American Journal of Respiratory and Critical Care Medicine

In 1992, inhaled NO (iNO) at low doses entered the practice of medicine for cardiopulmonary indications. Recently, iNO at higher doses has been tested in diverse pulmonary infections. However, nothing is known about the ability of exogenous NO gas to kill Mycobacterium tuberculosis (Mtb), the leading cause of death from infection between major viral pandemics. Here we mimicked exposure conditions used in recent human studies of high-dose iNO to explore the effects of NO gas against Mtb in vitro and in mice. We saw a profound bactericidal effect of NO gas in vitro against Mtb incubated in shallow, mildly acidic fluid. Mtb-infected mice tolerated inhaled NO well, except for developing more methemoglobinemia than humans at the same level of exposure. In Mtb-infected mice with poorly aerated pulmonary infiltrates, inhaled NO had an anti-inflammatory effect but did not reduce the bacterial burden. These results may help inform the decision whether to test inhaled NO as an adjunctive treatment for tuberculosis, and if so, in what settings and with what goals.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by excessive extracellular matrix deposition and irreversible architectural distortion of the lung. Fibrotic remodeling is driven by dynamic interactions among endothelial, fibroblast, epithelial and immune cells. Although pulmonary endothelial cells (ECs) are increasingly recognized as important contributors to IPF pathogenesis, the molecular and cellular events underlying endothelial dysfunction remains poorly understood. Using integrative multi-omics analyses of human IPF lungs combined with functional in vitro assays, we identify ACKR1-expressing venous endothelial cells (ACKR1+ VECs) as critical regulators of a pathogenic niche that promotes lung fibrosis. Single-cell RNA sequencing and spatial transcriptomics analyses reveal that ACKR1+ VECs exhibit a distinct pro-fibrotic and pro-inflammatory transcriptional program enriched for hypoxia responses, extracellular matrix remodeling, and immune cell recruitment. In both mouse and human fibrotic lungs, ACKR1+ VECs localize adjacent to fibroblastic foci and are surrounded by pro-fibrotic CD68+/CCR5+/SPP1+ macrophages-monocytes, suggesting a spatial organized cellular crosstalk supporting fibrotic remodeling. Consistent with these findings, in vitro co-culture assays using ACKR1+ VECs isolated from IPF lungs demonstrate that these cells drive myeloid recruitment and fibroblast activation through ACKR1 dependent mechanisms. Silencing of ACKR1 in IPF-derived VECs suppressed inflammatory and fibrotic transcriptional programs, and pharmacological inhibition of ACKR1 attenuated stromal and immune remodeling and reduced bleomycin-induced lung fibrosis in vivo. Together, these findings identify ACKR1+ VECs as key orchestrators of fibrosis progression and establish ACKR1 and the pathogenic vasculature as promising therapeutic targets for IPF. Clinical RelevanceIdiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited treatment options. We identify ACKR1-expressing venous endothelial cells as key drivers of inflammatory and fibrotic remodeling and show that pharmacologic inhibition of ACKR1 attenuates experimental lung fibrosis. These findings establish endothelial ACKR1 as a promising therapeutic target and highlight the pulmonary vasculature as a novel avenue for disease-modifying therapies in IPF.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease associated with failed alveolar epithelial repair with an expansion of aberrant airway-like epithelium in the alveolar space leading to lung function decline usually resulting in death within 3-5 years of diagnosis. While single-cell and spatial transcriptomic approaches have been used to characterize disease-emergent cell populations, less is known about the regulation of transcriptional programs that drive failed alveolar epithelial cell repair in IPF. DNA methylation is a fundamental layer of gene regulation that stabilizes differentiated cell identity; however, changes in methylation in the epithelial compartment in IPF have not been studied. To identify novel epigenetic mediators of epithelial cell dysfunction, we performed high-resolution DNA methylation profiling of purified distal lung epithelial cells from 10 age-matched control and 14 IPF lungs using Oxford Nanopore Technologies (ONT) whole-genome, long-read sequencing. We identified widespread methylome remodeling in the IPF lung epithelium, with 84% of differentially methylated regions (DMRs) hypomethylated. DMRs were largely found outside of promoters, with 88% outside of 3 kb from the transcription start site (TSS), consistent with altered distal regulatory element activity. DMRs were enriched for transcription factor (TF) binding site motifs and gene associations consistent with dysregulation of polycomb repressive complex 2 (PRC2) and increases in p63 activity. DNA methylation at DMRs associated with p63 target genes, including KRT5, and genes implicated in failed epithelial repair, including MUC5B, MMP7, and S100A2 inversely correlated with gene expression. Consistent with the observed dysregulation of the IPF epithelial methylome, an experimental co-culture model of alveolar type II epithelial cell (AT2s) to basal-like epithelial cell transdifferentiation revealed widespread hypomethylation. Further, sites associated with NKX2.1 and FOXA1/2 binding, TFs involved in alveolar fate maintenance, were hypermethylated, suggesting loss of epigenetic regulation of alveolar identity. Together, these data implicate DNA methylation in the failed alveolar epithelial repair processes in IPF, potentially providing future therapeutic strategies by identifying putative regulatory elements associated with aberrant transcriptional programs.

