Small airway fibroblasts from patients with chronic obstructive pulmonary disease exhibit cellular senescence

Abstract

Small airway disease (SAD) is a key early-stage pathology of chronic obstructive pulmonary disease (COPD). COPD is associated with cellular senescence whereby cells undergo growth arrest and express the senescence-associated secretory phenotype (SASP) leading to chronic inflammation and tissue remodeling. Parenchymal-derived fibroblasts have been shown to display senescent properties in COPD, however small airway fibroblasts (SAFs) have not been investigated. Therefore, this study investigated the role of these cells in COPD and their potential contribution to SAD. To investigate the senescent and fibrotic phenotype of SAF in COPD, SAFs were isolated from nonsmoker, smoker, and COPD lung resection tissue (n = 9-17 donors). Senescence and fibrotic marker expressions were determined using iCELLigence (proliferation), qPCR, Seahorse assay, and ELISAs. COPD SAFs were further enriched for senescent cells using FACSAria Fusion based on cell size and autofluorescence (10% largest/autofluorescent vs. 10% smallest/nonautofluorescent). The phenotype of the senescence-enriched population was investigated using RNA sequencing and pathway analysis. Markers of senescence were observed in COPD SAFs, including senescence-associated β-galactosidase, SASP release, and reduced proliferation. Because the pathways driving this phenotype were unclear, we used cell sorting to enrich senescent COPD SAFs. This population displayed increased p21^CIP1 and p16^INK4a expression and mitochondrial dysfunction. RNA sequencing suggested these senescent cells express genes involved in oxidative stress response, fibrosis, and mitochondrial dysfunction pathways. These data suggest COPD SAFs are senescent and may be associated with fibrotic properties and mitochondrial dysfunction. Further understanding of cellular senescence in SAFs may lead to potential therapies to limit SAD progression.NEW & NOTEWORTHY Fibroblasts and senescence are thought to play key roles in the pathogenesis of small airway disease and COPD; however, the characteristics of small airway-derived fibroblasts are not well explored. In this study we isolate and enrich the senescent small airway-derived fibroblast (SAF) population from COPD lungs and explore the pathways driving this phenotype using bulk RNA-seq.

Keywords: COPD; fibroblast; senescence; small airway disease.

Graphical abstract

Figure 1.

Expression of fibrotic and senescence markers in fibroblasts derived from small airway and parenchymal tissue from matched donors. Fibroblasts were grown from airway and parenchymal lung tissue of the same donors (n = 18 donors). Samples isolated from COPD lungs are shown in red and control in black. Gene expression of senescence markers CDKN2A (p16) and CDKN1A (p21) (A) and fibrotic markers COL1A1, COL3A1, and TGFβ1 (B) was assessed using qPCR in parenchymal (●) and small airway fibroblasts (■). Data were analyzed using a Wilcoxon test: **P < 0.01. COPD, chronic obstructive pulmonary disease; PF, parenchymal fibroblast; SAF, small airway fibroblast.

Figure 2.

Cellular senescence markers in COPD SAFs. A: representative images of SA-β-Gal staining and quantification for percent positive SAF for SA-β-Gal NS (●), S (▴), and COPD (■) SAFs (n = 4–6 donors). B: proliferation rate of NS, S, and COPD SAFs as measured by iCELLigence technology (n = 4–7 donors). C: representative flow cytometry plots of propidium iodide staining and quantification for stage of cell cycle arrest (n = 5–7 donors). D: representative image of zymography gelatin gel and quantification for pro and active-MMP2 (n = 5 donors). E: release of SASP markers PAI-1, IL-6, and CXCL8 in SAF (n = 4–7 donors). Data are represented as individual data points and/or means ± SE. iCELLigence data were analyzed using a repeated-measures two-way ANOVA. Cell cycle data were analyzed using a Mann–Whitney U test. Comparisons between NS, S, and COPD groups are with a Kruskall–Wallis and Dunn’s post hoc analysis. *P < 0.05, **P < 0.01 compared with NS. #P < 0.05, ##P < 0.01 compared with S. Scale bars are 100 µm. COPD, chronic obstructive pulmonary disease; MMP2 S, matrix metalloproteinase-2 standard; NS, nonsmoker; S, smoker; SAF, small airway fibroblast; SA-β-Gal, senescence-associated β-galactosidase; SASP, senescence-associated secretory phenotype.

Figure 3.

