Background:A smaller relative volume of the peripheral pulmonary vasculature in asthma, termed "vascular pruning", has been linked to greater disease severity, exacerbations, and airflow obstruction. It is not known whether this is reversible. Therefore, the study objective was to determine whether "loss" of computed tomography (CT) measures of peripheral pulmonary vasculature is a modifiable feature of asthma pathophysiology by assessing its response to dupilumab. Methods:27 adults with moderate-to-severe asthma received dupilumab (n=19) or placebo (n=8) every 2 weeks for 16 weeks. Chest CT scans acquired at baseline and follow-up were analysed to quantify pulmonary blood vessel volumes categorised by cross-sectional area (≤5 mm2 as BV5 and ≥10 mm2 as BV10). These volumes were reported relative to the total blood volume (TBV) as BV5% and BV10%. Change in blood vessel volumes from baseline to follow-up were evaluated and correlated with changes in patient-reported outcomes, type-2 inflammatory biomarkers and measurements of airway structure and function. Results:Following dupilumab treatment (but not placebo), BV5% increased by 1.0% (95% CI -0.2% to 4.5%, p=0.02) and BV10% decreased by 1.0% (95% CI -3.6% to 0.7%, p=0.03). The between-group difference in median change was 3.0% for BV5% (95% CI 0.6% to 5.5%; p=0.008) and -3.0% for BV10% (95% CI -5.2% to -1.0%; p=0.007). In dupilumab-treated participants, the increase in BV5% and decrease in BV10% was associated with improved Asthma Control Questionnaire-5, forced expiratory volume in 1 s, fraction of exhaled nitric oxide and sputum eosinophil percentage (all p<0.05). Conclusion:A redistribution of pulmonary blood volume toward smaller vessels was observed following dupilumab, suggesting that CT-derived pulmonary vascular measurements may be modifiable in asthma.

Background Chronic lung allograft dysfunction (CLAD) is the leading cause of late mortality after lung transplantation. Bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS) are the main underlying clinical entities. Their molecular and cellular signatures are unclear and, therefore, we aimed to identify molecular programmes associated with morphological disease severity in CLAD. Methods We performed high-resolution imaging-based gene expression profiling of 128 lung samples from explanted CLAD and donor lungs, using weighted gene co-expression network analysis, cellular and pathway enrichment, and hub gene identification. Findings were validated across four datasets, including a murine transplant model, human BOS lungs, transbronchial biopsies, and bronchoalveolar lavage fluid of lung transplant recipients. Results Unsupervised clustering revealed two transcriptomic CLAD endotypes aligning with mild-fibrotic (BOS, mild RAS) and advanced-fibrotic disease (moderate/severe RAS). Samples from the same patient often diverged molecularly, underscoring intra-patient heterogeneity and limitations of current phenotypical classification. Five molecular programmes emerged: (1) epithelial stress and innate immunity in early-fibrotic CLAD, (2) progressive adaptive immunity and cytotoxicity in advanced CLAD, (3) transient extracellular matrix remodelling, (4) progressive endothelial loss/dysfunction, and (5) progressive loss of homeostasis, wherein multiple potential druggable targets were detected. Finally, we identified a 26 CLAD hub gene-panel, that showed robust diagnostic performance to discriminate CLAD. Conclusion CLAD is a spatially heterogeneous, yet molecularly continuous disease process, wherein BOS and RAS represent variable stages of a shared immunopathological continuum. Our findings support the development of lung-specific molecular classifiers to guide diagnostics and reveal novel targets for personalised therapies in transplantation.