Extracellular vesicles (EVs) are potential sources of circulating biomarkers, but disease-associated EVs are often scarce and their nanoscale dimensions limit analysis by conventional flow cytometry. To improve the detection of disease-associated EV signals using widely available instruments, we developed a workflow combining preparation of serum EV-enriched fractions by size-exclusion chromatography, CHL1-initiated enzyme-mediated activation of radical sources (EMARS) proximity labeling, and conventional flow cytometry. EMARS deposits fluorescein on molecules proximal to antibody-bound CHL1, thereby increasing target-initiated fluorescence. In a representative comparison, EMARS increased the signal-to-background ratio from 1.21 with conventional antibody labeling to 6.28. Because unlabeled EVs and debris interfered with analysis, we used an AI-assisted procedure to explore FITC-H cutoffs for retained P1 events. ChatGPT compared 65 candidate cutoffs across statistical and event-retention metrics and recommended 1000; the authors reviewed the complete results before adopting this value. At this data-selected cutoff, mean fluorescence intensity was higher in interstitial lung disease (ILD) than in non-ILD samples among 28 evaluable samples (P = 0.002; area under the receiver operating characteristic curve [AUC] = 0.846); neither value was adjusted for cutoff selection. Post hoc nested leave-oneout cross-validation (LOOCV), in which cutoff selection was repeated within each outer fold, yielded an AUC of 0.716 with 29 of 34 samples evaluable. The association with ILD requires validation in independent cohorts, but these exploratory findings suggest that proximity labeling may enhance measurable fluorescence contrast in serum EV-enriched fractions and support further evaluation of disease-associated EV signals using conventional flow cytometers.

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.

Rationale: Conventional CT-derived pulmonary vascular metrics quantify vascular pruning but do not assess the spatial heterogeneity of vascular architecture. In COPD, peripheral microvascular pruning coexists with compensatory central vessel dilation, paradoxically preserving total vessel volume while reducing effective vascular penetration into lung parenchyma. Objectives: To develop and evaluate Vascular Proximity Coverage (VPC), a distance-based CT biomarker quantifying the spatial efficiency of pulmonary vascular distribution. Methods: This retrospective analysis included 7,881 COPDGene participants [PHASE] with complete vascular segmentations. VPC was estimated by integrating the area under the cumulative lung tissue coverage curve at distances of 0-10 mm from segmented vessel voxels. Multivariable models assessed associations with lung function, six-minute walk distance (6MWD), St. George's Respiratory Questionnaire (SGRQ) score, mMRC dyspnea score, and all-cause mortality over a median follow-up of [10.8] years, adjusting for demographics, smoking, emphysema, scanner type, and BV5. The cohort was stratified at median BV5 and VPC into concordant (high/high, low/low) and discordant (high-BV5/low-VPC, low-BV5/high-VPC) vascular phenotypes. Results: VPC declined across disease severity (5.82 {+/-} 0.50 [UNITS] in non-smokers to 5.21 {+/-} 0.64 in GOLD 4; p < 0.001). Among spirometrically normal smokers, current smokers had lower VPC than former smokers (5.31 vs. 5.68; p < 0.001). Each standard deviation decrease in VPC was independently associated with reduced 6MWD ({beta} = -16.8 m, 95% CI: -19.5 to -14.0), worse SGRQ ({beta} = 1.9, 95% CI: 1.4 to 2.4), and greater dyspnea (RR = 1.05, 95% CI: 1.03 to 1.08; all p < 0.001). The lowest VPC quartile was associated with 38% higher mortality than the reference quartile (HR = 1.38, 95% CI: 1.18 to 1.62; p < 0.001). The high-BV5/low-VPC phenotype showed 24% higher mortality than high-BV5/high-VPC (HR = 1.24, 95% CI: 1.08 to 1.42; p = 0.002) despite preserved vessel volume. Conclusions: VPC captures spatial heterogeneity of vascular architecture independent of vessel volume, is reduced in smokers without airflow obstruction, and identifies excess mortality risk among individuals whose total vessel volume appears preserved.

