RATIONALE:Prevention of bronchopulmonary dysplasia (BPD) remains a critical unmet need. OBJECTIVES:We continued evaluating aerosolized vitamin A in a neonatal rat hyperoxia-injury model of BPD, testing whether lung-targeted inhalation yields superior outcomes compared to enteral or intramuscular (IM) delivery. METHODS:Rat pups were exposed to continuous 95% hyperoxia for seven days to induce lung damage. Vitamin A was administered from post-natal day (PD) 1-7 by inhalation, enteral or IM dosing. Persisting effectiveness of inhaled vitamin A was examined at PD 21, dwelling in normoxia after all interventions were discontinued at PD 7. MEASUREMENTS AND MAIN RESULTS:Comprehensive analyses include alveolar morphometrics, in vivo pulmonary function testing, assessment of hepatic vitamin A level, and quantitating gene and protein expression of relevant biomarkers of lung maturation, inflammation and damage/repair, employing singlex and multiplexed assays and whole-genome gene expression. CONCLUSIONS:Inhaled vitamin A suppressed hyperoxia-induced lung damage, with key hallmarks of BPD, including lung morphometrics and biomarkers associated with lung damage and inflammation, being effectively indistinguishable from healthy controls at both PD 7 and 21, Natural reparative processes were enhanced, with normal alveolarization and pulmonary function at PD 21. Despite raising hepatic vitamin A levels, enteral dosing was ineffective in restoring alveolar morphology at PD 7. Consistent with previous reporting, IM dosing yielded modest effect at PD 7 but with little evidence of benefit to pulmonary function at PD 21. Observation of dose-dependent effects with aerosolized vitamin A strengthens the evidence of the benefits of lung-targeted delivery, supporting further development of inhaled vitamin A as a BPD preventive strategy.
RATIONALE:Sickle cell trait (SCT), carried by millions worldwide, is generally considered benign. However, SCT is increasingly linked to life‑threatening complications under environmental stress. OBJECTIVES:We tested whether sickle cell trait confers heightened vulnerability to oxidant inhalation injury and whether post-exposure haptoglobin mitigates injury. METHODS:Using humanized SCT (AS) and control hemoglobin (AA) mice carrying human hemoglobin knock‑in genes, we examined physiologic, biochemical, histologic, mitochondrial, and proteomic responses to chlorine (Cl₂) inhalation at concentrations encountered near chemical accidents. Pulmonary and kidney injury were assessed 24 h post‑exposure. MEASUREMENTS AND MAIN RESULTS:Compared with AA controls, SCT mice exhibited markedly increased mortality, severe acute lung and kidney injury, hypoxemia, and exaggerated hemolysis/rhabdomyolysis following Cl2 exposure. Global lung proteomics revealed widespread remodeling, with modifications to lung proteins. Systems biology analysis revealed that neutrophil degranulation, acute‑phase, and oxidative/mitochondrial injury pathways were uniquely intensified in SCT. To test whether scavenging extracellular hemoglobin mitigates injury, mice received haptoglobin 30 min after Cl2 exposure. Post‑exposure haptoglobin significantly improved survival, oxygenation, renal function, and epithelial barrier integrity, while reversing key inflammatory and mitochondrial injury signatures and restoring proteomic architecture to near-baseline levels. CONCLUSIONS:Our data showed that Cl2 inhalation triggers hemolysis-driven multiorgan injury in SCT, establishing SCT as a previously unrecognized high-risk phenotype for toxic environmental exposures. Haptoglobin administered after exposure provides robust protection across physiologic, molecular, and mitochondrial endpoints, supporting hemoglobin scavenging as a practical, rapidly deployable countermeasure for chemical inhalational injury in individuals with SCT.