Background: Small airway disease (SAD) is a defining feature of chronic obstructive pulmonary disease (COPD), but its functional consequences across the whole bronchial tree remain incompletely quantified. We determined how progressive SAD alters lung airflow dynamics during inspiration and expiration, and whether simplified models reproduce the results of anatomically realistic 3D simulations. Methods: Three lung explants, healthy control, moderate (GOLD II), and end-stage (GOLD IV) COPD, were imaged by micro-CT and segmented from the main-bronchus to the small airways. Inspiratory and expiratory computational fluid dynamics (CFD) simulations used matched main-bronchus-flow and terminal-pressure scenarios. A linear Poiseuille model on the same anatomy benchmarked simplified airway representations. Findings: Airways (0·5–2·5 mm) decreased by 22% in GOLD II and 71% in GOLD IV relative to control. At matched inspiratory flow, whole-lung resistance rose from 0·33 (control) to 0·58 (GOLD II) and 2·15 cm H₂O·s/L (GOLD IV). Expiratory resistance exceeded inspiratory resistance by 19% (control), 38% (GOLD II), and 79% (GOLD IV), an asymmetry that widened with severity. Wall shear stress in GOLD IV rose 8-fold during inspiration and 5-fold during expiration vs control. Simplified linear models underestimated CFD-derived driving pressure 3·8- to 6·8-fold and overestimated expiratory outflow 12·6- to 14·3-fold. Interpretation: Whole-lung CFD revealed a severity-dependent airflow burden in COPD that is amplified during expiration and strongly underestimated by linear flow models and simplified airway geometries. These findings provide a quantitative bridge between distal airway pathology and expiratory flow limitation, and underline the need for anatomy-based 3D modelling when assessing SAD.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic injury and oxidative stress leading to progressive lung tissue destruction. Emerging evidence suggests that regulated cell death pathways, particularly ferroptosis, contribute to COPD pathology. We previously identified decreased expression of the stress response protein and known ferroptosis inhibitor nuclear protein 1 (NUPR1) in alveolar type 2 cells from COPD patients. Here, we demonstrate that NUPR1 inhibition exacerbates iron accumulation, enhances lipid peroxidation, impairs mitochondrial function, disrupts cellular metabolism, and increases oxidative stress in lung epithelial cells. Furthermore, Nupr1 -/- mice exhibit mitochondrial abnormalities, increased oxidative damage, and lung tissue changes consistent with emphysema. Collectively, our findings establish NUPR1 as a critical regulator of ferroptosis, stress responses, mitochondrial integrity, as well as metabolic balance in the lung and suggest reduced NUPR1 contributes to COPD pathogenesis.

Age is a major risk factor for lung disease. We characterized the changing cellular, transcriptional, and genomic landscape of human lung aging using single-cell RNA sequencing. We find that lung aging is cell-type dyssynchronous, with alveolar epithelial and endothelial cells exhibiting the greatest transcriptional changes. Among alveolar epithelial cells, aging is associated with a decreased relative proportion of surfactant-expressing SPChigh AT2 cells. Among alveolar capillary cells, we observed loss of differentiation and capillary function. Analysis of somatic mutations called from single-cell data revealed an increase with aging, with alveolar epithelial and endothelial cell types exhibiting greater mutation burdens. Transcriptional entropy was increased with aging and was an independent predictor of age. Notably, cells expressing commonly accepted senescence signatures did not increase with age. Our results reveal cell type dyssynchrony in human lung aging with age-related changes concentrated in alveolar epithelial and endothelial cells. The changing cellular, transcriptional, and genomic landscape of human lung aging can be characterized using single-cell RNA sequencing. Here, the authors show that lung aging is cell-type dyssynchronous, with alveolar epithelial and endothelial cells exhibiting the greatest changes in gene expression, transcriptional entropy, and a high level of somatic mutations.

Tissue repair requires a highly coordinated cellular response to injury. In the lung, alveolar type 2 cells (AT2s) act as stem cells to replenish both themselves and alveolar type 1 cells (AT1s); however, the complex orchestration of stem cell activity after injury is poorly understood. Here, we establish longitudinal imaging of AT2s in murine intact tissues ex vivo and in vivo in order to track their dynamic behavior over time. We discover that a large fraction of AT2s become motile following injury and provide direct evidence for their migration between alveolar units. High-resolution morphokinetic mapping of AT2s further uncovers the emergence of distinct motile phenotypes. Inhibition of AT2 migration via genetic depletion of ArpC3 leads to impaired regeneration of AT2s and AT1s in vivo. Together, our results establish a requirement for stem cell migration between alveolar units and identify properties of stem cell motility at high cellular resolution.

