American Journal of Respiratory Cell and Molecular Biology

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with limited treatment options. Our group previously identified the antifibrotic potential of thyroid hormone, triiodothyronine (T3); however, clinical translation of thyroid hormone therapy is limited by its systemic adverse effects. In this study, we investigate whether sobetirome, a selective and well tolerated thyroid hormone receptor beta (THRB) agonist, offers antifibrotic benefits of thyroid hormone while minimizing systemic toxicity. Our study reveals that sobetirome, administered via intraperitoneal or inhalational routes, effectively mitigates bleomycin-induced pulmonary fibrosis in mice, with no evidence of toxicity. We identified that sobetirome restores mitochondrial homeostasis via activating the THRB-PPARGC1a axis. This protects alveolar type II epithelial cells from injury-induced apoptosis while selectively inducing apoptosis and metabolic reprogramming in apoptosis resistant IPF fibroblasts. Cell-specific deletion of Ppargc1a in either alveolar epithelial cells or fibroblasts abolishes sobetirome-mediated protection, establishing PPARGC1a as an essential mediator of therapeutic response. Importantly, sobetirome reverses fibrosis-associated transcriptional programs in human IPF lung tissue, reducing expression of key fibrosis-associated genes, including collagen I alpha 1 (COL1A1), collagen III alpha 1 (COL3A1), periostin (POSTN), cathepsin K (CTSK), and Chitinase 3 Like 1 (CHI3L1), while promoting extracellular matrix remodeling, epithelial restoration, and tissue homeostasis. Collectively, our findings identify THRB activation as a novel metabolic strategy for reversing pulmonary fibrosis. Across complementary in vitro, in vivo, and human ex vivo models, sobetirome restores mitochondrial function, modulates apoptotic pathways in pathogenic cells, and promotes fibrosis resolution, highlighting its potential as a lung-targeted therapeutic approach for IPF and other fibrotic lung diseases.

Background: COPD is associated with persistent airway epithelial mucous cell metaplasia (MCM), mucin hypersecretion, and airway obstruction. Cytokines, such as IL-13, are well-known inflammatory drivers of airway MCM development. However, less is known about the factors that contribute to the impaired resolution of MCM in COPD. We hypothesized that MTOR activation in COPD airway epithelial cells (AECs) impairs autophagy and contributes to the delayed resolution of MCM. Methods: AEC from COPD and non-diseased donors were grown under air-liquid interface (ALI) conditions and treated with IL-13 to promote MCM. Airway sections from COPD lung explants or non-diseased lung donors were utilized for immunohistochemical and immunostaining. Lysosomes were assessed using molecular probes and immunostaining. Results: There was persistent MTOR-dependent MUC5AC immunostaining and epithelial hypertrophy in COPD AEC, which did not resolve after IL-13 withdrawal. MTOR substrate phosphorylation of RBS6, S6K1, and ULK1 was significantly elevated under baseline conditions, and at multiple timepoints independent of IL-13. Pharmacologic activation of MTOR led to increased IL-13-mediated MUC5AC levels in normal AEC but less robustly in COPD derived AEC. We hypothesized that persistent MTOR activation would reduce lysosome function and abundance in disease. COPD airways had reduced lysosome markers, LAMP1 and LAMP2 and fewer functional lysosomes by live-cell reporter probes. This lysosome deficiency in COPD AEC was partially rescued with MTOR inhibition. Conclusions: We provide evidence to support an axis of autonomous MTOR activation and impaired lysosome function in the COPD airway epithelium. Persistent MTOR signaling is a molecular driver that contributes to persistent MCM.

