jaymin Kathiriya, Tatsuya Tsukui, Michael Matthay, Paul Wolters, Dean Sheppard, Hal Chapman & Tien Peng

  2. Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA

    Peri Matatia & Ari Molofsky

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  1. Monica Cassandras
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Contributions

M.C. and T.P. conceived the experiments. M.C., C.W., J.K., T.T., P.M., M.M. and P.W. performed the experiments and data analyses. D.S., A.M. and H.C. provided expertise and feedback. M.C. and T.P. wrote the manuscript.

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Correspondence to Tien Peng.

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Extended data

Extended Data Fig. 1 Epithelial progenitors expand distally in fibrotic repair.

(a, b) Time course showing appearance of metaplastic airway epithelium in the alveoli after 1, 2, 3 or 4 doses of weekly bleomycin injury. (n = 3 per group; each data point represents one animal). Data are expressed as mean ± SD. (c, d) Contribution of Sox2 Lin+ cells to the various epithelial lineages in the alveoli after bleomycin injury. (n = 6 per group; each data point represents one animal; one-tailed unpaired Student’s t-test). Data are expressed as mean ± SD. (e) Lack of Sox2 Lin+ cells in corn oil-treated SoxcreERT2:R26mTmG lung. (2 animals). (f) Co-localization of Sox2 Lin+ cells and SOX2 staining. (n = 3 animals). Data are expressed as mean ± SD. (g) Extended washout after tamoxifen (>4wks) followed by bleomycin demonstrates similar expansion of Sox2 Lin+ cells into the alveoli (3 animals). AW = airway. Scale bars, 100 μm.

Source data

Extended Data Fig. 2 Lineage tracing of diverse airway progenitors after bleomycin injury.

(a, b) p63 Lin+ cells also expanded into the alveoli after bleomycin but did not contribute significantly to KRT5 + basal cells in the alveoli. (n = 3 animals). Data are expressed as mean ± SD. (c, d) Comparison of p63 Lin+ and Sox2 Lin+ cells that expand into the alveoli after bleomycin injury. (n = 3 for p63 Lin, n = 6 for Sox2 Lin; each data point represents one animal; one-tailed unpaired Student’s t-test). Data are expressed as mean ± SD. (e, f) Contribution of Scgb1a1 Lin+ cells to the various epithelial lineages in the alveoli after bleomycin injury. (n = 3 per group; each data point represents one animal). Data are expressed as mean ± SD. AW = airway. Scale bars, 100 μm.

Source data

Extended Data Fig. 3 Gli1 Lin + mesenchyme expands distally in fibrotic repair.

(a) Lack of Gli1 Lin+ cells in corn oil-treated Gli1creERT2:R26YFP lung (2 animals). (b, c) Comparison of Gli1 Lin+ cell localization with Gli1 + cells in Gli1LacZ/+ lung. These experiments were each repeated three times independently with similar results. (d, e) Extended washout after tamoxifen (>4wks) followed by bleomycin demonstrates similar expansion of Gli1 Lin+ cells into the alveoli. (n = 3 per group; each data point represents one animal; one-tailed unpaired Student’s t- test). Data are expressed as mean ± SD. (f, g) Deletion of Gli1 + cells does not alter airway epithelial composition during normal homeostasis. (n = 3 per group; each data point represents one animal; one-tailed unpaired Student’s t-test). Data are expressed as mean ± SD. (h–j) Gli1-deleted lungs treated with bleomycin demonstrate reduced expression of Gli1 along with changes in fibrotic markers (n = 6 for control, n = 5 for Gli1- deleted; each data point represents one animal; for (i), Col1a1 P = 0.0862; Snai1 P = 0.3126; Cdh2 P = 0.0310; one-tailed unpaired Student’s t-test for (h–j)). Data are expressed as mean ± SD. (k) Weight loss after bleomycin injury in control and Gli1- deleted animals (n = 6 for control, n = 5 for Gli1-deleted; each data point represents one animal; one-tailed unpaired Student’s t-test). Data are expressed as mean ± SD. AW = airway. Scale bars, 100 μm.