Rationale: Cystic fibrosis (CF) is characterized by bronchiectasis on imaging, while functionally evolving toward obstructive impairment. Despite its assumed importance in CF, small airway remodeling and its relation to bronchiectasis remains poorly understood. Objectives: The aim of our study was to explore both large and small airway disease morphometrically, by using detailed imaging techniques, such as ex vivo high-resolution computed tomography (HRCT) and micro-computed tomography (μCT), and histological analysis in advanced CF. Methods: On HRCT (600 μm; CF, n = 21; control, n = 6) and μCT (150 μm; CF, n = 3; control, n = 1) scans of inflated explanted lungs, the ratio of visible airway volume to total lung volume (AV%) was calculated as a marker of bronchiectasis, while airway segmentation was used for generation analysis. Clinical data were retrospectively collected. On μCT (8.5 μm) images of lung cores (±2.8 cm3), extracted randomly from each lobe (three per lobe), distal airway (DA) diameter, number of airway collapses, and number of open terminal bronchioles per milliliter were analyzed. Morphometric analysis was supplemented with histological analysis of DA collapse. Results: AV% on HRCT was heterogeneous among CF lungs (0.7-4.6%), overlapping with controls (0.4-1.2%). However, the pattern of airway loss on μCT was homogeneous among CF lungs and most pronounced from generations 9-16. AV% did not correlate with the number of open terminal bronchioles per milliliter or percentage predicted forced expiratory volume in 1 second, which correlated with each other. Open DAs in CF lungs were narrowed compared with DA in controls. On the other hand, collapsed DAs in CF lungs showed varying degrees of proximal dilation, with DA diameter correlating with AV%. On histology, collapsed CF DAs showed constrictive bronchiolitis. Conclusions: Airway remodeling in end-stage CF is heterogeneous, ranging from minimal bronchiectasis, overlapping with control lungs, to extensive bronchiectasis with small airway dilation. However, the degree of bronchiectasis is unrelated to functional impairment or the amount of small airway loss, underscoring the importance of small airway disease.

In this research, we delve into the association between epigenetic aging and idiopathic pulmonary fibrosis (IPF), a debilitating lung disease that progresses over time. Utilizing the Illumina MethylationEPIC array, we assessed DNA methylation levels in donated human lung tissue from patients with IPF, categorizing the disease into mild, moderate, and severe stages based on clinical assessments. We used seven epigenetic clocks to determine age acceleration, which is the discrepancy between biological (epigenetic) and chronological age. Our findings revealed a notable acceleration of biological aging in IPF tissues compared with healthy controls, with four clocks-Horvath's, Hannum's, PhenoAge, and DunedinPACE-showing significant correlations. DunedinPACE, in particular, indicated a more rapid aging process in the more severe regions within the lungs of IPF cases. These results suggest that the biological aging process in IPF is expedited and closely tied to the severity of the disease. The study underscores the potential of DNA methylation as a biomarker for IPF, providing valuable insights into the underlying methylation patterns and the dynamics of epigenetic aging in affected lung tissue. This research supports the broader application of epigenetic clocks in clinical prognosis and highlights the critical role of biological age in the context of medical research and healthcare.NEW & NOTEWORTHY Using epigenetic clocks, we found a notable acceleration of biological aging in IPF tissues, particularly in DunedinPACE, suggesting that the biological aging process in IPF is accelerated and closely related to the severity of the disease. The study also underscores DNA methylation's potential as a biomarker for IPF, as well as the dynamics of epigenetic aging and the need to consider biological age in medical research and healthcare.

