Evidence for a lipofibroblast-to- Cthrc1 + myofibroblast reversible switch during the development and resolution of lung fibrosis in young mice

Abstract

Background: Fibrosis is often associated with aberrant repair mechanisms that ultimately lead to organ failure. In the lung, idiopathic pulmonary fibrosis (IPF) is a fatal form of interstitial lung disease for which there is currently no curative therapy. From the cell biology point of view, the cell of origin and eventual fate of activated myofibroblasts (aMYFs) have taken centre stage, as these cells are believed to drive structural remodelling and lung function impairment. While aMYFs are now widely believed to originate from alveolar fibroblasts, the heterogeneity and ultimate fate of aMYFs during fibrosis resolution remain elusive. We have shown previously that aMYF dedifferentiation and acquisition of a lipofibroblast (LIF)-like phenotype represent a route of fibrosis resolution.

Methods: In this study, we combined genetic lineage tracing and single-cell transcriptomics in mice, and data mining of human IPF datasets to decipher the heterogeneity of aMYFs and investigate differentiation trajectories during fibrosis resolution. Furthermore, organoid cultures were utilised as a functional readout for the alveolar mesenchymal niche activity during various phases of injury and repair in mice.

Results: Our data demonstrate that aMYFs consist of four subclusters displaying unique pro-alveologenic versus pro-fibrotic profiles. Alveolar fibroblasts displaying a high LIF-like signature largely constitute both the origin and fate of aMYFs during fibrogenesis and resolution, respectively. The heterogeneity of aMYFs is conserved in humans and a significant proportion of human aMYFs displays a high LIF signature.

Conclusion: Our work identifies a subcluster of aMYFs that is potentially relevant for future management of IPF.

Overview of the study.

FIGURE 1

Lineage tracing of Acta2⁺ cells in saline, tamoxifen-bleomycin (Tam-Bleo) and bleomycin-tamoxifen (Bleo-Tam) conditions. a) 8-to-12-week-old female Tg(Acta2-CreERT2)/⁺;tdTomato^flox/flox mice are used to lineage label Acta2⁺ cells in saline, Tam-Bleo and Bleo-Tam conditions. Control and experimental lungs are collected at day 14 following saline or bleomycin administration. b and c) Corresponding low and high magnification of haematoxylin and eosin (H&E) staining showing fibrosis formation at day 14 following bleomycin injury both in Tam-Bleo and Bleo-Tam. d) Corresponding Ashcroft score confirming mild-to-moderate fibrosis formation upon fibrosis injury: saline (0±0 score, n=5), Tam-Bleo (4.8±0.4 score, n=5), Bleo-Tam (5±0.6 score, n=5). e and f) Corresponding low and high magnification of immunofluorescent staining against red fluorescent protein (RFP), α smooth muscle actin (α-SMA/Acta2) and 4′,6-diamidino-2-phenylindole (DAPI), indicating the presence of tdTom⁺ cells in the bronchiolar region as well as in the alveolar region in saline conditions. Note the minor contribution of tdTom⁺ cells in the fibrotic region in Tam-Bleo conditions and the presence of abundant tdTom⁺ cells in the fibrotic region in Bleo-Tam conditions. g) Quantification of the percentage of tdTom⁺/total DAPI in the fibrotic areas of Tam-Bleo (15%±1.5%; n=5) and Bleo-Tam (52.2%±4.5%; n=5). h) Gating strategy to sort lineage-labelled tdTom⁺ cells. i) Representative quantification of sorted tdTom⁺ cells from saline, Tam-Bleo, Bleo-Tam out of resident mesenchymal cells (rMCs). No significant difference (p=0.233) was observed between Tam-Bleo (5.6%±0.4%, n=5) and saline (3.9%±0.3%; n=5). By contrast, there is a stark increase in Bleo-Tam (9.6%±1.6%, n=5) versus Tam-Bleo (5.6%±0.4%, n=5) (p=0.013) or between Bleo-Tam and saline (p=0.003). i.p.: intraperitoneal. Scale bars: b) 500 µm, c) 50 µm, e) 75 µm, f) 10 µm. Statistical analysis was performed using d and i) one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons; g) unpaired two-tailed t-test. *: p<0.05, **: p<0.01, ***: p<0.001.

