Chronic Obstructive Pulmonary Disease (COPD) is a progressive and heterogeneous condition characterized by varying combinations of emphysema, small airway disease, chronic bronchitis, and exacerbations. Although multiple symptomatic therapies exist, no disease-modifying treatments are available. This gap highlights the need for improved preclinical models with greater translational relevance. Large human cohorts and single-cell/multi-mics studies have informed the development of current COPD models. We provide a state-of-the-art review of the major experimental platforms-in vivo (small and large animals, genetic and injury models, environmental exposures), ex vivo (precision-cut lung slices, organoids, co-cultures, lung-on-chip systems), and in silico (aerosol dispersion and computational tools). While each approach has yielded important mechanistic insights, none fully captures the complexity of COPD progression, comorbidities, gene-environment interactions, or heterogeneous clinical endotypes. Future progress will depend on the development of more integrated, human-relevant modeling systems, alongside advanced exposure platforms and AI-driven multi-omics integration to identify biologically meaningful endotypes and speed the creation of phenotype-specific therapies. We propose a phenotype-driven, cross-platform framework in which hypotheses emerging from human clinical and omics data, are validated in in vitro and ex vivo systems, evaluated in phenotype-specific in vivo models, and ultimately confirmed through clinical studies.

Chronic obstructive pulmonary disease (COPD) was the third leading cause of global mortality in 2011 but receives limited attention and research funding. This Review describes the current knowledge on COPD risk factors, including genetic and epigenetic determinants and their interactions with the microbiome and environmental exposures. Preclinical models are being refined and single-cell transcriptomic, metabolomic, and proteomic technologies are being implemented to investigate the molecular mechanisms of disease progression. Patient cohorts to define biomarkers of early disease and the latest approaches to diagnose pre-COPD are essential to accelerate the development of novel and effective therapeutic interventions and translate new findings into clinical trials. This Review is a summary of topics covered by a symposium organized by the COPD-iNET consortium, an international network of researchers who have established a platform that facilitates collaboration of this multidisciplinary group of preclinical, translational, and clinical researchers.

Menopause associated asthma impacts a subset of women and is less responsive to current treatments. Mechanisms driving this late-onset asthma are unknown. We recently developed a mouse model of menopause associated asthma using a combination of 4-vinylcyclohexene diepoxide (VCD) and house dust mite (HDM) exposures. The goal of this study was to determine how hormone replacement therapy during perimenopause impacts lung function and inflammation. The experimental groups included menopausal mice (VCD) with and without exposure to HDM (to model allergic airways disease) and menopausal mice with and without hormone replacement therapy (HRT; via estrogen pellet implantation). Lung function during methacholine challenge was assessed by flexiVent. Serum, bronchoalveolar lavage fluid (BALF), and histological samples were collected for assessment. Mice that received HRT during perimenopause had enhanced airway hyperresponsiveness (AHR) detected by total airway resistance (R-rs), tissue damping (G), and downward shifts in pressure-volume (PV) curves compared with controls, independent of HDM challenge. Although HRT in perimenopause resulted in decreased eosinophils in the HDM model, neutrophil levels and mucus production were unchanged. Mice receiving HRT in perimenopause also had significantly increased collagen production and inflammation associated with large and small airways, independent of HDM challenge. HRT given during perimenopause may be detrimental to lung responses, including increased AHR and decreased lung function, as well as increased tissue inflammation and airway remodeling. NEW & NOTEWORTHY Menopause-associated asthma is a subtype of asthma that is still largely unexplored and difficult to manage. Women experiencing menopause-associated asthma often have more severe exacerbations, higher rates of exacerbation, and most importantly, poor response to standard treatments. This study examined the impact of estrogen replacement given during the perimenopause phase on lung inflammation and function after menopause. While decreasing eosinophil recruitment, estrogen replacement actually led to worse lung function and more airway remodeling.