Pulmonary hypertension (PH) is a progressive and fatal disease characterized by pulmonary vascular remodeling, inflammation, and immune dysregulation. Myeloid-derived suppressor cells (MDSCs) and macrophages contribute to PAH pathobiology; however, the molecular regulators of their pathological activation remain poorly defined. The triggering receptor expressed on myeloid cells 2 (TREM2) is an immunomodulatory receptor that shapes myeloid cell metabolism, survival, and immunosuppressive function, yet its role in PH has not been investigated. Methods: Single-cell RNA sequencing (scRNA-seq) data from human pulmonary artery tissue (GSE210248; n=3 PAH, n=3 donors) were analyzed to characterize TREM2 expression across cell populations. Wild-type (WT) and global TREM2 knockout (TREM2 KO) mice were exposed to chronic hypoxia (10% FiO2, 28 days). Hemodynamic, histological, flow cytometric, and ex vivo functional assessments were performed. TREM2 expression was measured by flow cytometry in circulating MDSCs from PAH patients (n=22) and healthy controls (n=13). Results: TREM2 was markedly enriched in monocyte/macrophage populations in PH pulmonary arteries and TREM2-high immune cells exhibited transcriptional downregulation of chemotaxis programs and upregulation of antigen processing and MHC II presentation pathways. TREM2 deficiency significantly attenuated hypoxia-induced RVSP increase, right ventricular dysfunction, pulmonary inflammation, and vascular remodeling. MDSCs from TREM2 KO mice exerted significantly less suppression of CD4+ and CD8+ T cell proliferation. TREM2 was significantly elevated in circulating MDSCs from PAH patients and showed a directional association with hemodynamic severity. Conclusions: TREM2 is a novel regulator of myeloid-driven immunosuppression and vascular remodeling in PAH and warrants investigation as a therapeutic target and biomarker.

Redox homeostasis requires both inducible antioxidant defense and constitutive machinery that continuously regenerates cellular reducing capacity. How these systems are altered across lung cell populations in chronic obstructive pulmonary disease (COPD) remains unclear. We integrated donor-level single-nucleus lung transcriptomes with smoke-exposure models, independent cohorts, and large-scale pharmacologic perturbation data. Both redox programs were reduced in established COPD, but constitutive redox support showed the greater loss, concentrated in fibroblasts and lymphatic endothelium. In fibroblasts, PGC-1 target-program output showed the most negative effect among 515 regulator programs despite preserved PPARGC1A expression. Smoke exposure activated inducible antioxidant defense across experimental models, whereas this adaptive program was reduced in established COPD. Pharmacologic perturbations further separated the two arms of redox homeostasis, with redox-directed interventions producing larger transcriptional responses in inducible defense and a distinct set of compounds increasing constitutive support together with PGC-1 target output and movement away from an independently defined COPD fibroblast state. These findings identify a cell-selective loss of constitutive redox support in COPD and implicate reduced PGC-1 target-program output in the fibroblast redox phenotype.