FIGURE 2

Acta2⁺ cells contribute to multiple lineages in saline and their labelling before bleomycin injury indicate their minimal commitment to the Cthrc1⁺ myofibroblast lineage. a) Integrated uniform manifold approximation and projection (UMAP) of Acta2⁺ cells isolated from saline and tamoxifen-bleomycin (Tam-Bleo) lungs at day 14 showing 18 distinct clusters including alveolar fibroblasts (Alv. F.), peribronchial fibroblasts (Peribr. F), adventitial fibroblasts (Ad. F.) and Cthrc1⁺ myofibroblasts. b) Heatmap showing genes enriched for each cluster. c) Distribution of the different clusters in saline and Tam-Bleo at day 14. Note that the Cthrc1⁺ myofibroblasts already exist in saline and are minimally amplified in Tam-Bleo. The alveolar fibroblasts and adventitial fibroblasts are decreased in Tam-Bleo, while the peribronchial fibroblasts are increased. d) Violin plot for Cthrc1 expression indicates that the Ct2 cluster displays the highest expression level. e) Integrated UMAP of the Cthrc1 clusters from saline and Tam-Bleo lungs. Ct2 represents 45.7% of the overall Cthrc1⁺ cells. Cthrc1⁺ cells are amplified in the context of fibrosis formation. f) Heatmap showing the main differentially expressed genes for each Cthrc1 subclusters. Note that Ct1 contains Limch1, a canonical lipofibroblast (LIF) marker. The signature of this cluster is not significantly impacted by bleomycin. Ct2 in saline is enriched in the Ct1 signature. However, upon bleomycin exposure, this Ct1 signature is lost and an Ct2 signature is increased. The Ct3 signature contains Hhip and Lgr6, markers of peribronchial fibroblasts. The Ct4 cluster, is only observed in the context of Tam-Bleo and displays high levels of the fibrotic marker Spp1. g) Expression of Cthrc1, Limch1, Penk and Spp1 on feature plots. h) Integrated UMAP of the alveolar fibroblasts subclusters from saline and Tam-Bleo lungs. Note that the Al1 represents 63.5% of the total alveolar fibroblasts cluster. i) Heatmap showing the main differentially expressed genes for each alveolar fibroblasts subcluster. Al1 in saline displays both the upper and lower part of the alveolar fibroblast signature. Upon Bleo treatment, the lower part of the signature is decreased and the upper part is increased. Al2 in saline does not express the upper part of the signature. Upon Bleo injury, the upper signature is induced and the lower part is reduced. The lower part of the signature contains Scube2, Ces1d and Npnt. j) Expression of the LIF marker Inmt, Ces1d and the differentially expressed gene Rbp4 on feature plots. k) Expression of the LIF signature in the different Cthrc1 subclusters indicates enrichment in Ct1. This signature is also enriched in Al2 of the alveolar fibroblasts. Prolif.: proliferating; F: fibroblasts; APP: acute phase proteins; IFN: interferon; VSMC: vascular smooth muscle cell; ASMC: airway smooth muscle cell.

FIGURE 3

Acta2⁺ cells captured during fibrosis formation (bleomycin-tamoxifen (Bleo-Tam) condition) massively contribute to the Cthrc1⁺ myofibroblast lineage. a) Integrated uniform manifold approximation and projection (UMAP) of Acta2⁺ cells isolated from saline and Bleo-Tam lungs at day 14 post-bleomycin injury showing 18 distinct clusters including alveolar fibroblasts (Alv. F.), peribronchial fibroblasts (Peribr. F), adventitial fibroblasts (Ad. F.) and Cthrc1⁺ myofibroblasts. b) Heatmap showing genes enriched for each cluster. c) Distribution of the different clusters in saline and Bleo-Tam at day 14. Note that the Cthrc1⁺ myofibroblasts are drastically increased in Bleo-Tam and include the proliferating Cthrc1⁺ cells. The percentage of alveolar fibroblasts is not changed; adventitial fibroblasts are decreased in Bleo-Tam, and the peribronchial fibroblasts are increased. d) Violin plot for Cthrc1 expression indicates that all Cthrc1⁺ fibroblast express high level of Cthrc1 with Ct2 showing the maximum expression. e) Integrated UMAP of the Cthrc1⁺ clusters from saline and Bleo-Tam lungs. Ct2 represents 35.1% of the overall Cthrc1⁺ cells. Cthrc1⁺ cells are amplified in the context of fibrosis formation. f) Heatmap showing the main differentially expressed genes for each Cthrc1 subclusters. Note that Ct1 contains Limch1 and Apoe, two canonical lipofibroblast (LIF) markers. The signature of this cluster is not significantly impacted by bleomycin. Ct2 in saline is enriched in the Ct1 signature. However, upon bleomycin exposure, this Ct1 signature is lost and an Ct2 signature is increased. The Ct4 is only observed in the context of Bleo-Tam. g) Pathway analysis comparing the different Cthrc1⁺ subclusters suggesting that Ct4 represents the most activated cluster. h) Integrated UMAP of the alveolar fibroblast subclusters from saline and Bleo-Tam lungs. Note that the newly formed Al3 represents 40.4% of the total alveolar fibroblasts cluster. i) Heatmap showing the main differentially expressed genes for each alveolar fibroblasts subcluster. Note the presence of new alveolar fibroblast subcluster, Al3, which is characterised by the expression of the fibrotic markers Spp1, Eln, Ltbp2 and Sfrp1. j) Expression of LIF marker Inmt, the differentially expressed gene Rbp4, the fibrotic gene Sfrp1 and the LIF marker Ces1d on feature plots. k) Expression of the LIF signature in the different Cthrc1 subclusters indicates enrichment in Ct1. This signature is also enriched in Al2 of the alveolar fibroblasts. Prolif.: proliferating; F: fibroblasts; APP: acute phase proteins; IFN: interferon; VSMC: vascular smooth muscle cell; ASMC: airway smooth muscle cell.