Background Idiopathic Pulmonary Fibrosis (IPF) is a fatal chronic lung disease with limited therapeutic options. While alveolar epithelial injury and fibroblast activation are well-studied, endothelial-mesenchymal transition (EndoMT) is emerging as a critical pathogenic mechanism. The regulatory role of exosomal miRNAs in pulmonary fibrosis remains unclear. This study investigates serum exosomal miRNAs, particularly let-7a-5p, in modulating EndoMT during the onset of pulmonary fibrosis. Methods Clinical cohorts of IPF patients and healthy controls were enrolled. Serum exosomal miRNAs were profiled, followed by differential expression and functional enrichment analyses. In vitro experiments involved human pulmonary artery endothelial cells (HPAECs) transfected with let-7a-5p mimic or inhibitor. Dual-luciferase reporter assays confirmed the binding between let-7a-5p and TGFBR1. HPAECs were co-cultured with lung epithelial cells to examine paracrine signaling. In vivo studies used a bleomycin-induced mouse model with let-7a-5p agomir administration. Assessments included histopathological staining, hydroxyproline content, Western blot, qPCR, micro-CT, and pulmonary function tests. Results Let-7a-5p was significantly downregulated in serum exosomes from IPF patients, correlating with clinical indicators. Mechanistically, let-7a-5p directly bound the TGFBR1 3'UTR to inhibit its expression. Inhibition of let-7a-5p upregulated -SMA, FN1, smad2/3 phosphorylation, and collagen I, while downregulating CD31 and VE-cadherin. Therapeutically, let-7a-5p mimic reversed bleomycin-induced EndoMT and suppressed epithelial-mesenchymal transition (EMT) via paracrine signaling. Mice administered agomir showed reduced fibrosis, improved lung function, and suppressed TGF-{beta}/Smad signaling. Conclusion Serum exosomal let-7a-5p suppresses pulmonary fibrosis by targeting TGFBR1 to inhibit EndoMT. Its downregulation in IPF patients correlates with disease progression, highlighting its biomarker potential.

Goblet cell hyperplasia (GCH) is a hallmark of chronic obstructive pulmonary disease (COPD) and contributes to morbidity and mortality. We investigated the cellular and molecular origins of GCH in COPD using single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and in vitro models. We identified "basal-to-goblet transitional cells" (BGTC) which are transcriptionally and physically located between basal and goblet cells and are characterized by expression of clade B serpins. In vitro studies indicate that inflammatory cytokines, including IL-1{beta}, stimulate basal-to-goblet differentiation through a SERPINB3/4+ intermediate and SERPINB3 overexpression induces differentiation of airway basal cells into IL1B-producing inflammatory goblet cells. In addition, we identified a subset of SERPINB4+ "goblet-variant basal cells" from lungs of COPD patients that express goblet cell and inflammatory genes ex vivo. Together, these results indicate clade B serpins contribute to altered epithelial differentiation and inflammatory signaling in COPD and support therapeutic targeting of clade B serpins to reduce GCH.

Rationale: The role of DNA methylation (DNAm) markers in the upper-airway microbiome related to asthma exacerbations despite inhaled corticosteroid (ICS) treatment is unexplored. Objectives: To identify whole-blood DNAm markers associated with the upper-airway microbiome related to asthma exacerbations despite ICS treatment. Methods: We analyzed salivary, nasal, and pharyngeal microbiome in 242 asthma patients (GEMAS) for discovery. We tested associations between whole-blood DNAm at 703,505 CpGs and 21 exacerbation-related microbiome traits. Replication for the nasal microbiome was evaluated in 272 (Project Viva) and 66 (SysPharmPediA) children with and without asthma. We analyzed differentially methylated regions (DMRs), gene enrichment, associations with blood gene expression (RNA-seq, n=119), and DNAm predictors of plasma proteins. Measurements and Main Results: Whole-blood DNAm at 73 CpGs and 550 DMRs was associated with 17 exacerbation-related microbiome traits (false discovery rate [FDR]<0.05), including genes implicated in immune and inflammatory responses and the 17q12-q21 asthma locus ORMDL3. Most CpGs were associated with Campylobacter (pharyngeal) and Leptotrichia (nasal) abundance. Ten DNAm markers were replicated (p<0.05), including the corticosteroid insensitivity-related gene PTPRR associated with nasal bacterial richness. Enrichment analyses identified genes related to diabetes, obesity, smoking, TGF-{beta}, and Wnt signaling and regulated by trichostatin A, NF-{kappa}B, and the glucocorticoid receptor (FDR<0.05). The CpG cg18994033, associated with pharyngeal Tannerella, was associated with ITM2C whole-blood expression. Nine DNAm-predicted plasma proteins involved in inflammation, tissue remodeling, and TGF-{beta} signaling were associated with seven exacerbation-related microbiome traits, including Streptococcus and Selenomonas. Conclusions: Whole-blood DNAm markers implicated in inflammation, immunity, and asthma comorbidities are associated with the upper-airway microbiome linked to asthma exacerbations.