ObjectivesThe preferential phosphodiesterase 4B (PDE4B) inhibitor nerandomilast was recently approved for treatment of idiopathic pulmonary fibrosis (IPF) and progressive pulmonary fibrosis. Its proposed immunomodulatory, anti-fibrotic, and endothelial-stabilising actions target all three cardinal features of SSc, yet PDE4B expression has not been systematically characterised in SSc tissue. We aimed to define PDE4B expression across fibrotic organs and cellular compartments in SSc. MethodsPDE4B expression was profiled in SSc lung, peripheral blood mononuclear cells (PBMCs) and skin on the transcript level using single-cell RNA sequencing data and on the protein level using immunohistochemistry, immunofluorescence and multiplexed immunofluorescent stainings. ResultsPDE4B was consistently dysregulated in immune cells across SSc tissue and PBMCs, with compartment-specific direction and distribution. In SSc-ILD lung, expression was increased in CD8 and CD4 memory T-cells. In PBMCs, expression was increased in B cells, monocytes, and CD8 T-cells, and stratified patients into three endotypes (PDE4B//hi) not distinguishable by clinical variables. In skin, bulk RNA-seq showed a significant global increase, which localized to myeloid cells in scRNA-seq data. Approximately 90% of FAP activated fibroblasts co-expressed PDE4B at the protein level in SSc skin, identifying the activated fibroblast compartment as a candidate target for PDE4B inhibition. No PDE4B dysregulation was detected in vascular cell types. ConclusionsThis first cell-type-resolved characterisation of PDE4B in SSc demonstrates consistent immune-cell dysregulation across tissues and protein-level enrichment in activated fibroblasts. This provides a human-tissue rationale for the immunomodulatory and anti-fibrotic effects of PDE4B inhibition and supporting PDE4B as a disease-relevant therapeutic target in SSc. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LINerandomilast (BI 1015550), a PDE4B-preferential inhibitor, was approved for idiopathic pulmonary fibrosis and progressive pulmonary fibrosis. C_LIO_LIPre-clinical studies indicate that PDE4B inhibition may act on all cardinal features of SSc. C_LI What this study addsO_LIFirst cell-type-resolved characterization of PDE4B expression across SSc-affected lung, PBMCs, and skin. C_LIO_LIPBMC PDE4B expression is heterogeneous, stratifying patients into PDE4B// endotypes independent of standard clinical variables. C_LIO_LIscRNA-seq shows increased myeloid PDE4B expression in SSc skin, while [~]90% of FAP activated fibroblasts in SSc skin express PDE4B protein. C_LI How this study might affect research, practice or policyO_LIThe study strengthens the human-level evidence underpinning the target rationale for PDE4B inhibition in SSc. C_LI

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.

BackgroundAsthma exacerbations are caused by dysregulated cellular interactions between airway and immune cell populations. Circulating microRNAs (miRNAs) are potential biomarkers for asthma exacerbations; however, their target airway cells remain poorly defined. ObjectiveTo identify the cell types that are regulated by the circulating microRNAs linked to asthma exacerbations and the extent to which the cells are regulated by miRNAs. MethodsWe integrated a curated panel of exacerbation-associated circulating miRNAs with single-cell RNA sequencing (scRNA-seq) profiles from induced sputum of 16 asthma patients and 8 healthy controls. Experimentally validated miRNA-target interactions were combined with cell-type-specific differential expression. Elastic Net regression and SHAP analysis quantified gene-level regulatory contributions, yielding a composite Regulation Strength metric. Findings were validated against four independent GEO datasets. ResultsImmune cells, including monocytes, dendritic cells, and macrophages, demonstrated the strongest statistically significant miRNA regulatory signals, in contrast to airway epithelial cells.hsa-miR-222-3p showed opposing regulatory effects in mature versus alveolar macrophages, indicating differentiation-state-dependent activity, while B_Plasma cells showed no detectable regulatory effect from any miRNA tested. Independent GEO validation confirmed higher expression of protective miRNAs (hsa-miR-126-3p, hsa-miR-146b-5p) in healthy individuals, consistent with prior CAMP cohort associations. ConclusionCirculating miRNAs show cell-type-specific regulatory activity, strongest in monocytes, dendritic cells, and macrophages. hsa-miR-222-3p showed opposing regulatory directions between macrophage subtypes, while B_Plasma cells showed no effect, validated across independent GEO cohorts.