RATIONALE:Pathogen-associated molecular patterns (PAMPs) are known to initiate pro-inflammatory responses, but emerging evidence has revealed their roles in inducing macrophage-dependent immunomodulatory pathways that dampen inflammation. OBJECTIVES:We explored the interactions between the asthma-associated type 2 immunopathology and immune response to poly I:C, a PAMP mimicking double-stranded RNA, in ovalbumin (OVA) sensitized and challenged mice. METHODS:Following intranasal co-administration of Poly I:C during OVA challenges, we performed immunophenotyping of the lung and measured airway responses to inhaled methacholine (MCh). We characterized the pulmonary macrophages by flow cytometry and examined the effects of monocyte-derived (Ly6C+) macrophage depletion and c-c motif chemokine receptor 2 (CCR2) deficiency on inflammation and responses to MCh. The role of IL-10 was assessed by antibody inhibition of the IL-10 receptor. MEASUREMENTS AND MAIN RESULTS:Poly I:C attenuated allergen-induced type 2 immunopathology and airway hyperresponsiveness. Unexpectedly, the poly I:C receptors and poly I:C-induced cytokines were suppressed in the co-administered group compared to poly I:C only group. We observed an enrichment of lung Ly6C+ macrophages in co-administrated mice, which featured alternatively activated- (M2) and a myeloid-derived suppressor cell-like phenotype, expressed immunoregulatory factor IL-10, and inhibited in vitro T cell activation. Poly I:C did not inhibit Th2 immunopathology in mice depleted of Ly6C+ macrophages or deficient in CCR2, which mediates monocyte recruitment. Lastly, the blockade of the IL-10 receptor reversed the Th2-suppressing effect of co-administered poly I:C. CONCLUSIONS:Concurrent poly I:C and allergen exposure at the airway mucosa evokes macrophage-mediated immunomodulation that suppresses allergic airway reactions.
Advances in GPCR biology and drug-discovery technology are simultaneously reshaping what can be targeted in asthma and how those strategies must be validated. Recent insights into biased signaling, receptor dimerization and signaling microdomains have revealed new therapeutic opportunities beyond the limitations of traditional β₂-agonist bronchodilation and cytokine-targeted biologics. At the same time, innovations in structural biology, computational modeling, machine learning, biosensor-based pharmacology, and physiologically relevant airway models have transformed the interrogation of GPCRs and the engineering of therapeutics that target them. This review synthesizes these developments, emphasizing how emerging GPCR concepts inform modern discovery platforms, how receptor- and post-receptor signaling control points can be targeted, and how validation across artificial and physiologic systems improves translational fidelity. We further discuss how GPCR therapeutics may reshape clinical development and complement existing biologic therapies in asthma.
Rapid advances in single-cell technologies now allow measurement of thousands of transcripts and other molecular features of individual cells offering unprecedented insight into lung biology in homeostasis and in disease. The accelerated generation of multimodal data has, however, been accompanied by the reporting of putatively "novel" cell types described without consensus regarding their ontogeny, identity, function, or defining markers. To fully realize the value of the technological advances and to enable rigorous comparison across studies, respiratory research will benefit from standardized, quantitative, and biologically grounded cell classifications and nomenclature. Achieving the transformative potential of the multimodal data will depend on common, machine- and human-readable nomenclature, structured and expandable dictionary and atlas resources, and clear methodological standards that ensure consistency as technologies evolve. The American Thoracic Society (ATS) recognized the importance of promoting a common nomenclature to enhance equitable access and utility of the vast amounts of multimodal data generated by the lung research community. The Collaborative Cross-Consortium and Country Lung Cell Nomenclature Project (C3LCN) was adopted as an ATS Assembly Project in 2024. This is the consensus report outlining the goals and framework of the Project to foster coordinated progressive lung cell research to include: 1) providing best practices for analysis, publication and reporting of lung single-cell transcriptomic datasets; 2) establishing a contemporary lexicon for healthy adult human cells of the lower respiratory tract with structured, persistent, and resolvable identifiers; 3) defining a scalable taxonomy to organize a common lung cell nomenclature; 4) offering tools to support collaboration, knowledge dissemination, and translational advances rooted in modern lung biology augmenting, not replacing, pre-genomic biological knowledge; and 5) describing an infrastructure capable of incorporating new ontological refinements as higher-resolution, multimodal single-cell and spatial datasets emerge, cellular heterogeneity is better defined, and disease-associated abnormal cell types and reactive cell states are increasingly recognized and mechanistically interrogated. Together, this coordinated effort aims to provide the foundation necessary for a robust, harmonized, and expandable nomenclature for lung science.