Background: Chronic obstructive pulmonary disease (COPD) is a complex, heterogeneous disease influenced by genetic and environmental factors. However, the interactions between COPD risk genes and their collective role in COPD susceptibility remain largely elusive. We hypothesize that the protein-protein interaction (PPI) network of genes associated with COPD risk can provide molecular insight into the mechanisms of COPD pathogenesis. Methods: We used affinity purification mass spectrometry (AP-MS) to detect protein-protein interactions of six well-established COPD GWAS gene products (AGER, FAM13A, FBXO38, HHIP, IREB2, MFAP2) in two relevant lung cell lines (IMR90, 16HBE). These genes are located in significant COPD GWAS regions, and their physiological role in COPD has been previously confirmed by functional studies. We analyzed the impact of the newly identified interactions in contextual, cell type-specific PPI networks built from the combination of publicly available PPI data (HUBRIS) and cell type-specific gene expression data. Results: Using AP-MS, we identified 482 unique interactors across 701 newly identified protein interactions with the six GWAS gene products in at least one of the cell lines (AGER: 122, FAM13A: 83, FBXO38: 71, HHIP: 108, IREB2: 8, MFAP2: 309). Subsequent network analysis incorporating these new PPIs with public PPI databases revealed: (1) the new interactions significantly reduce the network distance between known COPD GWAS gene products; (2) 5.2% of the newly identified interactors are differentially abundant between COPD cases and controls in lung proteomic data from the Lung Tissue Research Consortium (LTRC); and (3) 41 (14.3%) of the newly identified interactors were directly connected to more than one of the six COPD GWAS genes. We identified a tightly connected core network of COPD GWAS gene products and protein biomarkers with intersecting signals; this network consists of 8 proteins, including 5 of the 6 GWAS gene products (AGER, FAM13A, HHIP, FBXO38, and MFAP2) as well as CAVIN1, TGM2, and HSPA1A. Conclusions: By combining AP-MS experimental data, multiple types of "omics" data, and network analysis, we have constructed a disease network module for COPD. This module can be a foundation for understanding the collective influence of COPD GWAS gene products in disease pathogenesis.

BackgroundReactive oxygen species (ROS) overproduction, inflammatory cytokines, and plasma mediators disrupt alveolar tight junction integrity and perpetuate oxidative injury in acute respiratory distress syndrome (ARDS). Biological heterogeneity of ARDS, reflected by hyper- and hypoinflammatory subphenotypes with distinct plasma mediator profiles, may contribute to heterogeneity of treatment effect. Stem cell-derived EVs have been shown to exert regenerative and immunomodulatory effects by horizontal transfer of proteins, lipids and genetic material to injured target cells. This study investigated whether extracellular vesicles derived from bone marrow (MSC-EVs) and adipose tissue (ASC-EVs) mesenchymal stem cells restore barrier integrity and redox homeostasis, and whether efficacy differs across subphenotypes. MethodsBronchial epithelial cells (BEAS-2B), microvascular endothelial cells (HMEC-1), and small airway epithelial cells (SAEC) were challenged with plasma from patients with hyperinflammatory or hypoinflammatory ARDS subphenotypes, including those treated with venovenous ECMO. MSC-EVs or ASC-EVs were added concomitantly with plasma exposure or six hours later, at a dose of 5,000 particles/cell. After 24 h, the expression of actin and tight junction proteins, zonula occludens (ZO)-1, occludin, and vascular endothelial (VE)-cadherin, was evaluated by immunofluorescence. Oxidative stress parameters, antioxidant defenses, and antioxidant enzyme activities, were measured. ResultsPlasma from patients with the hyperinflammatory ARDS subphenotype induced marked disruption of ZO-1, occludin, and VE-cadherin expression, together with actin cytoskeletal rearrangement, across BEAS-2B, HMEC-1, and SAEC cells. Barrier disruption was accompanied by increased oxidative stress markers and altered antioxidant enzyme activity. Both MSC-EVs and ASC-EVs restored tight junction protein expression and cytoskeletal organization when administered either concurrently with ARDS plasma or 6 hours after exposure, and both effectively modulated redox parameters. Delayed administration 6 hours after injury produced greater effects for several oxidative stress endpoints, particularly in cells challenged with hypoinflammatory plasma. ConclusionARDS-associated plasma mediators induce subphenotype-dependent impairment of epithelial-endothelial barrier integrity through oxidative mechanisms. Both MSC-EVs and ASC-EVs mitigate this injury, with efficacy that varies according to ARDS subphenotype and timing of administration.