FIGURE 4

Evidence for lipofibroblast (LIF) to activated myofibroblast (aMYF) differentiation in the human idiopathic pulmonary fibrosis (IPF) lungs. a) Uniform manifold approximation and projection (UMAP) plot for the integration datasets of Habermann et al. [21] and Adams et al. [20] gathering all control and IPF stromal cells. b) Dotplot for the cluster markers of the UMAP of all stromal cells. Colour scale corresponds to the scale gene expression and dot size represents the percentage of expressing cells. c) Characterisation of CTHRC1⁺ cells. UMAP on selected CTHRC1⁺ cells and corresponding expression of CTHRC1, ACTA2, LIMCH1 and APOE. d) The trajectory inferred for the fibroblast subset from IPF lungs was projected onto the UMAP dimension reduction plot (black line). The gradient colour indicates cell pseudotime (red is low, blue is high). e) Ordering of cells by pseudotime. f) Clustered heatmap of the smoothed expression of the 5126 genes significantly associated with pseudotime. A cluster of 401 genes was activated in the second part of the trajectory and is shown in the red frame. g) Heatmap of the cluster of 401 genes. h) Scatter plots for the gene expression measures against cell pseudotime and their respective smoothed expression values. i) Normalised expression across IPF stromal clusters. F.: fibroblast; VSMC: vascular smooth muscle cell; meso: mesothelial cell; Adv. F.: adventitial fibroblasts.

FIGURE 5

Characterisation of the fate of the Cthrc1⁺ clusters during fibrosis resolution. a) Integrated uniform manifold approximation and projection (UMAP) of Acta2⁺ cells isolated from bleomycin-tamoxifen (Bleo-Tam) lungs at days 14 and 60 post-bleomycin injury showing a strong decrease in the Cthrc1⁺ myofibroblasts. b) Heatmap showing genes enriched for each cluster. c) Distribution of the different clusters in Bleo-Tam lungs at day 14 and 60. Note that the Cthrc1⁺ myofibroblasts are drastically reduced at day 60. Ct3 and Ct4 clusters are barely detected at day 60. The percentage of alveolar fibroblasts is modestly increased, while the peribronchial fibroblasts populations are strongly increased. d) Violin plot for Cthrc1 expression. Clusters Ct1 and Ct2 display a strong reduction in Cthrc1 expression at day 60. e) Integrated UMAP of the Cthrc1 clusters from Bleo-Tam lungs at days 14 and 60. Ct2 represents 35.4% of the overall Cthrc1⁺ cells. Cthrc1⁺ cells from each subcluster are massively decreased in the context of fibrosis resolution. f) Heatmap showing the main differentially expressed genes for each Cthrc1⁺ subclusters. Note that Ct1 re-enforces its transcriptomic signature at day 60. Importantly, Ct2 re-acquires the Ct1 signature during fibrosis resolution (green box). Resolution cannot be analysed for Ct3 and Ct4, as these cells are minimally detected at day 60. g) Expression of Cthrc1, Limch1, Penk and Spp1 on feature plots. h) Integrated UMAP of the alveolar fibroblasts subclusters from Bleo-Tam lungs at days 14 and 60. Note that the newly formed Al3 represents 56.5% of the total alveolar fibroblasts cluster. i) Heatmap showing the main differentially expressed genes for each alveolar fibroblast subcluster. Note that Al3 drops its fibrotic markers Sfrp1, Col3a1, Col1a1 and Col5a2. j) Expression of lipofibroblast (LIF) marker Inmt, the differentially expressed gene Rbp4, the fibrotic gene Sfrp1 and the LIF marker Ces1d on feature plots. k) Expression of the LIF signature in the different Cthrc1⁺ subclusters indicates enrichment in Ct1 during fibrosis resolution. This signature is also enriched in Al2 of the alveolar fibroblasts. Prolif.: proliferating; F: fibroblasts; APP: acute phase proteins; IFN: interferon; VSMC: vascular smooth muscle cell; ASMC: airway smooth muscle cell.

