A Single-Cell Atlas of Small Airway Disease in Chronic Obstructive Pulmonary Disease: A Cross-Sectional Study.

Rationale: Emerging data demonstrate that the smallest conducting airways, terminal bronchioles, are the early site of tissue destruction in chronic obstructive pulmonary disease (COPD) and are reduced by as much as 41% by the time someone is diagnosed with mild (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 1) COPD. Objectives: To develop a single-cell atlas that describes the structural, cellular, and extracellular matrix alterations underlying terminal bronchiole loss in COPD. Methods: This cross-sectional study of 262 lung samples derived from 34 ex-smokers with normal lung function (n = 10) or GOLD stage 1 (n = 10), stage 2 (n = 8), or stage 4 (n = 6) COPD was performed to assess the morphology, extracellular matrix, single-cell atlas, and genes associated with terminal bronchiole reduction using stereology, micro-computed tomography, nonlinear optical microscopy, imaging mass spectrometry, and transcriptomics. Measurements and Main Results: The lumen area of terminal bronchioles progressively narrows with COPD severity as a result of the loss of elastin fibers within alveolar attachments, which was observed before microscopic emphysematous tissue destruction in GOLD stage 1 and 2 COPD. The single-cell atlas of terminal bronchioles in COPD demonstrated M1-like macrophages and neutrophils located within alveolar attachments and associated with the pathobiology of elastin fiber loss, whereas adaptive immune cells (naive, CD4, and CD8 T cells, and B cells) are associated with terminal bronchiole wall remodeling. Terminal bronchiole pathology was associated with the upregulation of genes involved in innate and adaptive immune responses, the interferon response, and the degranulation of neutrophils. Conclusions: This comprehensive single-cell atlas highlights terminal bronchiole alveolar attachments as the initial site of tissue destruction in centrilobular emphysema and an attractive target for disease modification.

Second harmonic generation imaging of collagen scaffolds within the alveolar ducts of healthy and emphysematous mouse lungs.

The alveolar ducts are connected to peripheral septal fibers which extend from the visceral pleura into interlobular septa, and are anchored to axial fibers in the small airways. Together these axial and septal fibers constitute a fiber continuum that provides tension and integrity throughout the lung. Building on the observations that alveolar ducts associated with sub-pleural alveoli are orientated perpendicular to the visceral pleura, and in parallel to each other, the goal of the present study was to investigate the nature of the collagen fiber organization within sub-pleural alveolar ducts in healthy control and elastase-induced emphysema murine lungs. Employing three-dimensional second harmonic generation imaging, the structural arrangement of fibrilar collagen fibers could be visualized in cleared murine lungs. In healthy control lungs, fibrilar collagen fibers within alveolar mouths formed the coiled collagen structure within the alveolar duct. In the elastase-treated emphysema lungs, there was loss of fibrilar collagen fibers (p < 0.01) and disruption of collagens structural organization as measured by the fibrillar collagen length (p < 0.01) and entropy (p < 0.01). Compared to the alveolar ducts from healthy controls, there was a significant increase in the area of cells (nm2, p < 0.001), and area of cells with cytoplasmic granules (nm2, p < 0.001) compared to emphysematous lungs. These results are consistent with the idea that one of the major contributors to the progressive loss of alveolar surfaces and elastic recoil in the emphysematous lung is loss of the structural integrity of the collagen scaffold that maintains the spatial relationships important for cell survival and lung function.

Airway remodeling heterogeneity in asthma and its relationship to disease outcomes.

Asthma affects an estimated 262 million people worldwide and caused over 461,000 deaths in 2019. The disease is characterized by chronic airway inflammation, reversible bronchoconstriction, and airway remodeling. Longitudinal studies have shown that current treatments for asthma (inhaled bronchodilators and corticosteroids) can reduce the frequency of exacerbations, but do not modify disease outcomes over time. Further, longitudinal studies in children to adulthood have shown that these treatments do not improve asthma severity or fixed airflow obstruction over time. In asthma, fixed airflow obstruction is caused by remodeling of the airway wall, but such airway remodeling also significantly contributes to airway closure during bronchoconstriction in acute asthmatic episodes. The goal of the current review is to understand what is known about the heterogeneity of airway remodeling in asthma and how this contributes to the disease process. We provide an overview of the existing knowledge on airway remodeling features observed in asthma, including loss of epithelial integrity, mucous cell metaplasia, extracellular matrix remodeling in both the airways and vessels, angiogenesis, and increased smooth muscle mass. While such studies have provided extensive knowledge on different aspects of airway remodeling, they have relied on biopsy sampling or pathological assessment of lungs from fatal asthma patients, which have limitations for understanding airway heterogeneity and the entire asthma syndrome. To further understand the heterogeneity of airway remodeling in asthma, we highlight the potential of in vivo imaging tools such as computed tomography and magnetic resonance imaging. Such volumetric imaging tools provide the opportunity to assess the heterogeneity of airway remodeling within the whole lung and have led to the novel identification of heterogenous gas trapping and mucus plugging as important predictors of patient outcomes. Lastly, we summarize the current knowledge of modification of airway remodeling with available asthma therapeutics to highlight the need for future studies that use in vivo imaging tools to assess airway remodeling outcomes.