The mechanisms underlying the progression of pulmonary fibrosis in idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases remain unclear. Increased extracellular matrix stiffness is a hallmark of fibrotic lung diseases. Monocyte-derived macrophages can promote fibrosis progression. However, there is limited understanding of how the mechanical properties of the fibrotic microenvironment influence macrophage phenotypes. Profibrotic macrophages are apoptosis-resistant, and this phenotype is modulated by enhanced mitochondrial bioenergetics. The objective of the study was to determine how lung tissue stiffness impacts macrophage phenotypes and fibrotic progression. We demonstrate that mechanoactivated macrophages exhibit apoptosis-resistance, increased expression of the antiapoptotic protein Bcl-xL and increased mitochondrial oxidative phosphorylation. Critically, the metabolic reprogramming observed in mechanoactivated macrophages is dependent on increased glutaminolysis. Inhibition of glutaminolysis attenuated apoptosis resistance in mechanoactivated macrophages. Moreover, inhibition of Bcl-xL in vivo protected mice against experimental pulmonary fibrosis. Lastly, mechanoactivated primary IPF macrophages produce more profibrotic cytokines and promote extracellular matrix production in precision-cut lung slices. We describe a mechanism for acquired macrophage apoptosis resistance dependent on metabolic reprogramming regulated by extracellular matrix stiffness. Our results identify mechanoactivated apoptosis-resistant macrophages as pro-fibrotic mediators, suggesting a novel therapeutic target in IPF and related fibrotic disorders.

Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease characterised by aberrant fibroblast function, extracellular matrix (ECM) remodelling and defective tissue repair. Protein kinase N2 (PKN2) is associated with accelerated forced vital capacity decline in IPF, but its functional role in pulmonary fibrosis remains unknown. We hypothesised that PKN2 regulates fibroblast phenotype and tissue repair. Methods PKN2 expression was assessed in human lung tissue, induced sputum and primary airway and parenchymal fibroblasts from non-fibrotic controls and patients with interstitial lung disease (ILD). DNA methylation was profiled using the Illumina HumanMethylationEPIC array. PKN2 function was investigated by siRNA-mediated depletion in primary human lung fibroblasts using transcriptomic, proteomic and functional analyses. Tissue repair was assessed following pharmacological PKN inhibition in zebrafish. Results PKN2 expression was reduced in ILD lung tissue and primary airway and parenchymal fibroblasts and further suppressed by TGF-{beta}1. Differential methylation was identified across the PKN2 locus in both fibroblast populations. Integrated transcriptomic and proteomic profiling following PKN2 depletion revealed coordinated remodelling of ECM, cell adhesion, non-canonical WNT and VEGF pathways, including dysregulation of COL1A1, WNT, VEGF and MMP1. PKN2 loss increased VEGF and MMP-1 secretion and accelerated fibroblast wound closure. PKN inhibition altered epithelial organisation and collagen fibre alignment during zebrafish wound repair. Conclusion PKN2 loss drives fibroblast reprogramming and aberrant ECM remodelling, establishing PKN2 as an important regulator of pulmonary fibroblast homeostasis and tissue repair.

Post-COVID-19 residual lung abnormalities (RLA) are associated with persistent respiratory symptoms and radiological changes, yet the underlying mechanisms remain unclear. We performed integrated multi-omic profiling of paired bronchoalveolar lavage and blood samples from patients with post-COVID-19 RLA and healthy controls, combining single-cell RNA sequencing, CITE-seq, single-cell T cell receptor sequencing, bronchoalveolar lavage fluid proteomics and functional fibroblast assays. In post-COVID-19 RLA lungs, we identified an increased abundance of profibrotic SPP1hi monocyte-derived alveolar macrophages, arising from an expanded circulating HLA-DRlowCD163+PDE4Dhi classical monocyte progenitor population, supporting a blood-lung myeloid axis. Cell-cell communication modelling positioned macrophages as central hubs of immune-stromal crosstalk, promoting monocyte recruitment with profibrotic priming, and fibroblast activation. Proteomic analysis of bronchoalveolar lavage fluid from post-COVID-19 RLA and idiopathic pulmonary fibrosis, compared with healthy controls, revealed shared and distinct signatures. These alveolar proteins in post-COVID-19 RLA were predominantly attributed to myeloid cells and predicted to engage fibroblast receptors. Bronchoalveolar lavage fluid induced fibroblast proliferation, differentiation and collagen deposition in vitro, with proliferation attenuated by the antifibrotic drug nintedanib. We also identified compartment-specific lymphoid dysregulation, including depletion of mucosal-associated invariant T (MAIT) cells in both the lung and blood, decreased natural killer (NK) cells with oligoclonal T cell expansion in the lung, and expansion of regulatory and cytotoxic T cells in the blood. These findings support a persistent monocyte-macrophage-fibroblast axis linking immune dysregulation to fibroproliferative remodelling after COVID-19 and highlights candidate therapeutic targets for post-viral lung fibrosis. We provide a publicly available atlas (on publication).