FIGURE 6

Evaluation of resident mesenchymal niche activity for alveolar epithelial type 2 cells (AT2s) stem cells during fibrosis formation and resolution. a) 8-to-12-week-old female Tg(Acta2-CreERT2)/⁺;tdTomato^flox/flox mice are used to lineage-label Acta2⁺ cells in saline day 14 and bleomycin-tamoxifen (Bleo-Tam) days 14 and 60. b) Corresponding low and high magnification of haematoxylin and eosin (H&E) staining showing fibrosis formation at day 14 and existence of fibrotic regions at day 60. c) Corresponding low and high magnification of immunofluorescent staining against red fluorescent protein (RFP) (tdTom), α smooth muscle actin (α-SMA/Acta2) and 4′,6-diamidino-2-phenylindole (DAPI), indicating the presence of tdTom⁺ cells in the bronchiolar region as well as in the alveolar region in saline condition at day 14 and the presence of abundant tdTom⁺ cells in the fibrotic region at day 14. Note the presence of tdTom⁺ cells in the fibrotic region at day 60, which suggest that fibrosis is not completely resolved. d) Gating strategy to sort Cd31/Cd45/Epcam triple negative (rMC) positive for Sca1 (rMC Sca1⁺ cells) as well as AT2s (Cd31/Cd45 negative Epcam⁺ Lysotracker⁺). AT2 and rMC Sca1⁺ cells are co-cultured in Matrigel for 14 days (alveolosphere assay). e) Bright-field pictures showing formation of organoids when rMC Sca1⁺ cells are isolated from saline day 14 lungs and co-cultured with AT2s from noninjured C57Bl6 mice. Please note that the mesenchymal niche activity is significantly reduced at the peak of fibrosis (Bleo day 14) and completely lost during fibrosis resolution (Bleo day 30 and day 60). f) Quantification of colony-forming efficiency (CFE) confirming the decrease in stem cell niche activity during fibrosis formation (Bleo day 14 0.77±0.09%, n=3) versus saline day 14 (2.2±0.20%, n=3). Note also the lack of recovery during fibrosis resolution (Bleo day 30 0±0%, n=3 and Bleo day 60 0±0%, n=3). i.p.: intraperitoneal; Scale bars: b) low magnification=500 µm, high magnification=50 µm; c) low magnification=75 µm, high magnification=10 µm; e) low and high magnification=100 µm. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons. *: p<0.05; **: p<0.01; ***: p<0.001.

FIGURE 7

Model of lipofibroblast (LIF) to myofibroblast (MYF) reversible switch during fibrosis formation and resolution. a) The main contributors during fibrosis formation to Cthrc1⁺ activated (a)MYF (Ct1 and Ct2) are Acta2⁻ LIF^high alveolar fibroblasts (Al3) which get activated and express a fibrotic signature and eventually differentiate into Cthrc1^low LIF^high aMYF belonging to the Ct1 cluster. These Ct1 cells further differentiate into Ct2 Cthrc1^high aMYF. During fibrosis resolution, the opposite occurs with Ct2 cells differentiating into Ct1 cells which will eventually give rise to Cthrc1⁻ LIF^high alveolar fibroblasts (Al3 and Al2). b) Pathways modulated in Ct2 cluster during fibrosis formation and resolution. IL: interleukin; sign: signature. Green: pathways upregulated in Ct2 during fibrosis formation. Note that some of these pathways are also upregulated during resolution, albeit at a lower level (lighter shade of green). Red: pathways specifically upregulated in Ct2 during fibrosis resolution.