Pulmonary lymphatic vessels are essential for interstitial fluid balance and macromolecular clearance, yet the endothelial mechanisms regulating lymphatic vessel function during chronic fibroproliferative lung injury remain undefined. Here, we demonstrate that the lineage-defining transcription factor ERG in lymphatic endothelial cells (LECs) governs junctional plasticity and fluid drainage in pulmonary fibrosis. In human idiopathic pulmonary fibrosis (IPF) lungs, lymphatic ERG expression is markedly downregulated. Lineage-specific inducible deletion of Erg in murine LECs (Erg-CKO) unexpectedly conferred robust protection against bleomycin-induced pulmonary fibrosis, significantly dampening acute inflammation, reducing edema, and preserving pulmonary compliance. Mechanistically, loss of ERG enhanced lymphatic drainage capacity in vivo. Transcriptomic profiling of isolated LECs revealed that ERG deficiency activates actomyosin contractile pathways and selectively upregulates Endothelin Receptor Type B (Ednrb), driving junctional remodeling from continuous zippers into discontinuous button-like configurations that facilitate interstitial fluid entry. Pharmacological blockade of EDNRB with BQ-788 abolished junctional reorganization, eliminated the enhanced lymphatic drainage, and completely reversed the anti-fibrotic protection in Erg-CKO mice. Together, our findings identify an endothelial ERG-EDNRB regulatory axis that controls lymphatic junctional architecture and demonstrate that promoting lymphatic clearance via endothelial junctional remodeling represents a viable therapeutic strategy for fibrotic vascular remodeling.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by airway remodeling, which may extend to the central airways and alter tracheal morphology. Whether tracheal shape relates to disease severity, quality of life, functional capacity, or survival is not established. Objective: To determine whether CT-derived tracheal lumen eccentricity is associated with cross-sectional and longitudinal clinical outcomes in COPD. Methods: We analyzed 8,383 participants from the multicenter COPDGene study. At each axial cross-section of the trachea, lumen eccentricity ({epsilon}) was computed from the major and minor axis lengths, and the participant-level value was taken as the arithmetic mean across all tracheal slices; {epsilon} = 0 denotes a circular lumen and values approaching 1 denote progressively elongated ellipses. Baseline outcomes were spirometry, 6-minute walk distance (6MWD), St. George's Respiratory Questionnaire (SGRQ) total score, and modified Medical Research Council (mMRC) dyspnea score. Longitudinal outcomes were annualized change in FEV1 and all-cause mortality. Multivariable linear and Cox proportional hazards models were adjusted for [age, sex, race, BMI, smoking status, pack-years, scanner model]. Results: Tracheal eccentricity rose monotonically with severity of airflow obstruction (0.60{+/-}0.07, 0.61{+/-}0.07, 0.62{+/-}0.07, 0.64{+/-}0.08, and 0.64{+/-}0.08 in GOLD stages 0 through 4, respectively). Higher eccentricity was independently associated with lower FEV1 ({beta} = -0.30 L, 95% CI -0.47 to -0.14; p < 0.001), lower FEV1/FVC ({beta} = -0.06, 95% CI -0.09 to -0.04; p < 0.001), shorter 6MWD ({beta} = -15.7 m, 95% CI -25.2 to -6.2; p = 0.001), higher SGRQ score ({beta} = 16.9, 95% CI 11.2 to 22.5; p < 0.001), higher mMRC dyspnea score ({beta} = 0.4, 95% CI 0.1 to 0.6; p = 0.007), and accelerated lung function decline ({beta} = -23.8 mL/year, 95% CI -45.13 to -2.48; p = 0.029). Eccentricity was also associated with all-cause mortality (adjusted HR 1.81, 95% CI 1.01 to 3.26; p = 0.047). Conclusions: Tracheal lumen eccentricity is associated with worse quality of life, reduced functional capacity, accelerated lung function decline, and mortality in COPD, supporting central airway morphology as a candidate imaging biomarker.