The contribution of reticular basement membrane proteins to basal airway epithelial attachment, spreading and barrier formation: implications for airway remodeling in asthma.

Rationale: In the healthy lung, the pseudostratified conducting airway epithelium is anchored to the reticular basement membrane (RBM) via hemidesmosome junction complexes formed between basal cells and the extracellular matrix (ECM). The RBM within the healthy lung is composed of the ECM proteins laminin and collagen-IV. In patients with asthma, the RBM is remodeled with collagen-I, -III and fibronectin deposition. The goal of this study was to assess the effect of RBM ECM proteins on basal airway epithelial cell attachment, spreading and barrier formation using real-time electrical cell-substrate impedance sensing (ECIS). Methods: ECIS 8-well arrays were coated with 50 µg/mL of fibronectin, collagen-I, collagen-III, collagen-IV, or laminin and compared to bovine serum albumin (BSA) or uncoated controls. The airway epithelial cell line (1HAEo-) was seeded 40, 50, 60, and 70 k cells/well and continuously monitored over 70 h to assess cell attachment, spreading and barrier formation using high (64 k Hz) and low (500 Hz) frequency resistance and capacitance. Data were analyzed using a one-phase decay model from which half-life (time cells cover half of the electrode area) and rate-constant (cell-spreading rate/h) were determined and a generalized additive mixed effect model (GAMM) was used to assess ECM proteins over the entire experiment. Results: High-frequency (64 kHz) capacitance measures demonstrated the half-life for 1HAEo-cells to attach was fastest when grown on fibronectin (6.5 h), followed by collagen-I (7.2 h) and collagen-III (8.1 h), compared to collagen-IV (11.3 h), then laminin (13.2 h) compared to BSA (12.4 h) and uncoated (13.9 h) controls. High-frequency (64 kHz) resistance measures demonstrated that the rate of 1HAEo- cell spreading was significantly faster on fibronectin and collagen-I compared to collagen-III, collagen-IV, laminin, BSA and the uncoated control. Low-frequency (500 Hz) resistance measures demonstrated that 1HAEo-cells formed a functional barrier fastest when grown on fibronectin and collagen-I, compared to the other ECM conditions. Lastly, the distance of 1HAEo-cells from the ECM substrates was the smallest when grown on fibronectin reflecting high cell-matrix adhesion. Conclusion: Airway epithelial cells attach, spread and form a barrier fastest on fibronectin, and collagen-I and these reticular basement membrane ECM proteins may play a protective role in preserving the epithelial barrier during airway remodeling in asthma.

The Role of the Dynamic Lung Extracellular Matrix Environment on Fibroblast Morphology and Inflammation.

The extracellular matrix (ECM) supports lung tissue architecture and physiology by providing mechanical stability and elastic recoil. Over the last several decades, it has become increasingly clear that the stiffness of the ECM governs many cellular processes, including cell-phenotype and functions during development, healing, and disease. Of all the lung ECM proteins, collagen-I is the most abundant and provides tensile strength. In many fibrotic lung diseases, the expression of collagen is increased which affects the stiffness of the surrounding environment. The goal of this study was to assess the effect on fibroblast morphology, cell death, and inflammation when exposed to 2D and 3D low (0.4 mg/mL) versus high (2.0 mg/mL) collagen-I-matrix environments that model the mechanics of the breathing lung. This study demonstrates that human fetal lung fibroblasts (HFL1), grown in a 3D collagen type-I environment compared to a 2D one, do not form cells with a myofibroblast morphology, express less F-actin stress fibers, exhibit less cell death, and significantly produce less pro-inflammatory IL-6 and IL-8 cytokines. Exposure to mechanical strain to mimic breathing (0.2 Hz) led to the loss of HFL1 fibroblast dendritic extensions as well as F-actin stress fibers within the cell cytoskeleton, but did not influence cytokine production or cell death. This dynamic assay gives researchers the ability to consider the assessment of the mechanodynamic nature of the lung ECM environment in disease-relevant models and the potential of mechano-pharmacology to identify therapeutic targets for treatment.

The Role of miRNAs in Extracellular Matrix Repair and Chronic Fibrotic Lung Diseases.