RATIONALE:Early-life lung function trajectories predict long-term respiratory health, including COPD risk. Club Cell protein 16 (CC16) is a key determinant of lung health, with low levels associated with impaired lung development, reduced lung function, and COPD. Cigarette smoking lowers CC16, but it is unknown whether maternal smoking leads to persistent CC16 deficiency from early life, thereby disrupting lung development and predisposing to COPD risk and progression. METHODS:CC16 expression was analyzed across 4 human cohorts, in plasma samples (COPDGene [n = 1062] and ECLIPSE [n = 2164]), nasal brushings (ALLIANCE [n = 63]), and peripheral lung sections (LTRC [n = 44]) from participants with and without a history of maternal smoking exposure. Lung histology and respiratory mechanics were assessed in WT and Cc16-/- mice with and without maternal smoking exposure. Recombinant human (rh)CC16 effects on lung maturation were assessed in embryonic murine lung explants. RESULTS:Maternal smoking was linked to reduced circulating and airway CC16 in COPD patients, controls, and a preclinical murine COPD model. In human adults, lower CC16 correlated with accelerated lung function decline and emphysema progression, while in children it was associated with obstructive physiology and early small airway impairment. In both mice and humans, maternal smoking-induced CC16 reduction was accompanied by greater epithelial injury (fibrosis, inflammation, apoptosis, and oxidative stress). In murine explants, smoking impaired lung branching, whereas rhCC16 restored branching via α2-integrin binding. CONCLUSIONS:Maternal smoking reduces CC16 levels, disrupting lung development in ways that predispose to lifelong impairment of lung function and worse COPD outcomes. Defining the mechanisms by which CC16 regulates lung maturation is essential for establishing reliable outcome measures and designing trials aimed at preventing early COPD.

BACKGROUND:Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality during viral pneumonia, including SARS-CoV-2 infections. Nevertheless, most patients with SARS-CoV-2 infections recover seamlessly without developing ARDS, suggesting the existence of endogenous pathways to protect the lungs. Since microRNAs (miRNAs) can regulate endogenous molecular pathways involved in lung protection, we hypothesized that alveolar miRNAs could function to dampen SARS-CoV-2-associated lung injury. METHODS:Screening studies in human alveolar epithelial cells and SARS-CoV-2-infected mice were performed to identify miRNAs induced during infection. Candidate miRNAs were confirmed via RT-qPCR. The role of hypoxia-inducible factor 1A (HIF1A) in regulating miRNA expression was examined in molecular studies. Loss- and gain-of-function approaches in a murine SARS-CoV-2 ARDS model were used to assess the physiological relevance of miRNA. Viral sequence analyses and site-directed mutagenesis were used to determine direct miRNA - viral RNA interactions. RESULTS:Screening studies identified miR-147b (hsa-miR-147b-3p or mmu-miR-147-3p) as the leading candidate during infection of human alveolar epithelia or mice with SARS-CoV-2. Functional and molecular studies implicate HIF1A in miR-147b induction during alveolar injury or SARS-CoV-2 infection. Studies in mice with induced deletion of miR-147b in alveolar epithelia (miR147fl/fl SPC-CreER mice) or nano-particle-mediated miR-147b overexpression revealed a protective role of alveolar-expressed miR-147b during murine SARS-CoV-2-associated ARDS. Moreover, we identified the ORF8 region within the SARS-CoV-2 template strand as a direct target of miR-147b, with an ORF8 silent mutation of the SARS-CoV-2 miR-147b-binding site abolishing the observed protection in vitro and in vivo. CONCLUSIONS:Alveolar epithelial cell-derived miR-147b serves as an endogenous lung protective miRNA against SARS-CoV-2-associated ARDS by directly targeting virus-encoded RNA, revealing a previously unrecognized antiviral and lung-protective mechanism.

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disorder with limited treatment options. Macropinocytosis is one of the key cellular processes involved in nutrient consumption from the extracellular environment under stress conditions. Here, we studied the role of macropinocytosis in lung fibroblast activation and experimental pulmonary fibrosis. We found that macropinocytosis is increased in human lung fibroblasts (HLFs) derived from IPF patients. The inhibition of macropinocytosis with 5-(n-ethyl-n-isopropyl)-amiloride (EIPA) significantly inhibited profibrotic responses in IPF-derived and TGF-β1-stimulated HLFs. EIPA exerted antifibrotic effects by regulating amino acid (AA) uptake, mammalian target of rapamycin complex 1 (mTORC1) activation and mesenchyme homeobox1 (MEOX1) expression in activated HLFs. Both genetic and pharmacological inhibition of macropinocytosis significantly ameliorated pulmonary fibrosis in bleomycin (Bleo)-injured mice. Using IPF-derived precision cut lung slices (PCLS), we observed robust repression of profibrotic gene expression programs in EIPA-treated PCLS across different fibroblast subpopulations. Finally, we found that imipramine (Imi), a tricyclic antidepressant approved by the Food and Drug Administration (FDA), effectively inhibited macropinocytosis and ameliorated profibrotic responses in lung fibroblasts, Bleo-injured mice and IPF-derived PCLS. Taken together, our results suggest macropinocytosis inhibition as a potential therapeutic strategy to treat pulmonary fibrosis.