Background: Obesity is associated with airway disease, but the contribution of specific thoracic adipose depots remains unclear. We examined whether intrathoracic adipose tissue (ITAT), defined as adipose tissue within the thoracic cavity excluding pericardial adipose tissue (PAT), was associated with airway phenotypes. Methods: We included Framingham Heart Study participants with CT-derived thoracic adipose tissue measures. Cross-sectional associations of ITAT with CT-based airway wall and lumen measurements, spirometry, and respiratory symptoms were evaluated using multivariable regression. Primary analyses used sex-specific ITAT z-scores and adjusted for waist circumference to account for central obesity. Secondary analyses examined sex-specific ITAT quintiles and compared ITAT and PAT in individual-depot and mutually adjusted models. Results: Among 2,825 adult participants (52.0% female), after adjustment for waist circumference, each 1-SD higher ITAT, relative to the sex-specific distribution, was associated with greater segmental ({beta} [95% CI]: 0.010 mm [0.006, 0.014]) and subsegmental airway wall thickness (0.006 mm [0.002, 0.009]), narrower segmental airway lumen diameter (-0.049 [-0.088, -0.011]), lower FEV1 percent predicted (-1.92 percentage points [-2.72, -1.12]), and higher odds of wheezing (OR [95% CI]: 1.16 [1.02, 1.31]) and dyspnea (1.22 [1.05, 1.42]). Associations were similar after further adjustment for PAT and across ITAT quintiles. In contrast, PAT was not independently associated with airway structural measures after adjustment for ITAT. Conclusion: Higher ITAT volume was associated with greater airway wall thickness and narrower airway lumen, as well as lower FEV1 and greater respiratory symptoms, independent of central obesity and pericardial adiposity. These findings support a depot-specific association of ITAT with airway disease.

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.

The alveolar sac architecture is essential for efficient gas exchange and must be precisely maintained throughout life; however, how this delicate structure is preserved during adult regeneration remains poorly understood. Using a mouse pneumonectomy model, we found that Lgr6+ Hhip+ ductal myofibroblasts, a poorly characterized mesenchymal population, are indispensable for maintaining alveolar integrity during lung regrowth. Comprehensive characterization using single-cell transcriptomics, mouse genetics, and pharmacological assays demonstrated that these ductal myofibroblasts secrete myogenic factors, most notably CCN4, to reactivate a myogenic program that converts them into contractile PA-DMFs, thereby preserving alveolar architecture. Lineage-tracing further revealed that these ductal myofibroblasts originate from embryonic MCAM- SMA+ distal progenitors via subepithelial TGF-{beta} signaling, serving as a lifelong guardian of alveolar structural integrity. Notably, cross-species analysis identified an analogous population of LGR6+ fibromyocytes in human respiratory bronchioles. Together, these findings indicate ductal myofibroblasts as a developmentally programmed cell population that reactivate a contractile program to structurally support the regeneration of adult lungs.

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.

BackgroundRight ventricular (RV) adaptation determines outcomes in pulmonary arterial hypertension (PAH), yet multicellular molecular programs associated with adaptive versus maladaptive RV remodeling in living humans remain incompletely defined. MethodsWe collected 32 human RV tissue biopsies from patients with idiopathic PAH, systemic sclerosis-associated PAH (SSc-PAH), systemic sclerosis without pulmonary hypertension, with 24 nonfailing donor RVs serving as controls. We performed single-nucleus RNA sequencing and integrated cell-type specific transcriptional programs with contemporaneously obtained multi-beat pressure-volume loop measurements of RV contractility (Ees, end-systolic elastance) and RV-pulmonary arterial coupling (the ratio of Ees to Ea, the effective arterial load). SSc modification of PAH-associated biology was assessed using interaction terms. Bulk RV proteomic pathway enrichment was performed to assess an orthogonal molecular layer, and exploratory cell-cell communication analyses alongside independent spatial transcriptomic analyses were performed to contextualize key findings. ResultsPAH was associated with broad depletion of biosynthetic, trafficking, and mitochondrial programs across cell types. SSc modified the magnitude of many PAH-associated transcriptional programs while largely preserving pathway directionality. Significant multicellular pathway enrichments were associated with RV-pulmonary arterial coupling. Joint analysis of Ees, Ea, and Ees/Ea identified biologic programs associated with different RV responses to varying loading conditions. Preserved coupling was characterized by enriched extracellular matrix, laminin-integrin, receptor tyrosine kinase, mitochondrial, and translational programs involving fibroblast, endothelial, endocardial, and cardiomyocyte compartments. Cell- cell communication analyses predicted coordinated stromal-vascular signaling networks involving laminin-integrin and endothelial-to-mural signaling in preserved coupling. Proteomic and spatial analyses supported recurrent multicellular themes. ConclusionsRV adaptation in PAH is associated with distinct, coordinated multicellular programs that vary with load and contractile response. RV-PA coupling in PAH is associated with multicellular remodeling that extends beyond cardiomyocytes and reflects organized vascular-stromal support architecture. These findings identify extracellular matrix, laminin- integrin signaling, mitochondrial, and translational programs as associated with adaptive RV remodeling in PAH. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LICell type-resolved molecular profiling of living human RV tissue identifies PAH-associated depletion of biosynthetic, trafficking, mitochondrial, and repair-associated programs across multiple cardiac cell types. C_LIO_LIIntegration with contemporaneously obtained pressure-volume loop physiology demonstrates that preserved RV-pulmonary arterial coupling under lower load was associated predominantly with cardiomyocyte mitochondrial and metabolic competency, whereas preserved coupling under higher load was associated with extracellular matrix remodeling and vascular-stromal signaling. C_LIO_LIProteomic, cell-cell communication, and spatial analyses provided orthogonal support for coordinated extracellular matrix and vascular-stromal programs associated with preserved RV-pulmonary arterial coupling. C_LI What are the clinical implications?O_LIThese findings shift the biology of RV adaptation from a predominantly cardiomyocyte- centered model toward a multicellular tissue model in which metabolic, matrix, and vascular support programs vary according to loading conditions and contractile states. C_LIO_LIExtracellular matrix-integrin signaling, endothelial-mural communication, and mitochondrial competency represent candidate pathways for mechanistic investigation toward RV-directed therapies in PAH. C_LI

