Airway-derived emphysema-specific alveolar type II cells exhibit impaired regenerative potential in COPD

Abstract

Emphysema, the progressive destruction of gas exchange surfaces in the lungs, is a hallmark of COPD that is presently incurable. This therapeutic gap is largely due to a poor understanding of potential drivers of impaired tissue regeneration, such as abnormal lung epithelial progenitor cells, including alveolar type II (ATII) and airway club cells. We discovered an emphysema-specific subpopulation of ATII cells located in enlarged distal alveolar sacs, termed asATII cells. Single-cell RNA sequencing and in situ localisation revealed that asATII cells co-express the alveolar marker surfactant protein C and the club cell marker secretaglobin-3A2 (SCGB3A2). A similar ATII subpopulation derived from club cells was also identified in mouse COPD models using lineage labelling. Human and mouse ATII subpopulations formed 80-90% fewer alveolar organoids than healthy controls, indicating reduced progenitor function. Targeting asATII cells or their progenitor club cells could reveal novel COPD treatment strategies.

Main findings of the study. In human emphysema and mouse models, club cells give rise to an emphysema-specific alveolar type II (ATII) cell subpopulation in enlarged alveolar sacs, characterised by impaired regenerative function. scRNAseq: single-cell RNA sequencing; RASC: respiratory airway secretory cell.

FIGURE 1

Alveolar epithelial cell heterogeneity in COPD. a) Sample information of single-cell RNA sequencing (scRNAseq) data. b) Schematic showing workflow of sampling parenchymal tissue from emphysematous (n=6) and healthy (n=4) lungs, viable epithelial cell isolation and scRNAseq using the 10X Genomics platform. Uniform manifold approximation and projection (UMAP) shows cells obtained from COPD patients and healthy donors. c) UMAP visualisation of annotated lung epithelial cell types. d) The cellular composition of epithelial cells from individual COPD patients and healthy donors. e) The numbers of genes upregulated (log₂ fold change (fc) >1, p<0.05) and downregulated (log₂fc <−1, p<0.05) in each cell type from COPD patients, compared to those of healthy donor. f) UMAP visualisation of alveolar type II (ATII) cells marked by expression of surfactant protein C (SFTPC). g) UMAP showing ATII cells from emphysema patients and healthy donors. h) Feature plots showing expression of secretoglobins SCGB3A2, SCGB3A1 and SCGB1A1 in ATII cells. NA: not applicable; GOLD: Global Initiative for Chronic Obstructive Lung Disease; MACS: magnetic-activated cell sorting; FACS: fluorescence-activated cell sorting; EpCAM: epithelial cell adhesion molecule; NEC: nonepithelial cells; DEGs: differentially expressed genes.

FIGURE 2

Identification of secretaglobin (SCGB^pos) alveolar type II (ATII) cell subpopulations in human COPD lungs. a) Haematoxylin and eosin (H&E) staining of emphysematous parenchymal lung tissue from COPD patient at low magnification. Scale bar=200 µm. Within thin alveolar sacs (inset), fluorescent RNAScope for SCGB3A2 (red), 1A1 (green), 3A1 (green) and immunofluorescence (IF) staining for surfactant protein C (SPC) (white) identifies 3A2⁺ ATII cells (arrowheads). Scale bars=40 µm. b) H&E staining of respiratory bronchiole region (inset) in COPD lung tissue. Scale bar=100 µm. Multiplex fluorescence RNAScope for SCGB3A2 (red), 1A1 (green), 3A1 (green), and co-IF for SPC (white) identifies ATII cells co-expressing multiple SCGB genes (arrowheads) in this region. Scale bars=40 µm. c) Quantification of percentages of 3A2⁺ and 1A1⁺/3A1⁺/3A2⁺ ATII cells detected by RNAScope and IF in alveolar sacs of healthy (n=5) and emphysematous (n=9) lung tissue. d) Uniform manifold approximation and projection (UMAP) showing annotation of ATII subclusters based on expressions of SCGB genes and tissue localisations. ATII cells dominantly presenting in alveolar sacs of COPD samples and expressing SCGB3A2 were identified as asATII. ATII cells expressing multiple SCGBs and localised in respiratory bronchioles were annotated as rbATII. The remaining of ATII cells, dominantly presented in healthy samples, were marked as hATII. e) Percentages of ATII subclusters in healthy and COPD samples. f) Dot plot of selected senescence marker genes in asATII, rbATII and hATII cells. g) Dot plot of selected marker genes identified in asATII and rbATII cells. ns: nonsignificant. ***: p<0.001. ns: p>0.05, Mann–Whitney test.