Acute and repetitive lung epithelial injury can lead to irreversible and even progressive pulmonary fibrosis; Idiopathic pulmonary fibrosis (IPF) is a fatal disease and quintessential example of this phenomenon. The composition of epithelial cells in human pulmonary fibrosis - irrespective of disease etiology - is marked by the presence of Aberrant Basaloid cells: an abnormal cell phenotype with pro-fibrotic and senescent features, localized to the surface of fibrotic lesions. Despite their relevance to human pulmonary fibrosis, the exotic molecular profile of Aberrant Basaloid cells has obscured their etiology, preventing insights into how or why these cells emerge with fibrosis. Here we identify cellular intermediaries between Aberrant Basaloid and normal alveolar epithelial cells in human IPF tissue. We track the emergence of Aberrant Basaloid cells from alveolar epithelial cells ex vivo and uncover a role for similar cells in epithelial regeneration under normal conditions. Lastly, we characterize the epigenetic changes that distinguish Aberrant Basaloid cells from their progenitors and identify hallmarks of AP-1 injury memory retention. This study elucidates the phenomenon of maladaptive epithelial plasticity and regeneration in pulmonary fibrosis and re-contextualizes therapeutic strategies for epithelial dysfunction.

BACKGROUND:Small airways (<2 mm diameter) are major sites of airflow obstruction in chronic obstructive pulmonary disease (COPD). This study aimed to quantify the impact of small airway disease, characterized by narrowing, occlusion, and obliteration, on airflow parameters in smokers and end-stage patients with COPDs. METHODS:We performed computational fluid dynamics (CFD) simulations of inspiratory airflow in three lung groups: control non-used donor lungs (no smoking/emphysema history), non-used donor lungs with a smoking history and emphysema, and explanted end-stage COPD lungs. Each group included four lungs, with two tissue cylinders. Micro-CT-scanned small airways were segmented into 3D models for CFD simulations to quantify pressure, resistance, and shear stress. CFD results were benchmarked against simplified linear and Weibel models. FINDINGS:CFD simulations showed higher pressures in COPD vs. controls (p = 0.0091) and smokers (p = 0.015), along with increased resistance (p = 0.0057 vs. controls; p = 0.0083 vs. smokers) and up to a tenfold rise in shear stress (p = 0.010 vs. controls). Narrowing and occlusion were shown to independently increase pressure, resistance, and shear stress, which were validated through segmentation corrections. Pressures and resistance assessed with simplified models were up to seven-fold higher for smokers and even 72 higher for COPD compared with CFD values. INTERPRETATION:These findings show that increased airflow parameters can explain the association between small airway disease and airflow limitation in COPD, underscoring small airway vulnerability. Additionally, they highlight the limitations of theoretical models in accurately capturing small airway disease. FUNDING:Supported by the KU Leuven (C16/19/005).

Rationale: The precise nature of small airway obstructions in chronic obstructive pulmonary disease (COPD) remains poorly understood, especially at early disease stages. Objectives: This study aimed to characterize small airway obstructions and numbers up to the terminal bronchioles (TBs) in smokers with limited emphysema and end-stage COPD. We hypothesized that obstruction subtypes would differ in morphology, nature, and number from early to end-stage COPD. Methods: Whole lungs were inflated and processed from seven control donors (control: declined for extrapulmonary reasons); from eight donors with a history of smoking, of whom three had <5% emphysema (smokers with no emphysema) and five had >5% emphysema (smokers with emphysema); and from eight patients with end-stage COPD. Micro-computed tomography of tissue was used to assess number of TBs, aerated TBs, and number and type of obstructions and was cross-correlated with histopathology. Measurements and Main Results: Obstructions were mainly present in smokers with emphysema and patients with COPD, resulting in less aerated TBs. On the basis of emphysema extent, more nonaerated TBs were present in regions with no emphysema than in regions with mild emphysema; however, destruction was more prominent in mild emphysema. Multiple types of obstructions were identified, comprising occlusions, webs, and collapses. In smokers with emphysema, obstructions primarily comprised webs and occlusions, whereas all obstruction types were present in COPD. On histopathology, obstructions were identified as mucus plugs. Conclusions: Multiple types of obstruction characterized as mucus plugs were identified in smokers with emphysema and patients with end-stage COPD. Their morphology, nature, and number evolved from smokers with emphysema to end-stage COPD. A shift from obstruction-dominant dysfunction to destruction-dominant pathology was found in smokers on the basis of emphysema presence.