Using routinely recorded ventilator data from 16 preterm infants with respiratory distress syndrome, dynamic compliance trajectories after surfactant administration were smoothed with a moving-median filter and characterized with a four-parameter logistic model; 10 infants showed sigmoidal improvement, and among 11 infants who received a second surfactant dose, 6 showed subsequent improvement, suggesting that this approach can visualize and quantify individual compliance recovery as a complementary tool to conventional clinical indicators.

Introduction: Idiopathic inflammatory myopathy-associated interstitial lung disease (IIM-ILD) is a major cause of morbidity and mortality. We tested whether quantitative myositis-specific autoantibodies and proteomic profiling capture biological heterogeneity and prognosis beyond categorical serology. Methods: Myositis-specific autoantibodies were quantified using the luciferase immunoprecipitation systems assay, and 184 serum proteins were measured in 226 IIM patients; 199 with higher-ILD-risk autoantibodies (Jo-1/MDA5/PL-7/PL-12/EJ), 27 with lower-ILD-risk autoantibodies (Mi-2/NXP2/TIF1{gamma}) and 35 healthy controls. We identified shared and subgroup-specific differences by comparing each subgroup with controls, then correlated quantitative autoantibody and protein levels within higher-risk subgroups. Additional analyses included pathway enrichment, unsupervised clustering, longitudinal lung-function change, and mortality. Results: Higher-ILD-risk subgroups shared interferon-responsive CXCR3 chemokine, IL-6/JAK/STAT3, and apoptosis signaling. Dominant autoantibody subgroup profiles differed: interferon/CXCR3 chemokine signaling with T-cell activation and monocyte recruitment in anti-Jo-1; proteostasis/antigen-processing and vascular/cellular stress signals in anti-MDA5; IL-6/macrophage and profibrotic signals in anti-PL-12; and apoptotic and innate immune activation with metabolic/redox-stress signals in anti-PL-7. Within higher-ILD-risk subgroups, autoantibody levels correlated with interferon-response, profibrotic, and metabolic/vascular proteins (r=0.40-0.74; nominal p<0.05). Unsupervised clustering identified four proteomic endotypes beyond autoantibody type, including an injury-stress endotype associated with worse lung function and poorer survival, and a chemokine/checkpoint-high endotype with relatively preserved lung function. Across 203 participants with 38 deaths, a weighted 10-protein score was associated with all-cause mortality (HR, 3.28; 95% CI, 2.12-5.08; p<0.001). Conclusions: Integrated quantitative autoantibodies and proteomic profiling revealed shared inflammatory biology, autoantibody-associated signatures, and an injury-stress endotype associated with poor survival in IIM-ILD, supporting risk stratification beyond categorical serology.