Source data

Extended Data Fig. 4 ITGB4 + airway progenitors give rise to KRT5 + and SFTPC + cells that are not from pre- existing Krt5 + and Sftpc + cells.

(a) Gating strategy for isolation of ITGB4 + airway progenitors and Gli1 + mesenchymal cells by FACS, presented in Fig. 2a, e; 3 f; 5e; 6 f and Extended Data Fig. 4d, h; 5h; 8g. (b) Cytospin of freshly sorted ITGB4 + confirms that majority of cells are SOX2 + , SCGB1A1 + , and TubIV + , with cells rarely positive for SFTPC, KRT5, p63, or NGFR (n = 1 for NGFR, n = 3 for rest; each data point represents one biological replicate). Data are expressed as mean ± SD. (b) Z-axis scan shows that Gli1 Lin+ mesenchyme settle to bottom of the Matrigel well. (d–g) Lineage trace of pre-existing Krt5 Lin + (tdTomato + ) cells followed by isolation of lung for ITGB4 + airway progenitor organoid culture with Gli1 + cells. Tracheal Krt5 Lin+ cells demonstrate expression of basal cell markers KRT5 and p63 (e), while few Krt5 Lin+ cells appear in ITGB4 + cell-derived organoid isolated from the lung of the same animal (f, g). The overwhelming majority of KRT5 + cells in the ITGB4-derived organoids are Krt5 Lin-negative. (n = 3 wells for (f, g); n = 1 sample for (e)). Data are expressed as mean ± SD. (h–j) Lineage trace of pre-existing Sftpc Lin + (membrane GFP + ) cells followed by isolation of the lung epithelium for organoid culture with Gli1 + cells. ITGB4 + (red box) or Sftpc Lin + (green box) cells were sorted from the same lung. The overwhelming majority of SFTPC + organoids in the ITGB4- derived organoids are Sftpc Lin-negative, while most SFTPC + cells in the Sftpc Lin+ derived organoids are Lin + (n= 3; each data point represents one well). Data are expressed as mean ± SD. Scale bars, 100 μm.

Source data

Extended Data Fig. 5 Characterization of Hh- activation during homeostasis and in fibrotic repair.

(a) RNAScope in situ of Shh shows expression in ectopic SOX2 + cells in the alveoli after bleomycin injury. This experiment was repeated independently twice with similar results. (b, c) Histology quantification shows no evidence of KRT5 + metaplasia in Hh-activated animals without injury when compared to controls (n = 3 per group; each data point represents one animal; one-tailed unpaired Student’s t- test). Data are expressed as mean ± SD. (d–g) After bleomycin injury, Hh-activated lungs show similar expansion of Gli1 + cells in distal alveoli compared to controls, a trend towards increased myofibroblasts (SMA + ) differentiation of the Gli1 Lin+ cells in the alveoli, and no difference in ectopic SCGB1A1 + cells in the alveoli (for (e), n = 4 for control, n = 6 for Hh-activated; for (f, g), n = 9 per group; each data point represents one animal; one-tailed unpaired Student’s t-test for (e–g)). Data are expressed as mean ± SD. (h) Model of 3D airway organoid assay using Hh-inducible mesenchyme whereby pretreatment of 4- hydroxytamoxifen (4OHT) induces Hh activation in the mesenchyme as shown by upregulation of Gli1 transcript compared to vehicle (ethanol) (n = 3 per group; each data point represents one biological replicate; one-tailed unpaired Student’s t-test). (i–l) Mesenchymal Hh activation in vitro reduces CFE and organoid size, but increases the expression of Krt5 and number of KRT5 + organoids while reducing Sftpc expression and number of SFTPC + organoids ((i–k) n = 3 per group; (l) n = 3 for Hh-activated, n = 5 for control; each data point represents one well; one-tailed unpaired Student’s t-test for (j–l)). Data are expressed as mean ± SD. Hh = hedgehog, 4OHT = 4-hydroxytamoxifen, AW = airway. Scale bars, 100 μm.