Chronic lung allograft dysfunction (CLAD) is a major cause of death after lung transplantation and manifests principally as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Although pulmonary macrophages are key regulators of lung injury and repair, their contributions to CLAD pathogenesis remain under-examined. We performed single-cell RNA sequencing-based transcriptomic analysis of CLAD (7 BOS, 7 RAS) and control lung tissue (n = 6), complemented by comparative and functional analyses. Two distinct macrophage populations were identified in CLAD: inflammatory macrophages expressing leukocyte immunoglobulin-like receptors (LILR), and fibrotic macrophages characterized by expression of osteopontin (SPP1). LILR macrophages were present in both BOS and RAS, whereas SPP1 macrophages were selectively enriched in RAS. Both populations were identified in idiopathic pulmonary fibrosis and cross-tissue comparisons. Surface marker-based sorting strategies were developed to isolate both populations. Functional studies demonstrated that LILR macrophages exhibited enhanced phagocytic activity, promoted T cell chemotaxis and secreted inflammatory mediators, whereas SPP1 macrophages produced soluble factors that drove fibroblast activation and contraction. These findings identify distinct inflammatory and fibrotic macrophage programs associated with CLAD, link RAS to conserved fibrotic macrophage states observed in pulmonary fibrosis and highlight macrophage populations as potential therapeutic targets for inflammatory and fibrotic allograft injury.

Objective: Lymphatic dysfunction has been implicated in exacerbating fibrosis in numerous diseases, yet the role of the lymphatic system in laryngotracheal injury has not previously been explored. This study aims to evaluate lymphatic vascular remodeling in a murine model of laryngotracheal stenosis (LTS) and determine how pharmacologic blockade of lymphangiogenesis impacts airway healing after mucosal injury. Methods: LTS was induced in C57BL6 mice using an established chemomechanical injury model. Lymphatic density was quantified using LYVE-1 immunohistochemistry. Mice were treated with the VEGFR-3-selective tyrosine kinase inhibitor SAR131675 to block lymphangiogenesis after injury. Outcomes assessed included survival, histopathology, immunohistochemistry, and Evans blue dye vascular leakage. Results: Laryngotracheal injury induced a substantial increase in subepithelial lymphatic vessel density concomitant with fibrotic remodeling. Pharmacologic inhibition of VEGFR-3 signaling with SAR131675 abrogated this lymphangiogenic response and resulted in markedly increased mortality, impaired epithelial repair with obstructive sloughing, increased edema, and persistent histopathologic evidence of tissue injury. A qualitative increase in pathologic fibrocellular remodeling was also observed, though with no measurable difference in lamina propria thickness. Conclusion: These findings establish lymphatic remodeling as an essential component of successful airway repair following mucosal injury. Lymphatic dysfunction is a common feature of known risk factors for LTS including diabetes, obesity, and prematurity, and can be exacerbated by positive pressure ventilation. Disruption of the lymphangiogenic response to airway injury may lead to stasis of pro-inflammatory factors that result in chronic inflammation, maladaptive remodeling, and pathologic tissue changes. The lymphatic vasculature is a viable target for future mechanistic study and potential therapeutic intervention following airway injury.

Muco-obstructive lung diseases are characterized by impaired airway clearance and altered mucin composition. MUC5B and MUC5AC are the primary gel-forming mucins in airway mucus, yet how their relative abundance influences the physical and functional properties of mucus remains poorly understood. Here, we investigated how compositional variations in MUC5B and MUC5AC within mucus impact mucociliary transport. Mucus enriched in either MUC5B or MUC5AC was generated using human airway epithelial models depleted for each mucin via CRISPR/Cas9-targeted knockout. Defined mixtures of these mucins were generated at physiologically relevant ratios, for assessment of their microrheological properties and mucociliary transport behavior in differentiated primary human airway epithelial tissue cultures. We found that increasing MUC5AC content reduced network pore size and increased microviscosity, concomitant with reduced mucociliary transport, demonstrating that mucin composition alters the biophysical and functional properties of the mucus barrier. In normal airway tissue cultures with MUC5B-predominant mucus, overlay of MUC5AC on the apical surface impaired mucociliary transport, whereas MUC5B had minimal effect. Conversely, MUC5B supplementation uniquely improved mucociliary transport in IL-13 stimulated cultures exhibiting mucostasis, whereas additional MUC5AC did not alter transport. Together, these findings demonstrate that the ratio of MUC5B to MUC5AC can shape mucus organization at the microscale, which in turn governs mucociliary transport at the tissue scale.