RATIONALE:Impaired alveolar regeneration is a central feature of chronic lung diseases. Type 2 alveolar epithelial cells (AT2) serve as lung stem/progenitor cells that differentiate into type 1 cells (AT1) to restore gas exchange following injury. However, the metabolic determinants governing this regenerative process remain poorly understood. OBJECTIVES:This study aimed to determine the role of glutamine metabolism via glutaminase 1 (GLS1) in regulating AT2-to-AT1 differentiation and alveolar regeneration after lung injury. METHODS:We used primary murine AT2 cells, alveolar organoids, and lineage-tracing mouse models of bleomycin-induced lung injury. The effects of AT2-specific GLS1 deletion on epithelial differentiation were assessed in vitro and in vivo. Chloroquine and bafilomycin A1, two autophagy inhibitors with distinct molecular mechanisms of action, were used for mechanistic rescue experiments. MEASUREMENTS AND MAIN RESULTS:GLS1 expression increased during AT2-to-AT1 differentiation and was accompanied by metabolic reprogramming characterized by enhanced glycolysis and increased glutamine entry into the TCA cycle. AT2-specific GLS1 deletion impaired differentiation in vitro and in vivo, resulting in defective alveolar repair and exacerbated pulmonary fibrosis. Mechanistically, GLS1 deficiency induced excessive autophagy and promoted degradation of the Hippo pathway effectors YAP and TAZ, key regulators of epithelial cell fate decisions. Inhibition of autophagy with either chloroquine or bafilomycin A1 restored YAP/TAZ levels, rescued AT2-to-AT1 differentiation, reduced fibrosis, and improved lung function. CONCLUSIONS:GLS1-mediated glutaminolysis is essential for alveolar stem/progenitor cell differentiation through regulation of autophagy and YAP/TAZ stability. Modulation of autophagy may represent a therapeutic strategy to enhance lung regeneration in fibrotic lung diseases.
Alveolar homeostasis depends on tissue-resident professional phagocytes known as alveolar macrophages (AMs) that catabolize pulmonary surfactant. Pulmonary alveolar proteinosis (PAP) arises from impaired surfactant clearance due to loss or dysfunction of AMs, most commonly from disrupted GM-CSF-dependent AM homeostasis and less frequently from congenital defects in surfactant synthesis or processing. PAP has also been reported as a pulmonary toxicity associated with mTOR inhibitor-based immunosuppressive therapy. Although mTOR activity regulates macrophage metabolism and proliferation, its requirement for AM survival and lipid homeostasis remains unclear. Here, we examined the role of mTOR in AM survival and surfactant homeostasis, using complementary genetic and pharmacologic approaches in vivo, and GM-CSF-driven AM-like cell culture models in vitro. Myeloid-specific deletion of mTOR caused progressive, preferential depletion of AMs among tissue-resident macrophage populations, accompanied by impaired phagocytosis, intracellular lipid accumulation, and development of PAP-like lung pathology. In vivo, pharmacologic mTOR inhibition with temsirolimus reproduced key features of genetic mTOR deletion, including AM depletion, apoptosis, lipid accumulation, and PAP-like pathology. In vitro, mTOR activity was required to sustain GM-CSF-dependent expansion, maturation, and survival of AM-like cells. Mechanistically, mTOR loss reduced expression of PPARγ and pro-survival Bcl-2 family members, linking mTOR activity to AM viability and lipid handling capacity. In summary, these findings identify mTOR as a nonredundant, cell-intrinsic regulator of alveolar macrophage survival and function required to maintain alveolar homeostasis. This work provides experimental support for AM-intrinsic mechanisms contributing to mTOR inhibitor-associated pulmonary toxicity, including PAP.