The lung extracellular matrix (ECM) plays a key role in the normal architecture of the lung, from embryonic lung development to mechanical stability and elastic recoil of the breathing adult lung. The lung ECM can modulate the biophysical environment of cells through ECM stiffness, porosity, topography and insolubility. In a reciprocal interaction, lung ECM dynamics result from the synthesis, degradation and organization of ECM components by the surrounding structural and immune cells. Repeated lung injury and repair can trigger a vicious cycle of aberrant ECM protein deposition, accompanied by elevated ECM stiffness, which has a lasting effect on cell and tissue function. The processes governing the resolution of injury repair are regulated by several pathways; however, in chronic lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary disease (IPF) these processes are compromised, resulting in impaired cell function and ECM remodeling. Current estimates show that more than 60% of the human coding transcripts are regulated by miRNAs. miRNAs are small non-coding RNAs that regulate gene expressions and modulate cellular functions. This review is focused on the current knowledge of miRNAs in regulating ECM synthesis, degradation and topography by cells and their dysregulation in asthma, COPD and IPF.

Epithelial-interleukin-1 inhibits collagen formation by airway fibroblasts: Implications for asthma.

In asthma, the airway epithelium has an impaired capacity to differentiate and plays a key role in the development of airway inflammation and remodeling through mediator release. The study objective was to investigate the release of (IL)-1 family members from primary airway epithelial-cells during differentiation, and how they affect primary airway fibroblast (PAF)-induced inflammation, extracellular matrix (ECM) production, and collagen I remodeling. The release of IL-1α/ß and IL-33 during airway epithelial differentiation was assessed over 20-days using air-liquid interface cultures. The effect of IL-1 family cytokines on airway fibroblasts grown on collagen-coated well-plates and 3-dimensional collagen gels was assessed by measurement of inflammatory mediators and ECM proteins by ELISA and western blot, as well as collagen fiber formation using non-linear optical microscopy after 24-hours. The production of IL-1α is elevated in undifferentiated asthmatic-PAECs compared to controls. IL-1α/ß induced fibroblast pro-inflammatory responses (CXCL8/IL-8, IL-6, TSLP, GM-CSF) and suppressed ECM-production (collagen, fibronectin, periostin) and the cell's ability to repair and remodel fibrillar collagen I via LOX, LOXL1 and LOXL2 activity, as confirmed by inhibition with ß-aminopropionitrile. These data support a role for epithelial-derived-IL-1 in the dysregulated repair of the asthmatic-EMTU and provides new insights into the contribution of airway fibroblasts in inflammation and airway remodeling in asthma.

Small airways disease in mild and moderate chronic obstructive pulmonary disease: a cross-sectional study.

BACKGROUND: The concept that small conducting airways less than 2 mm in diameter become the major site of airflow obstruction in chronic obstructive pulmonary disease (COPD) is well established in the scientific literature, and the last generation of small conducting airways, terminal bronchioles, are known to be destroyed in patients with very severe COPD. We aimed to determine whether destruction of the terminal and transitional bronchioles (the first generation of respiratory airways) occurs before, or in parallel with, emphysematous tissue destruction. METHODS: In this cross-sectional analysis, we applied a novel multiresolution CT imaging protocol to tissue samples obtained using a systematic uniform sampling method to obtain representative unbiased samples of the whole lung or lobe of smokers with normal lung function (controls) and patients with mild COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 1), moderate COPD (GOLD 2), or very severe COPD (GOLD 4). Patients with GOLD 1 or GOLD 2 COPD and smokers with normal lung function had undergone lobectomy and pneumonectomy, and patients with GOLD 4 COPD had undergone lung transplantation. Lung tissue samples were used for stereological assessment of the number and morphology of terminal and transitional bronchioles, airspace size (mean linear intercept), and alveolar surface area. FINDINGS: Of the 34 patients included in this study, ten were controls (smokers with normal lung function), ten patients had GOLD 1 COPD, eight had GOLD 2 COPD, and six had GOLD 4 COPD with centrilobular emphysema. The 34 lung specimens provided 262 lung samples. Compared with control smokers, the number of terminal bronchioles decreased by 40% in patients with GOLD 1 COPD (p=0·014) and 43% in patients with GOLD 2 COPD (p=0·036), the number of transitional bronchioles decreased by 56% in patients with GOLD 1 COPD (p=0·0001) and 59% in patients with GOLD 2 COPD (p=0·0001), and alveolar surface area decreased by 33% in patients with GOLD 1 COPD (p=0·019) and 45% in patients with GOLD 2 COPD (p=0·0021). These pathological changes were found to correlate with lung function decline. We also showed significant loss of terminal and transitional bronchioles in lung samples from patients with GOLD 1 or GOLD 2 COPD that had a normal alveolar surface area. Remaining small airways were found to have thickened walls and narrowed lumens, which become more obstructed with increasing COPD GOLD stage. INTERPRETATION: These data show that small airways disease is a pathological feature in mild and moderate COPD. Importantly, this study emphasises that early intervention for disease modification might be required by patients with mild or moderate COPD. FUNDING: Canadian Institutes of Health Research.