Recent evidence suggests that bronchial epithelial cells from individuals with asthma exhibit altered metabolic signatures. This metabolic shift of energetically demanding cells leads to increased inflammation, excessive reactive oxygen species production (ROS), and oxidative stress-all hallmarks of mitochondrial dysfunction. While mitochondrial dysfunction has been implicated in disruption in epithelial cell function in asthma, the mechanistic link between bronchoconstriction observed in asthma and these metabolic alterations remains poorly defined. Club cell secretory protein (CC16) is the most abundant protein found in the lung and exerts key anti-inflammatory and antioxidant functions contributing to protection against airway remodeling. Decreased levels of CC16 in both serum and bronchial alveolar lavage fluid (BALF) are characteristic of asthma and worsening respiratory disease. Using a well-established transmembrane compression system to model bronchoconstriction coupled with mass spectrometry and quantitative proteomics, we investigated how modeling bronchoconstriction in airway cells impacts CC16 expression and cell metabolic pathway changes over time. Using naive mouse tracheal epithelial cells (MTECs) and normal human bronchial epithelial cells (HBECs), we observed that recombinant (r)CC16 induces the expression of proteins related to various metabolic pathways, such as glycolysis, gluconeogenesis, and the pentose phosphate pathway and that compression of airway cells results in acute decreases in CC16 expression, as well as decreases in metabolic processes. MTECs deficient in CC16 (CC16-/-) had lower mitochondrial oxygen consumption rate (OCR) compared to WT cells. Exogenous addition of rCC16 significantly increased OCR of both WT and CC16 deficient MTECs. Our findings suggest a novel role for CC16 in mediating airway epithelial cell metabolic processes, which could be decreased by bronchoconstrictive events in human asthma. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD067703.

Survival after lung transplantation lags that of other solid organ transplants. Long-term survival is hampered primarily due to chronic lung allograft dysfunction (CLAD) development. It remains elusive how (chronic) rejection is organized within the lung graft over time post-transplant. Using a model of orthotopic left lung transplantation in major mismatched mouse strains with daily immunosuppression, we aimed to study the spatiotemporal dynamics of (chronic) rejection, using micro-computed tomography imaging, flow cytometric analyses and spatial proteomics. Endothelial cells demonstrated early activation and destruction (day 7 post-transplant). The accompanying early inflammation at the vascular compartment, progressed towards aberrant tissue repair resulting in irreversible bronchovascular fibrosis and chronic graft dysfunction. We provide new insights in the spatiotemporal dynamics of (chronic) rejection with a vascular-oriented onset that may have future implications for diagnosis and treatment in clinical lung transplantation.

Despite recent advances, the underlying mechanisms of the development and progression of many chronic respiratory diseases remain to be elucidated. Factors such as heterogeneity and complexity of human diseases and difficulty interpreting large datasets hinder research into chronic respiratory diseases. Omics assesses the changes in specific biological entities, such as mRNA expression, epigenetics/epigenomics, genomics, proteomics, metagenomics and metabolomics, and provides valuable insights into the roles of these processes in chronic respiratory diseases. High-throughput omics at bulk, single-cell and spatial levels empower the exploration of disease-related changes through untargeted data-driven statistical methods. Multi-omics is the exploration and integration of multiple biological processes, which compared to a single-omics, can provide a substantially greater and more holistic overview of the pathogenic mechanisms that underpin complex diseases. Multi-omics analysis can comprehensively characterise the mechanisms that drive chronic respiratory diseases, capturing unique biological signatures and cellular interactions at different omics levels. Use of these methods has begun to identify key factors and biomarkers in chronic respiratory diseases. Here, we review current omics approaches and highlight recent advances in respiratory research achieved using multi-omics and integrative methods. Our review provides a valuable resource for researchers and clinicians in this area.

Spatial transcriptomics shows that airway remodelling of severe COPD is characterised by increased epithelial cells, reduced vascularity and altered immune cells, providing new insights into the pathological changes in COPD small airways https://bit.ly/4bWF8VG.