Lung fibrosis, including idiopathic pulmonary fibrosis (IPF), represents a spectrum of progressive interstitial lung diseases characterized by disrupted epithelial repair, fibroblast activation, and excess extracellular matrix accumulation. A central feature of fibrotic progression is the loss of alveolar type 2 epithelial (AT2) cell identity and the emergence of aberrant transitional states that fail to support normal regeneration. Based on our re-analysis of multiple publicly available scRNA-seq datasets from the IPF Cell Atlas, S100A2 expression is tightly associated with basal-like reprogramming of AT2 cells and is a top upregulated gene at both mRNA and protein levels in IPF lungs. Furthermore, scRNA-seq data from human lung organoid models reveal that AT2 cells co-cultured with fibroblasts acquire a basal-like phenotype and express high levels of S100A2. Functionally, overexpression of S100A2 in human iPSC-derived lung alveolar epithelial type 2 (iAT2) cells leads to loss of AT2 cellular identity, increased generation of reactive oxygen species, and activation of RAGE signaling pathway. We demonstrate that pharmacologic inhibition of RAGE using Azeliragon preserves AT2 cell populations, associated with reduced oxidative stress in iAT2 cells, and significantly attenuates collagen deposition in bleomycin-induced lung fibrosis models. Collectively, our results indicate that S100A2 is a key driver of epithelial dysfunction in lung fibrosis, promoting loss of AT2 identity, aberrant basal fate acquisition, and persistent epithelial injury. Targeting the unrecognized S100A2-RAGE pathway may offer a new therapeutic strategy to restore epithelial homeostasis in lung fibrosis.
BACKGROUND:Lung cancer remains the most common cause of cancer-related mortality worldwide. The transforming growth factor-beta 1 (TGF-β1) pathway promotes epithelial-mesenchymal transition (EMT), invasion, and metastasis in advanced disease via mothers against decapentaplegic homolog 2 (Smad2) and Smad3. This study investigated how post-translational modifications regulate TGF-β1-Smad signaling. METHODS:Lung cancer cell lines and a mouse metastasis model were used to explore the roles of protein phosphatase magnesium-dependent 1A (PPM1A) palmitoylation, zinc finger DHHC-type palmitoyltransferase 11 (ZDHHC11), and ubiquitin-specific peptidase 5 (USP5) in regulating Smad2/3 signaling. Palmitoylation-deficient and wild-type PPM1A constructs were introduced by lentiviral transduction. Protein levels, modifications, and interactions were analyzed by western blot, immunoprecipitation, and immunofluorescence. Palmitoylation was measured using acyl-resin-assisted capture (Acyl-RAC). Cell invasion and migration were assessed using Transwell assays. RESULTS:Palmitoylation of PPM1A at cysteine 71 enhanced the interaction between PPM1A and Smad2/3, promoted Smad2/3 dephosphorylation, and suppressed lung cancer cell invasion and metastasis. ZDHHC11 was identified as the enzyme responsible for PPM1A palmitoylation; its loss diminished this modification and accelerated metastasis in vivo. TGF-β1 downregulated ZDHHC11 expression, weakened PPM1A-Smad2/3 binding, and increased cell migration. Additionally, USP5 stabilized Smad2/3 by preventing their ubiquitin-mediated degradation, and TGF-β1 further amplified this effect by limiting PPM1A access. CONCLUSION:This study identified a novel palmitoylation-dependent mechanism regulating TGF-β1-Smad signaling in lung cancer. Upregulating the ZDHHC11-PPM1A axis or targeting USP5 may offer new strategies to inhibit metastasis in lung cancer.
RATIONALE:Derived iPSCs airway epithelium are challenging given their dependency on the mesenchymal compartment. We hypothesized that growing vAFE cells on well-organized stiff matrix precolonized by adult pulmonary fibroblasts would improve epithelial differentiation yield and maturity. METHODS:Collagen-1/chitosan matrix were engineered to reach stiffness and scaffolding characteristics of subepithelial compartments. Primary fibroblasts derived from human lung samples were seeded for 45 days before the addition of vAFE cells differentiated from iPSCs, and comparisons made with iPSC-derived fibroblasts. Beads tracking was used to assess cilia beating efficiency. RESULTS:Primary human bronchial fibroblasts were able to enrich the CC Matrix with extracellular matrix components such as collagen, decorin and vimentin. In turn, iALI cultures performed in primary fibroblasts enriched CC matrix successfully led to high level of epithelial differentiation including rare cells (club, basal, neuroendocrine, ciliated, secretory). Large apical surfaces were covered by approximately 60% of ciliated cells able to generate mucociliary vortex. CONCLUSION:Primary human bronchial fibroblasts seeded into collagen-chitosan matrix dramatically improved iALI epithelial differentiation from vAFE cells, related to highly specific transcriptomic signatures when compared to iPSC derived fibroblasts.