Background: GINA-based clinical assessment of asthma control provides limited insight into the molecular mechanisms driving disease progression and treatment response. Circulating microRNAs (miRNAs) are implicated in immune regulation and airway remodeling, but their relationship to longitudinal, treatment-specific asthma control is not well characterized. We aimed to identify treatment-specific miRNAs associated with longitudinal asthma control and evaluate their ability to discriminate well-controlled from uncontrolled asthma. Methods: Baseline serum miRNA sequencing data from 491 children in the Childhood Asthma Management Program (CAMP), a randomized trial of budesonide versus placebo, were analyzed, with GINA-based composite symptom scores assessed at baseline and 2, 4, 8, and 12 months. Cumulative link mixed models were fitted across 266 miRNAs to identify associations with longitudinal ordinal asthma control, adjusting for time, baseline status, and treatment. Random Forest classifiers were trained within each treatment group using Group K-Fold cross-validation. Pathway enrichment of validated miRNA targets was performed with DAVID. Results: In the budesonide group, hsa-miR-1224-5p was associated with lower symptom severity and hsa-miR-199a-3p|hsa-miR-199b-3p with higher severity; both associations persisted at 12 months. The placebo group showed a broader pattern, with ten miRNAs associated with symptoms. Random Forest classifiers achieved mean AUC of 0.776 (budesonide) and 0.714 (placebo) for 12-month control status. Budesonide-associated targets were enriched for glucocorticoid-responsive and MAPK/Ras signaling, while placebo-associated targets showed broad enrichment for general regulatory processes. Conclusion: Treatment-specific circulating miRNAs distinguish asthma control over time and implicate distinct signaling pathways, supporting their potential as complementary molecular markers for asthma monitoring in children.

Rationale: Fibrotic interstitial lung diseases are progressive disorders characterized by lung scarring and declining respiratory function. Repeated injury and dysregulated epithelial-mesenchymal crosstalk are implicated in disease pathogenesis but remain incompletely understood. Three-dimensional (3D) organoids incorporating epithelial-mesenchymal crosstalk provide a physiologically relevant platform for investigating these mechanisms. Here, we present a 3D tri-co-culture alveolar organoid model and evaluate its responses to two profibrotic stimuli, TGF-{beta} and bleomycin. Methods: CI-huArlo, NCI-H441, and MRC-5 cells were co-cultured for 14 days to generate alveolar-like organoids. Cell marker expression was assessed by immunofluorescence (IF). To test injury response, organoids were exposed to TGF-{beta} (50 ng/mL) or bleomycin (20 g/mL) for 48 hours and characterized. Supernatants were collected to assess interleukin 8 (IL-8) and procollagen I by ELISA. qPCR assessed expression of CDKN1A and COL1A1 following treatments. Bulk RNA sequencing (RNA-seq) evaluated the transcriptomic responses to fibrotic stimulus. Results: Cellular marker expression was confirmed using IF for aquaporin-5 (alveolar type I cell marker) and TE-7 (fibroblast marker). Bleomycin exposure reduced viability and significantly increased IL-8 (126.3 {+/-} 16.86 vs. 48.88 {+/-} 4.470 pg/mL; p = 0.0002; N=6), with no change in secreted procollagen I compared to control. TGF-{beta} stimulation significantly increased secreted procollagen I (149.6 {+/-} 27.07 vs. 53.38 {+/-} 5.672 pg/mL; p <0.0001; N=6) without affecting viability or IL-8 release. qPCR resulted in no change in CDKN1A expression, while COL1A1 expression was increased with TGF-{beta} treatment compared with control and bleomycin. RNA sequencing demonstrated distinct and reproducible transcriptional responses to TGF-{beta} and bleomycin. TGF-{beta} induced 750 significantly upregulated and 641 downregulated genes, including increased COL1A1, COL4A1, FN1, and TGFB1, with enrichment of epithelial-mesenchymal transition and TGF-{beta} signalling programs. In contrast, bleomycin induced 953 significantly upregulated and 552 downregulated genes relative to untreated controls and was characterized by p53 signalling, DNA-damage responses, and reduced cell-cycle progression. Reference-state analysis further indicated reduced normal alveolar epithelial signatures following both treatments, with TGF-{beta} producing the strongest aberrant basaloid and myofibroblast-associated signatures. Conclusions: Tri-culture alveolar-like organoids exhibited stimulus-specific responses in a 3D multicellular system, supporting their use for mechanistic studies of epithelial-mesenchymal crosstalk, environmental exposures, and therapeutic responses.