Abstract Background Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and structural remodelling across different anatomical structures of the lung, including airways, parenchyma, and pulmonary vessels. However, compartment specific changes in immune cell composition and their communications with underlying structural cells remains incompletely defined. Methods Our study sought to assess the spatially resolved cellular organization of healthy and advanced end-stage COPD lungs using 10X Visium spatial transcriptomics. Non-negative matrix factorization was applied to identify transcriptional programs within spatially distinct areas. Data was validated through an integrative multi-modal approach, including flow cytometry, multiplex immunofluorescence, open access GeoMx Nanostring spatial transcriptomics and scRNA-seq datasets, and proof of concept in vitro co-culture experiments. Results Spatial mapping of advanced COPD lungs revealed compartment-specific immune and tissue remodelling programs. In the COPD parenchyma, molecular and immune signatures delineated macrophage-rich, remodelling–stress, and humoral B cell immune niches. Airway niches shifted from club cell/innate immunity in controls to goblet cell/adaptive immunity in COPD. Notably cytotoxic T cells colocalised with alveolar type 2 cells within the parenchymal remodelling–stress niche and goblet cells in COPD airways; predicted ligand–receptor interactions implicated these T cells in parenchymal cytotoxic injury and mucus-associated airway remodelling. Conclusions Our findings delineate compartment-specific cellular microenvironments and interaction networks that may orchestrate tissue remodelling in COPD. This work advances our understanding of immune-driven pathogenesis and pinpoints the cytotoxic T-cell axis as a candidate for targeted therapeutic strategies.

Objective:Neoadjuvant chemoimmunotherapy has improved outcomes in resectable non-small cell lung cancer (NSCLC), yet its real-world implementation is often challenged by surgical delays, immune-mediated fibrosis, and postoperative complications. Smokers with NSCLC, despite a high risk for surgical morbidity, show enhanced responses to neoadjuvant chemoimmunotherapy. This study aimed to identify smoking-associated immune cell determinants that could guide treatment strategies. Methods:Single-cell RNA sequencing (scRNAseq) was performed on 61 lung tissues from non-smokers and smokers to identify smoking-related immune compositions. The scRNAseq data from 19 invasive lung adenocarcinomas were used to validate their presence in the tumor-immune microenvironment. Bulk RNA sequencing data from 102 resected NSCLC and 24 NSCLC patients treated with neoadjuvant chemoimmunotherapy followed by surgery were used for in silico cellular deconvolution and outcome analyses. Results:Among 135 lung cellular phenotypes, two natural killer (NK) cell subsets were strongly associated with smoking and chronic obstructive pulmonary disease (COPD) severity. "Stress-responsive" NK (NKSR) cells exhibited immature features and cytokine-responsive features, and "Adaptive and immunoregulatory NK" (NKAIR) cells showed mature features and elevated multiple immune checkpoint expression. High intratumoral NKSR cells correlated with improved survival after surgery, particularly in current smokers. Conversely, tumors with low NKSR and high NKAIR cells responded more favorably to neoadjuvant chemoimmunotherapy. Conclusions:Intratumoral NK cell phenotyping may aid in therapeutic stratification in patients with NSCLC. NKSR cell preservation predicts benefit from upfront surgery, while NKAIR cell enrichment indicates improved response to neoadjuvant chemoimmunotherapy. These NK cell profiles may help optimize treatment by balancing therapeutic benefit and risk.

Chronic obstructive pulmonary disease (COPD) is clinically and molecularly heterogeneous. To investigate COPD heterogeneity, we profiled lung tissue by single-nucleus RNA sequencing from 141 study participants (1,516,727 nuclei) and identified shifts in cell composition and emergent cell states that correlated with lung function, emphysema and composite symptom scores. Epithelial regenerative states peaked in early COPD and declined thereafter, whereas inflamed nonimmune cells and profibrotic/remodeling states, together with select immune populations, expanded with disease progression. Clustering study participants by the proportion of pathologic cells coupled with spatial transcriptomics identified distinct patterns of cellular co-occurrence within spatially localized niches. Proteomic analyses identified plasma biomarkers of cell states and their impact on the extracellular matrix. Mediation and cell communication analyses revealed cell-autonomous and intercellular communication networks associated with disease. These data define the cellular landscape of COPD heterogeneity, revealing molecular drivers and biomarkers that could inform therapeutic strategies. This study uses single-nucleus RNA sequencing to analyze lung tissue from 141 individuals with chronic obstructive pulmonary disease. Distinct patterns of spatially resolved changes in cell composition and cell states that correlate with clinical features are highlighted.