Bronchopulmonary dysplasia (BPD) is a major complication of prematurity, characterized by impaired alveolar epithelium regeneration and long-term respiratory morbidity. The cellular basis of this defect and the underlying epithelial-mesenchymal signals remain unclear. To define alveolar type 2 (AT2) cell heterogeneity and regenerative capacity, we analyzed lineage-traced AT2 cells in neonatal mice exposed to hyperoxia (85% O2, postnatal days 1-14). We assessed cellular states and function by flow cytometry, transcriptomics, and organoid assays. During normal lung development, AT2 cells gradually segregated into two transcriptionally distinct subpopulations, TomHigh and TomLow. TomHigh cells exhibited canonical AT2 identity, lipid metabolism, and enhanced mitochondrial function, showing strong organoid-forming and differentiation potential. TomLow cells displayed metabolic restriction and limited regenerative capacity. Hyperoxia caused alveolar simplification, expanding TomLow cells at the expense of TomHigh cells. AT2 cells entered a proliferative state and became Krt8+ transitional cells, but RNA velocity and lineage analyses showed impaired maturation toward AT1 cells, leading to persistent AT1 deficiency. Hyperoxia induced Sfrp1 and Frzb expression in a rare but detectable Sftpc+Acta2+ AT2 subset, and more prominently in Pdgfra+ fibroblasts, which adopted a myofibroblast-like phenotype. Fibroblast-epithelial co-culture and fibroblast-specific Sfrp1/Frzb overexpression impaired organoid formation and reduced AT2-to-AT1 differentiation. These findings identify a fibroblast-dominated Sfrp1/Frzb-mediated niche that constrains AT2 differentiation into AT1 cells, providing a cellular basis for impaired alveolar regeneration in BPD and suggesting potential therapeutic targets.
Alveolar type 2 (AT2) cells are the resident progenitors of the distal lung. They maintain tissue homeostasis and regenerate the alveolar surface after injury through coordinated self-renewal and differentiation into alveolar type 1 (AT1) cells. When this differentiation program fails, AT2 cells accumulate in aberrant intermediate states, now recognized as the epithelial "transitional state" or alveolar differentiating intermediate (ADI), a defining feature of idiopathic pulmonary fibrosis (IPF) and other fibrotic lung diseases. Although the signaling pathways and transcription factors governing AT2 cell fate have been extensively characterized, the metabolic requirements for successful AT2-to-AT1 differentiation remain poorly understood. Emerging evidence indicates that AT2 cells undergo dynamic metabolic reprogramming during repair, with fatty acid oxidation, glucose metabolism, and glutamine catabolism each playing temporally distinct and mechanistically integrated roles. In IPF, AT2 cells exhibit profound mitochondrial structural abnormalities that compromise oxidative metabolism and likely underlie, at least in part, the broader pattern of metabolic dysregulation observed in diseased epithelium. This mitochondrial dysfunction, together with Warburg-like glycolytic reprogramming and impaired fatty acid oxidation, may function not only as a consequence of epithelial injury but also as a driver of differentiation failure. This review synthesizes current evidence, evaluates causal relationships among metabolic pathways, integrates metabolism with established signaling networks, and proposes a translational framework for metabolism-directed restoration of alveolar repair capacity in fibrotic lung disease.
Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants increasingly detected in maternal serum, breast milk, drinking water, and indoor environments, raising concern for fetal and early postnatal exposure during critical periods of lung development. Although epidemiologic and experimental studies link early-life PFAS exposure with altered immune function, impaired lung maturation, and later respiratory vulnerability, the molecular pathways underlying these effects remain incompletely defined. In this mini-review, we propose peroxisome proliferator-activated receptor gamma (PPARγ) as a central integrator of developmental lung reprogramming by PFAS. PPARγ regulates key processes required for normal alveolar maturation, including epithelial-mesenchymal interactions, lipofibroblast differentiation, epithelial barrier integrity, macrophage polarization, and inflammatory homeostasis. Recent evidence, including our own, indicates that in the developing lung PFAS exposure is associated with suppression of pulmonary PPARγ expression and signaling while relatively sparing PPARα, and that pharmacologic modulation of PPARγ significantly alters PFAS-induced epithelial inflammatory responses. We discuss how PFAS-mediated disruption of PPARγ signaling, potentially through direct pulmonary effects as well as indirect placental and endocrine mechanisms, may link structural and immune reprogramming by altering lipofibroblast identity, impairing alveolar maturation, altering epithelial permeability, and promoting macrophage programs that amplify later allergic susceptibility. We further highlight developmental timing, exposure mixture, and sex-divergent outcomes as emerging modifiers of this pathway. Defining how PFAS perturbs PPARγ-dependent developmental programs may provide a practical mechanistic framework for understanding environmentally programmed respiratory disease.
RATIONALE:Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants exposed to ventilatory support and hyperoxia, involves alveolar and vascular simplification and is linked to cellular senescence. We previously found senescence predominantly in alveolar macrophages (AMs) after neonatal hyperoxic exposure, but the specific mechanisms driving this and its role in neonatal lung injury remain poorly understood. OBJECTIVES:This study investigated the mechanisms driving hyperoxia-induced AM senescence, the effects of senescence/hyperoxia on AM function and how the resulting secretome contributes to lung injury. METHODS:scRNA-seq datasets from hyperoxia-exposed neonatal mice were used to score AM senescence and characterize senescent/hyperoxia-induced AM clusters. Hyperoxia-induced metabolic shifts were analyzed alongside AM motility and phagocytosis. Proteomics characterized the senescent AM secretome. Finally, the effects of senescent and hyperoxic secretomes on lung injury were assessed with and without p38MAPK inhibition. RESULTS:scRNA-seq analysis confirmed that hyperoxia significantly increases senescence markers in AMs. This induction occurs via increased glycolysis and leads to reduced AM motility and phagocytosis. Proteomics identified p38MAPK-regulated proteins in the AM secretome including upregulated pro-fibrotic factors and downregulated structural regulators. Intranasal administration of senescent and/or hyperoxia-conditioned media caused alveolar and vascular simplification in neonatal mice, which was attenuated by p38MAPK inhibition. CONCLUSIONS:Hyperoxia causes glycolytic reprogramming in AMs, increasing senescence markers and impairing function. This also leads to a secretome that drives lung injury. Inhibiting macrophage glycolysis and p38MAPK pathways represent novel, promising therapeutic approaches to prevent lung injury.
Emphysema, a heterogeneous and destructive disorder of the lung parenchyma driven by genetic and environmental factors, has long been considered irreversible. The 67th Aspen Lung Conference challenged this paradigm, highlighting emerging cellular, molecular, and structural findings that advance our understanding of emphysema pathogenesis and provide new mechanistic insights into alveolar regeneration. Presentations addressed monogenic forms of lung disease, environmental and sex-related modifiers of disease susceptibility, and pathways linking emphysema progression to impaired regenerative capacity, cellular senescence, and immune dysregulation. The meeting addressed four central themes: shared mechanisms in emphysema across chronic obstructive pulmonary disease and rare lung diseases with similar phenotypes; dynamic cellular and extracellular matrix remodeling underlying distal airspace destruction; innovative approaches to early detection, phenotyping, and endotyping emphysema; and emerging molecular and cellular targets for therapeutic intervention. This report synthesizes key concepts from presentations and discussions among experts regarding the critical remaining gaps in developing disease-modifying therapies.