Rationale: Plasma proteins are used as biomarkers of inflammation and injury in acute respiratory distress syndrome (ARDS). Whether they vary with body size before ARDS is not well established. Objectives: To determine whether ARDS biomarkers vary with body mass index in adults without acute illness. Methods: We analyzed 8,680 ambulatory adults in the All of Us Research Program with proteomics covering 5,420 protein assays. We grouped 35 proteins associated with ARDS risk or mortality into five compartments by the pathophysiological role assigned in a published systematic review, registered in advance. We modeled body mass index as a restricted cubic spline and reported the difference in each protein between 35 and 25 kg/m2 in standard deviations, adjusted for age, sex, smoking, thirteen chronic conditions and ten genetic principal components. Each estimate was ranked against all 5,420 protein assays. Results: Median age was 50 years, and median body mass index was 27.2 kg/m2. Associations with body mass index across all assays were predominantly positive with a median of 0.065 standard deviations. All four epithelial proteins were lower at higher body mass index, each below at least 92% of assays. Soluble receptor for advanced glycation end products was lower by 0.350 standard deviations and surfactant protein A2, surfactant protein D and club cell secretory protein 16 were lower by 0.081 to 0.084 standard deviations. Interleukin-6 and E-selectin were 0.534 and 0.465 standard deviations higher, above 99% of assays. Differences across body mass index extended to lung enriched proteins outside the prespecified panel. Compartments accounted for 49% of the variance in the 35 protein estimates, against 10% under random reassignment (P < 0.001). At fixed body mass index, type 2 diabetes, hypertension and dyslipidemia showed smaller differences in ARDS biomarkers and were not associated with lower epithelial proteins. Conclusions: Plasma proteins used as biomarkers of inflammation and injury in ARDS vary with body size even in the absence of acute illness. Differences during ARDS may not be attributable to injury alone and may be misinterpreted in patients with obesity due to antecedent variation.

Background: Moderate-to-severe asthma affects 15-30% of asthma patients, but accounts for over 50% of healthcare costs and disproportionate morbidity. However, the genetic mechanisms underlying moderate-to-severe versus mild asthma remain poorly understood. Methods: We performed separate pooled multi-ancestry GWAS of mild (N = 14,372) and moderate-to-severe asthma (N = 7,096) versus non-asthma controls (N = 28,816) among adult participants in the NIH All of Us Research Program (version 8). European, African, and Latino/admixed ancestry-specific GWAS were combined using fixed-effect meta-analysis. Lung expression quantitative trait loci (eQTL) and pathway analyses were performed to identify candidate genes and biological pathways across asthma phenotypes. Results: Mild and moderate-to-severe asthma shared susceptibility loci at IL1RL1, IKZF3, and the GTF3AP1-IL33, GTF3AP1-RANBP6, LINC02757-EMSY and HLA-DRB1-HLA-DQA1 regions. Shared lung eQTLs implicated IL18R1, IL18RAP, HLA genes, IL33, LRRC32, GRB7, MIEN1 and GSDMB. In contrast, the phenotypes exhibited distinct genetic architectures. Mild asthma was characterized by broader HLA class II signals and associations at SMAD3 and GSDMB, with enrichment of antigen presentation, T-helper cell differentiation, and TGF-{beta} regulation, consistent with adaptive immune mechanisms. Moderate-to-severe asthma was characterized by WDR36 and PTCH1, with lung eQTLs implicating TSLP, CAMK4, and FANCC. Pathway analysis identified enrichment of IL-13, IL-6, and IL-10 production, myeloid leukocyte differentiation, and oxidative stress responses, consistent with innate inflammation and pathways implicated in steroid resistance. The IL33 signals showed a severity-gradient effect, with stronger associations in moderate-to-severe asthma. Conclusion: Mild and moderate-to-severe asthma exhibit shared and distinct genetic architectures that support existing biologic targets and suggest novel therapeutic candidates.