Background: Although cigarette smoke induces lung inflammation in all smokers, only a subset develops COPD. Inflammation often persists even after smoking cessation, suggesting a self-sustaining pathogenic process similar to autoimmune responses. Increased B cell activity and autoantibodies have been associated with emphysema severity, yet their specific role in COPD progression remains underexplored in a well-defined COPD cohort. Methods: We assessed the association of 502 autoantibodies (IgG, IgA, IgM, and IgE) targeting 100 common lung antigens in plasma from 100 COPDGene participants, with emphasis on their relationship to emphysema. Emphysema was measured using volume noise-bias-adjusted lung density. Statistical models adjusted for age, sex, smoking history (pack-years), current smoking status, and lung function parameters (FEV1% predicted and FEV1/FVC ratio). To control for multiple testing, we used immunoglobulin correlation-based adjustments as described by Galwey et al. Additionally, we analyzed associations without lung function adjustments and explored other emphysema measures. Results: The study cohort had an average age of 66, was approximately 50% female, and included both non-Hispanic White and African American participants, with 65% of individuals classified as GOLD 2 or higher for COPD severity. Findings revealed that most elevated autoantibodies in COPD were IgM, notably Myeloperoxidase-IgM (MPO-IgM), an anti-neutrophil cytoplasmic antibody (ANCA). Lung density showed a significant association with MPO-IgM (B = 0.6, P adjusted = 5.6x10⁻³), which was even stronger in analyses without lung function adjustments (P = 1.7x10⁻⁸). This MPO-IgM association suggests a link between autoimmunity and emphysema. Conclusions: Our study identifies autoreactive IgM levels in COPD patients, with MPO-IgM showing a significant inverse association with emphysema. IgM antibodies are part of the innate immune response, potentially clearing dead cells and cellular debris and thus reducing inflammation. However, excessive or dysregulated IgM may shift from beneficial clearance functions to targeting healthy tissues. The specific role of MPO-IgM in milder emphysema forms presents an intriguing finding, as ANCAs are generally associated with systemic autoimmune diseases. Further research is warranted to clarify whether MPO-IgM contributes directly to emphysema progression or represents an immune response byproduct, potentially offering new insights into the autoimmune aspects of COPD pathogenesis.

RATIONALE: Early detection of lung cancer is a key issue to overcome its poor prognosis. In a pilot study we showed the potential of exosomes, a subtype of extracellular vesicles, as biomarkers for diagnosis and prognosis of lung adenocarcinoma (LUAD). This study aims to validate the value of one of the identified exosomal microRNAs (miR), miR-885, as a potential biomarker in LUAD. METHODS: Patients were selected from the LuCEx cohort (Lung Cancer Extracellular Vesicles). All underwent surgery due to a suspected malignant solitary pulmonary nodule, and long-term post-surgery follow-up (25 non-cancer -NCa- and 100 LUAD). Blood samples were collected prior to surgery. LUAD patients were followed up for a minimum of one year, with an average follow-up period of 2.4 years after surgery. Exosomes were isolated by precipitation and characterized by dot blot and transmission electron microscopy. miR-885 levels were analyzed by RT-qPCR. The predictive value of this miR was assessed using univariable ROC analyses, and a Kaplan-Meier test was performed to evaluate survival in relation to high (ΔCt<4.2), medium (ΔCt=4.2-7.4) and low levels of miR-885 (ΔCt>7.4). RESULTS: The NCa and LUAD groups were similar in age (66 and 69 years, respectively) and sex distribution (60% and 40% women, respectively). After exosome characterization, miR-885 levels were increase in LUAD patients compared to NCa (Fold Change -FC- = 6.48; p<0.001). The predictive value assessed by ROC analysis, with an area under the curve (AUC) of 0.844 (p<0.001). The positive predictive value (PPV) was 85 % and the negative predictive value (NPV) was 56%. Additionally, miR-885 levels were significantly higher in LUAD patients who died during follow-up (FC=7.24; p<0.001). ROC analysis using death as the outcome yielded an AUC of 0.828 (p<0.001), with a PPV of 50% and an NPV of 85%. Finally, survival analysis showed a significantly higher risk of death in LUAD patients with increased miR-885 levels compared to those with medium (HR=5.09; p=0.0003) or low levels (HR=11.43; p<0.0001). CONCLUSIONS: Exosomal miR-885 is confirmed as a promising biomarker for LUAD diagnosis. Furthermore, after resected surgery, low levels of miR-885 are associated with better prognosis, while elevated levels correlate with poorer survival outcomes in LUAD patients.