RATIONALE: Age is a major risk factor for lung disease. Accumulation of somatic mutations has been implicated in both aging and cellular senescence. Somatic mutations can affect transcriptional stability by mechanisms including affecting gene-regulatory networks and generating aberrant transcripts. We sought to examine the role of somatic mutations in the development of age-related lung dysfunction. METHODS: Single-cell RNAseq was performed on lung tissue cores from healthy donors (32 samples, 11-72 years, 21M/11F). Following standard pre-processing and cell type annotation, mutations were called using SComatic with the default parameters. Mutation burden was calculated by dividing the number of mutations by the number of callable sites. We calculated transcriptional entropy as a means to quantify loss of cell differentiation or phenotype. This was modeled using K-L divergence, with raw count data for each cell being compared to a uniform reference distribution. RESULTS: Our scRNAseq dataset consisted of 199,400 cells, which were clustered annotated to 25 distinct cell types. Mutation burden was positively correlated with age (r=0.28, p<0.001). Globally, the top genes correlated with mutation burden included ubiquitin ligase genes (AMBRA1, ANAPC1, SEL1L, USP25, USP33) and DNA damage response genes (RAD50, PRKDC). Notably, mutation burden also correlated with expression of senescence marker CDKN2A (r=0.48, p<0.05). In capillary endothelial cells, mutation burden was correlated with decreased expression of cell type differentiation markers (Il7R, VIPR1, FCN3), and mitochondrial genes. Similarly, in AT2 cells, mutation burden was associated with reduced expression of differentiation markers (SFTPC) and increased expression of chromatin organization (H2AFJ, ACTL6A) and damage repair genes (MACROD1, RAD17, BCCIP). Transcriptional entropy was increased with aging in most cell types, except for lymphocytes and myeloid cell types [Fig A]. The greatest age-associated difference in transcriptional entropy occurred in gCap cells (p<0.05) and AT2 cells (p<0.05). Finally, transcriptional entropy was strongly positively correlated with mutation burden (r=0.51, p<0.05) [Fig B]. CONCLUSIONS: Our analysis suggests that somatic mutation accumulation is associated with heterogenous transcriptional changes in different cell types, including loss of differentiation markers and increased transcriptional entropy. This process may contribute to phenotypic changes that predispose to age-related lung disease.

Human diseases are characterized by intricate cellular dynamics. Single-cell transcriptomics provides critical insights, yet a persistent gap remains in computational tools for detailed disease progression analysis and targeted in silico drug interventions. Here we introduce UNAGI, a deep generative neural network tailored to analyse time-series single-cell transcriptomic data. This tool captures the complex cellular dynamics underlying disease progression, enhancing drug perturbation modelling and screening. When applied to a dataset from patients with idiopathic pulmonary fibrosis, UNAGI learns disease-informed cell embeddings that sharpen our understanding of disease progression, leading to the identification of potential therapeutic drug candidates. Validation using proteomics reveals the accuracy of UNAGI's cellular dynamics analysis, and the use of the fibrotic cocktail-treated human precision-cut lung slices confirms UNAGI's predictions that nifedipine, an antihypertensive drug, may have anti-fibrotic effects on human tissues. UNAGI's versatility extends to other diseases, including COVID, demonstrating adaptability and confirming its broader applicability in decoding complex cellular dynamics beyond idiopathic pulmonary fibrosis, amplifying its use in the quest for therapeutic solutions across diverse pathological landscapes.

Purpose: Computational fluid dynamics (CFD) enables quantitative measurements of fluid-flow phenomena based on the conservation laws (conservation of mass, momentum, and energy) governing fluid motion. We applied CFD for the first time in a pilot study on human explant lungs for modeling the airflow and its impact throughout the airways in bronchiolitis obliterans syndrome (BOS) and healthy control lungs. We aimed to demonstrate its usefulness in identifying regions of airflow alteration (stagnation, turbulence), and excess pressure in relation with remodeled airways in BOS.