Background: Sex differences in asthma are well recognized, but sex-associated airway DNA methylation (DNAm) patterns and their relationships with gene expression remain incompletely characterized. We therefore conducted cis- and trans-expression quantitative trait methylation (eQTM) analyses using sex-stratified and CpG-by-sex interaction models. Methods: We analyzed nasal airway epithelial DNAm and transcriptomic data from 298 participants in the CAAPA discovery cohort. Asthma-associated differentially methylated CpGs (DMCs) were identified using sex-combined and sex-stratified models. Cis- and trans-eQTM analyses were then performed in participants with asthma using sex-combined, sex-stratified, and CpG-by-sex interaction models. To prioritize asthma-associated regulatory relationships, eQTMs that were also significant in non-asthmatic controls were excluded from downstream analyses. Replication in an independent bronchial epithelial cohort was defined by concordant effect direction and P < 0.05. Replicated targets underwent pathway and network analyses. Findings: Asthma-associated DMCs were predominantly hypomethylated in females and hypermethylated in males. Among trans-eQTMs, 379 associations were shared across all four models, whereas the majority were model-specific, indicating extensive sex-dependent regulatory architecture. We replicated 3, 1, 9, and 2 cis-eQTMs and 96, 84, 216, and 41 trans-eQTMs in the sex-combined, female-stratified, male-stratified, and interaction models, respectively. Sex-combined targets implicated innate immune and inflammatory signaling (TNF, IRF8); female-stratified targets implicated cytoskeletal remodeling, focal adhesion, and PI3K-Akt signaling (ITGA10); male-stratified targets implicated transcriptional regulation and post-translational modification (MOV10, HDAC7, CSF1, KAT2A); and interaction targets implicated lipid-mediator metabolism and inflammatory signaling and resolution (CYP2C9, LTB4R, CCL19). Our sex-dependent analyses showed that the majority of eQTMs resided within the trans-regulatory architecture, suggesting that sex-dependent gene expression differences are associated predominantly with trans rather than cis regulatory mechanisms. Pathway analyses revealed enrichment of innate immunity in the sex-combined model, structural remodeling in females, transcriptional control in males, and inflammatory resolution in the sex-interaction model. Interpretation: Sex-specific and sex-dependent airway methylation-expression associations were largely undetected by sex-combined analysis. Sex-aware analytical models may help resolve epigenetic mechanisms underlying sex differences in asthma biology.

Background: Although the lung is of low biomass, it harbors a diverse and dynamic microbiome that may influence disease and healing. Existing studies have used diverse sampling methods with high propensities for contamination and sampling error, leading to diverse and unclear results. Here, we characterized the lung microbiome via airway and parenchymal samples to determine variation across patients and sampling methods. Methods: We recruited adult patients undergoing lung resection for suspected or confirmed malignancy. After resection and under sterile conditions, a 1 cm cubic piece of non-cancerous lung parenchyma and a swab from the specimen's bronchus were collected and sent for microbiome analysis via 16S rRNA gene amplicon (V4) sequencing on an Illumina platform. An established bioinformatics pipeline was used to determine taxonomic identification. Baseline clinical and demographic data were compared to microbiome composition. Results: A total of 86 patients were included in the study. Beta diversity (microbial composition) varied significantly by sampling method (biopsy of lung parenchyma versus airway swabs), so all further results were analyzed within sample types. Further analyses revealed significant differences in beta diversity by lobe of the lung, indicating a different microbial composition by anatomic location. Analyses of patient demographics revealed significant differences by age and comorbidities, including chronic obstructive pulmonary disease and atrial fibrillation. Conclusions: The lung harbors a diverse microbiome that differs by anatomic location and patient characteristics. This study provides a framework for more accurate future lung microbiome sampling and characterization.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal lung disease with a median survival of 3-5 years after diagnosis. Current antifibrotic therapies slow disease progression, but do not halt or reverse fibrosis, underscoring the need for new therapies. We identified a dysregulated oxysterol-GPR183 axis as a driver of IPF. Oxidized cholesterols were elevated in lungs from IPF patients, with myofibroblasts representing the dominant source of 7,25-hydroxycholesterol (7,25-OHC), the endogenous high affinity ligand for the oxysterol-sensing receptor GPR183. IPF patients had increased GPR183 expression in interstitial and monocyte-like macrophages compared to controls. In a bleomycin-induced model of pulmonary fibrosis genetic deletion of GPR183 reduced disease severity characterized by reduced fibrosis, inflammation, and accumulation of macrophages and myofibroblasts. Pharmacological inhibition of GPR183 with the antagonist NIBR189 attenuated fibrosis when administered preventatively from day 1-7 after bleomycin exposure. Notably, therapeutic treatment with the GPR183 antagonist after commencement of fibrosis development at day 10 post-bleomycin also significantly reduced fibrotic pathology, achieving efficacy comparable to the approved antifibrotic nintedanib. However, the GPR183 antagonist was more potent in reducing inflammation and myofibroblast activation compared to nintedanib. Together, these findings identify an oxysterol-GPR183 signaling axis that contributes to pulmonary fibrogenesis and provide a strong preclinical rationale for targeting GPR183 as a novel therapeutic strategy for IPF. One Sentence SummaryTargeting GPR183 reduced lung fibrosis and inflammation in a preclinical model, supporting GPR183 as a promising new therapy.