Source data

Extended Data Fig. 6 Expansion of Gli1 Lin + mesenchyme into the alveoli.

(a) Multi-colour stochastic recombination of Gli1 Lin+ cells tagged with one of four possible fluorescent proteins (GFP, YFP, RFP, or CFP) shows clones of single color clustering around the airway in PBS treated lungs, and clones of single color spanning the airway and alveoli following bleomycin injury (4 animals per group) (b) Violin plots showing retention of Gli1 expression in Gli1 Lin+ cells following bleomycin injury as they migrate into the alveoli. Each black dot represents one cell (n values listed in source data for Fig. 4d). AW = airway. Scale bars, 100 μm.

Extended Data Fig. 7 Spatial regulation of BMP activation.

(a, b) pSMAD1/5/8 staining of Sox2 Lin+ cells in the alveoli after bleomycin injury demonstrates that the majority of pSMAD1/5/8 + /Sox2 Lin+ cells are SCGB1A1 + or SFTPC + , and rarely KRT5 + (n = 3 for KRT5 quantification, n = 4 for SCGB1a1 and SFTPC quantification; each data point represents one animal). Data are expressed as mean ± SD. (c) qPCR of Hh-inducible cultured mesenchyme isolated from UbccreERT2/+:R26SmoM2/+ lungs treated with 4OHT + /-TGF-β, showing effect on various BMP antagonists expression (n = 3; each data point represents one biological replicate). Wnt5a: Control v TGF-β P = 0.0094, Control v Hh-activated P = 0.1066, Control v TGF-β + Hh-activated P= 0.002; Fst: Control v TGF-β P = 0.0598, Control v Hh- activated P = 0.0389, Control v TGF-β + Hh-activated P = 0.3833; Fstl1: Control v TGF-β P = 0.2051, Control v Hh- activated P = 0.4620, Control v TGF-β + Hh-activated P = 0.028; Fstl3: Control v TGF-β P = 0.0005, Control v Hh- activated P = 0.0704, Control v TGF-β + Hh-activated P = 0.0003; Grem1: Control v TGF-β P = 0.4845, Control v Hh- activated P = 0.2422, Control v TGF-β + Hh-activated P = 0.1417; Fzd1: Control v TGF-β P = 0.3341, Control v Hh- activated P = 0.2689, Control v TGF-β + Hh-activated P = 0.3320; Sfrp1: Control v TGF-β P = 0.4103, Control v Hh- activated P = 0.4614, Control v TGF-β + Hh-activated P = 0.1093; ordinary one-way ANOVA test. Data are expressed as mean ± SD. Scale bars, 100 μm.

Source data

Extended Data Fig. 8 Effect of rhBMP4 in vivo and in vitro.