FIGURE 3

Club cells give rise to emphysema-specific alveolar type II (ATII) cell subpopulations in human and mouse COPD lungs. a) Principal curve representing lineage between non-mucin club cells and alveolar sac (as)ATII cells using Slingshot, and partition-based graph abstraction (PAGA) analysis of asATII, respiratory bronchiole (rb)ATII, and nonmucin club cells from COPD lungs. Arrows mark the predicted trajectory from nonmucin club cells to asATII cells. b) Data from a) are merged with respiratory airway secretory cells (RASCs) and secretory/goblet (Sec/Gob) cells published by Basil et al. [16] to perform Slingshot analysis. c) Experimental schematics of lineage tracing of club cells in the porcine pancreatic elastase (PPE)-induced mouse emphysema model. d) Uniform manifold approximation and projection (UMAP) visualisation and annotation of sorted tdTomato⁺ epithelial cells from saline- (n=2) and PPE- (n=3) treated Scgb1a1-CreERT;Rosa-tdTomato mice, respectively. e) Percentage of ATII-6 subcluster cells in tdTomato⁺ ATII cells in (d). f) Similarity measurement of human (h)ATII subpopulations and mouse ATII subclusters based on expression of common markers genes. Higher matchSCore indicates higher similarity. g) Scatter plots showing marker genes driving similarity between human asATII cells and mouse ATII-6 in (f). h) Scatter plot showing a lack of common marker gene in human hATII and mouse ATII-5 cells. scRNAseq: single-cell RNA sequencing.

FIGURE 4

Identification of emphysema-specific alveolar type II (ATII) cells in a porcine pancreatic elastase (PPE)-induced mouse COPD model. a) Representative images of organoids from tdTomato⁺ (red) epithelial cells of saline- and PPE-treated Scgb1a1-CreERT;Rosa-tdTomato mice on day 21 post-treatment. Scale bar=1000 µm. b) Quantification of alveolar organoids in c). Saline-treated mice (n=4), PPE-treated mice (n=5). Welch's t-test. c) Feature plot showing lymphocyte antigen 6 complex i (Ly6i) as a unique marker gene of mouse ATII-6 cells. d) Detection of ATII-6 cells in alveolar sacs of emphysematous region (black square in left picture with haematoxylin and eosin staining) by co-immunofluorescence for Ly6i (right, green), tdTomato (right, red), and surfactant protein C (SPC) (right, white). Scale bar=20 µm. e) Detection of tdTomato⁺/Sca1⁻/Ly6i⁺ ATII cells in viable epithelial cells (see gating strategy in supplementary figure S7a) from PPE-treated mouse lungs by flow cytometry. f) Percentages of Ly6i⁺ cells in the tdTomato⁺/Sca1⁻ and tdTomato⁻/Sca1⁻ ATII cells from mice treated with saline or PPE on days 21 and 28 after treatment. Mann–Whitney test. g) Representative pictures of organoids from fluorescence-activated cell sorted ATII-6 (tdTomato⁺/Sca1⁻/Ly6i⁺, left) and the rest (tdTomato⁺/Sca1⁻/Ly6i⁻, middle) ATII cells from PPE-treated mice at day 21 post-treatment and ATII (tdTomato⁺/Sca1⁻) cells from saline-treated mice. Whole-mount immunofluorescence staining for pro-SPC (green) is used to identify alveolar organoids. Endogenous tdTomato is in red. 4′,6-Diamidino-2-phenylindole (DAPI) in blue shows nuclei. Scale bar=1000 µm. h) Quantification of alveolar (SPC⁺) organoid-forming efficiency in g). ****: p<0.0001, Mann–Whitney test.

FIGURE 5

Decreased progenitor cell function of emphysema-specific alveolar type II (ATII) cells in human COPD lungs. a) Detection of asATII cells in alveolar sacs of emphysematous region (black square in left picture with haematoxylin and eosin (H&E) staining) by co-immunofluorescence (co-IF) for CD74 (green), secretaglobin-3A2 (SCGB3A2) (red), and surfactant protein C (SPC) (white). Scale bars=200 µm, 50 µm and 10 µm. b) Detection of rbATII cells in respiratory bronchiole of emphysematous region (black square in left picture with H&E staining) by co-IF for intercellular adhesion molecule 1 (ICAM1) (green, top), TM4SF1 (green, bottom), SCGB3A2 (red), and SPC (white). Scale bars=50 µm, 20 µm and 10 µm. c) Flow cytometry analysis of asATII and rbATII using CD74 (centre), TM4SF1 and ICAM1 (right) in healthy control sample (n=6, top) and emphysematous parenchymal tissue from COPD patients (n=6, bottom). d) Quantification of the percentages of asATII (CD74⁺) and rbATII (ICAM1⁺/TM4SF1⁺) cells in total ATII cells (HTII280⁺/epithelial cell adhesion molecule (EPCAM)⁺/4′,6-diamidino-2-phenylindole (DAPI)⁻) in a). Mann–Whitney test. e) Organoids formed by fluorescence-activated cell sorted asATII (HTII280⁺/CD74⁺) and rbATII (HTII280⁺/ICAM1⁺/TM4SF1) cells from human emphysematous parenchymal tissue (n=5) and by ATII cells (HTII280⁺) from healthy control (n=3). Scale bar=100 µm. f) Quantification of organoid forming efficiency of c). Values are presented as mean±sem. Mann–Whitney test. *: p<0.05.