*Corresponding author. Department of Chronic Diseases, Metabolism and ageing (CHROMETA), Laboratory of Respiratory Diseases and Thoracic Surgery (BREATHE), KU Leuven and University Hospitals Leuven, Herestraat 49, B-3000, Leuven, Belgium.

Rationale: G-protein coupled receptor 87 (GPR87), an alternative lysophosphatidic acid (LPA) receptor previously implicated in cancer, is highly expressed in basal and aberrant basaloid cells in idiopathic pulmonary fibrosis (IPF). We sought to determine whether signaling through GPR87 is important to the development of pulmonary fibrosis. Methods: Reanalysis of bulk and single cell RNA sequencing dataset was performed to confirm the increased expression of GPR87 in pulmonary fibrosis. The role of GPR87 in fibrosis in-vivo was assessed using global GPR87 knockout (GPR87-/-) and wildtype mice in the bleomycin model of pulmonary fibrosis, in-vitro in induced pluripotent stem cells (iPSCs) derived airway basal cells (iBC) using GPR87 siRNAs, and ex-vivo in human precision cut slices using disease free tissues in the fibrotic cocktail model as well as IPF tissues treated with GPR87 siRNA. Results: GPR87 is highly expressed in IPF lungs, and its expression correlates with disease severity. Furthermore, It is highly expressed in basal and aberrant basaloid cells. GPR87-/- mice are protected against bleomycin induced pulmonary fibrosis. GPR87 knockdown is protective against fibrosis development in normal PCLS treated with fibrotic cocktail and leads to fibrosis regression in IPF PCLS. In iBC, GPR87 knockdown leads to decreased expression of fibrosis related genes, proteins and microRNAs. GPR87 stimulation with LPA leads to the opposite results. The main downstream pathways are PI3K, mTOR, and TNF/NFkB; stimulation or inhibition of PI3K pathway mimics GPR87 stimulation or inhibition responses, respectively. Conclusion: GPR87 is highly expressed in basal and aberrant basaloid cells in IPF lungs and seems to mediate profibrotic effects based on in-vivo, ex-vivo and in-vitro models of disease, suggesting that it should be studied as a potential epithelial specific therapeutic target in pulmonary fibrosis. ### Competing Interest Statement The authors have declared no competing interest.

Age prediction based on single cell RNA-Sequencing data (scRNA-Seq) can provide information for patients’ susceptibility to various diseases and conditions. In addition, such analysis can be used to identify aging related genes and pathways. To enable age prediction based on scRNA-Seq data, we developed PolyEN, a new regression model which learns continuous representation for expression over time. These representations are then used by PolyEN to integrate genes to predict an age. Existing and new lung aging data we profiled demonstrated PolyEN’s improved performance over existing methods for age prediction. Our results identified lung epithelial cells as the most significant predictors for non-smokers while lung endothelial cells led to the best chronological age prediction results for smokers.

Small airway disease including obstruction is an important contributor to the decline in lung function observed in COPD. However, the mechanisms underlying small airway disease are not fully understood. We investigated the fluid dynamic alterations resulting from small airway deformation and obstruction in the lower airway zones up to the functional terminal bronchioles (TB) using computational fluid dynamics (CFD). Small cylinders (1.4cm diameter) obtained from explanted donor (n=1) and COPD (n=1) lungs frozen at TLC were µCT scanned (resolution 10 µm). Samples were matched for number of generations and segments. Small airways were segmented and modeled for CFD simulation. Under the same inlet flow rate and outlet pressure conditions, the COPD small airways showed a significantly higher magnitude of pressure drop and wall shear stress compared to the donor. Additionally, to understand the mechanism contributing to TB obstruction in the COPD sample, a new model was created by allowing flow out of the occlusion regions in order to simulate the flow before obstruction. A higher pressure drop (27%) was observed compared to donor, while the value of wall shear stress peaked. Results demonstrate(fig1) that airflow in COPD experiences higher resistance due to deformations and occlusions downstream. The corrected model suggests that flow parameters, particularly wall shear stress, could be a contributing factor to TB obstruction in COPD.