Change in fibroblast cell size and autofluorescence in senescence and gating strategy for enrichment via FACS. Fibroblasts from nonsmokers were treated with 500 µM H₂O₂ or untreated for 48 h. H₂O₂ induced expression of senescence markers. A: representative images of senescence-associated β-galactosidase staining. B: representative Western and quantification of p21 expression after H₂O₂ treatment (n = 4 donors). C: representative flow cytometry plots from vehicle (water) and H₂O₂-treated cells gated into “non-senescent” and “senescent” by their size and autofluorescence in the FITC channel. D: quantification of the change in the percentage of cells in the “non-senescent” and “senescent” gates with H₂O₂ treatment (72 h). E: sorting strategy to enrich for senescent COPD fibroblasts based on cell size and autofluorescence. Cells are gated to exclude debris and sorted into 10% smallest and least autofluorescence (FSC^lowFITC^low) and 10% largest and most autofluorescence (FSC^highFITC^high). F: after sorting, cells were checked for sorting accuracy by recording 5,000 events. Representative images showing differences in cell size (G) and autofluorescence (H) post sort. Data are represented as individual data points and/or means ± SE. Western blot data were analyzed using a repeated-measures two-way ANOVA and changes in FACS populations were analyzed using a Wilcoxon test. *P < 0.05 and **P < 0.01 compared with vehicle. Scale bars are 100 µm. COPD, chronic obstructive pulmonary disease; H₂O₂, hydrogen peroxide; SAF, small airway fibroblast.

Figure 4.

Expression of senescence markers in enriched senescent COPD SAF. SAF from COPD donors were sorted using FACS and sorted into 10% smallest and least autoflourescence (FSC^lowFITC^low, ●) and 10% largest and most autofluorescence (FSC^highFITC^high, ■). Sorted populations were assessed for senescence markers. A: shows staining for senescence-associated-β-galactosidase. B: proliferation rate of sorted populations using iCELLigence. Samples were also collected for qPCR, where the expression of antiaging protein SIRT1 (C) and cell cycle inhibitors (D) CDKN2B (p15), CDKN2A (p16), and CDKN2A (p21) was assessed (n = 6 donors). E: protein expression of p21^CIP1 and p16^INK4a in sorted COPD SAF (n = 8 donors). F: gene expression of SASP markers IL-6 and CXCL8. Data are represented as individual data points. Data were analyzed using a Wilcoxon test. *P < 0.05, **P < 0.01. Scale bars are 100 μm. COPD, chronic obstructive pulmonary disease; NS, nonsenescent (FSC^lowFITC^low); SAF, small airway fibroblast; SE, senescence-enriched (FSC^highFITC^high).

Figure 5.

Mitochondrial function of senescent-enriched COPD SAFs. OCR (oxidative phosphorylation measure) and ECAR (glycolysis) traces from Cell Mito stress test in NS (●) and COPD (■) SAF (A) and FSC^lowFITC^low (●) and FSC^highFITC^high (■) (B) sorted COPD SAFs. C: measurements of mitochondrial function parameters: basal respiration, maximal respiration, proton leak and ATP production in sorted COPD SAF populations. D: plot from ATP rate Seahorse assay showing distribution of ATP from glycolysis and oxidative phosphorylation (n = 6 donors) in sorted COPD SAF populations. E: mitochondrial ROS production in sorted COPD SAF populations (n = 5 donors). Data are represented as individual data points and/or means ± SE. Data are analyzed using a Wilcoxon test or a Friedman test with Dunn’s post hoc analysis. *P < 0.05. ATP, adenosine triphosphate; COPD, chronic obstructive pulmonary disease; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; O, oligomycin; OCR, oxygen consumption rate; R/A, rotenone and antimycin; SAF, small airway fibroblast.

Figure 6.

Altered pathways in senescence-enriched COPD SAF. Samples from FSC^lowFITC^low (●) and FSC^highFITC^high (■) sorted COPD SAF were analyzed by RNA sequencing (n = 6 donors). A: volcano plot of differentially expressed genes in sorted senescence-enriched populations. Labeled are some of the top genes most upregulated and downregulated in senescence-enriched SAF. Horizontal dashed line shows Padj = 0.05. Vertical dashed lines show a fold change of 1.5 (n = 6 donors). B: summary of top 5 upregulated and downregulated genes based on Log2 fold change. C: ingenuity pathway analysis of significantly enriched canonical pathways based on differentially expressed genes from RNA-seq data (n = 6 donors). D: enrichment for previously published gene sets altered in senescence in this RNA dataset. E: differentially expressed genes involved in the fibrosis pathway enrichment. F: expression of fibrosis-related genes TGFβ1, COL1A1, and COL3A1 in sorted COPD SAF (n = 6 donors). Data are represented as individual data points and/or mean. Data were analyzed using a Wilcoxon test. *P < 0.05. COPD, chronic obstructive pulmonary disease; SAF, small airway fibroblast.