(a–d) Effect of rhBMP4 treatment on fibrotic marker expression, myofibroblasts in the alveoli, weight change, and presence of ectopic SCGB1A1 + cells, in bleomycin injured C57BL/6 animals (n = 8 per group; each data point represents one animal). For (a), Col1a1 P = 0.0015; Snai1 P= 0.0769; Cdh2 P = 0.1771; Acta2 P = 0.0934; Tagln P = 0.3782.; one-tailed unpaired Student’s t-test for (a–d). Data are expressed as mean ± SD. (e) Effect of rhBMP4 on the expression of markers of chondrogenesis, adipogenesis, and osteogenesis in the lung (n = 8 per group; each data point represents one animal). Acan P = 0.0021; Col2a1 P = 0.2363; Col10a1 P = 0.4201; Plin2 P = 0.0578; Pparg P = 0.4343; Fabp4 P = 0.0006; Spp1 P = 0.3117; Alpl P = 0.0474; Ibsp P = 0.3371; Runx2 P < 0.0001; one-tailed unpaired Student’s t-test. Data are expressed as mean ± SD. (f) Effect of rhBMP4 on the total number of Sox2 Lin+ cells in the alveoli after bleomycin injury (n = 4 per group; each data point represents one animal; one-tailed unpaired Student’s t-test). Data are expressed as mean ± SD. (g–i) Airway progenitor organoid co-cultured with Gli1 + mesenchyme treated with BMP4 demonstrates increased CFE and enhanced Sftpc expression with reduced Krt5 (n = 4 for solvent, n = 3 for BMP4; each data point represents one well; one-tailed unpaired Student’s t-test for (h, i)). Conversely, the BMP antagonist GREM2 acts to suppress CFE and Sftpc expression while enhancing Krt5 (n = 4 per group; each datapoint represents one well; one-tailed unpaired Student’s t-test for (h,i)). Data are expressed as mean ± SD.

Source data

Extended Data Fig. 9 Metaplastic KRT5 + basal cells and SMA + fibroblastic foci in the alveoli of IPF lungs.

(a) UMAP projection of mesenchyme from each individual donor that was used in single cell RNA-seq analysis with contributing cell number. (b) Expression of basal cell markers NGFR, p63, and KRT14, in KRT5 + basal cells in human IPF, along with mouse bleomycin injured lungs and murine airway progenitor- derived organoids. This experiment was repeated three times independently with similar results. (c) Ectopic expression of KRT5 and SMA in the alveoli of IPF lungs form honeycomb cysts and fibroblast foci, respectively. This experiment was repeated independently twice with similar results. (d) Average cell-to-cell distance shows equivalent proximity of metaplastic KRT5 and endogenous SFTPC cells to SMA + fibroblastic foci (n = 3,280 KRT5 cells, n = 2,215 SFTPC cells; each datapoint represents an individual distance measurement with all measurements plotted for 3 samples; one-tailed unpaired Student’s t-test). (e, f) Unsorted epithelial organoids derived from dissociated IPF lungs grew KRT5 + organoids, and BMP4 attenuated the number and size of IPF-derived organoids. (n = 5; each data point represents one well; one-tailed unpaired Student’s t-test) Data are expressed as mean ± SD. Scale bars, 100 μm.

Source data

Extended Data Fig. 10 Gli1 + mesenchyme modulates SOX2 + progenitor fate.

Model of Gli1 + mesenchyme integrating Hh activation as a rheostat that controls local BMP activation to determine SOX2 + airway epithelial progenitor fate.

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Supplementary Table 1

List of qPCR primers (mouse) used in this study.

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Statistical source data for histological quantifications.

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Statistical source data for histological quantifications.

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Statistical source data for histological quantifications and arterial oxygen measurement raw data.

Source Data Fig. 4

Statistical source data for cluster information and Gene Ontology raw data.

Source Data Fig. 5

Statistical source data for histological quantifications and qPCR.

Source Data Fig. 6

Statistical source data for histological quantifications, arterial oxygen measurements and qPCR.

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Gene Ontology raw data and statistical source data for histological quantifications.

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Statistical source data for histological quantifications.

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Statistical source data for histological quantifications.

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Statistical source data for histological quantifications, qPCR and weight change.

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Statistical source data for histological quantifications.

Source Data Extended Data Fig. 5

Statistical source data for histological quantifications and qPCR.

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Statistical source data for histological quantifications and qPCR.

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Statistical source data for histological quantifications, qPCR and weight change.

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Statistical source data for histological quantifications.

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Cassandras, M., Wang, C., Kathiriya, J. et al. Gli1+ mesenchymal stromal cells form a pathological niche to promote airway progenitor metaplasia in the fibrotic lung. Nat Cell Biol 22, 1295–1306 (2020). https://doi.org/10.1038/s41556-020-00591-9

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