American Journal of Respiratory Cell and Molecular Biology

Cigarette smoking induces complex signaling disruptions that contribute to diseases such as COPD and lung cancer, yet the molecular mechanisms underlying these effects remain incompletely understood. To address this gap, we analyzed peripheral blood from 3190 COPDGene participants using LIONESS and PUMA and constructed miRNA-mRNA regulatory networks associated with smoking status. Comparing networks for active versus former smokers uncovered a striking shift in regulatory architecture: active smokers exhibited elevated miRNA targeting of the mitochondrial complex I protein NDUFA12. This finding was validated in lung tissue expression data from the Lung Genomics Research Consortium (LGRC), where we observed that ever-smokers showed consistent dysregulation of Ndufa12-targeting miRNAs compared to never-smokers. This allowed us to identify a set of smoking-defined circulating and tissue-associated miRNAs. To investigate the specific cellular compartment, we analyzed cell-type deconvoluted expression data from COPDGene blood and LTRC (Lung Tissue Research Consortium) lung tissue, as well as lung transcriptomics data from cigarette smoke-exposed mice, and identified the monocyte/macrophage compartment as a principal site of NDUFA12/Ndufa12 expression. Human THP-1 macrophages treated with cigarette smoke extract demonstrated selective inhibition of NDUFA12 by network-defined miRNAs. These distinct, NDUFA12-targeting, smoking-associated miRNA signatures, revealed through network analysis, describe new smoking-mitochondrial interactions that may serve as novel targets for therapeutic intervention.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive, chronic lung disease with limited therapeutic options. Tryptophan metabolism is significantly dysregulated during lung fibrogenesis, with the metabolite kynurenine (kyn) accumulating in lung tissue and driving pathology via the aryl hydrocarbon receptor (AHR). This study evaluates the cell-specific contributions of kyn-mediated AHR signaling across different pulmonary cell types to clarify its role in disease progression. MethodsUsing a murine model of bleomycin-induced pulmonary fibrosis, lung tryptophan metabolites were profiled via liquid chromatography-mass spectrometry. The functional and transcriptomic impacts of kyn administration and AHR modulation were subsequently characterized across three distinct cellular compartments: CD103+ dendritic cells (DCs), fibroblasts, and alveolar epithelial cells (AECs). ResultsKyn levels were elevated in fibrotic lungs, and exogenous kyn selectively exacerbated collagen deposition during the fibrogenic phase rather than altering acute injury. In vitro monocultures of primary lung fibroblasts and AECs revealed negligible functional responses to kyn or AHR inhibition regarding myofibroblast differentiation, migration, or epithelial barrier disruption. Intriguingly, primary tissue-resident CD103+ DCs exhibited a hyperinflammatory, non-canonical AHR signaling profile in vivo. While ex vivo monoculture rapidly reverted these DCs to an anti-inflammatory, canonical AHR state, directly co-culturing DCs with fibrotic primary lung fibroblasts successfully restored the pathogenic, non-canonical signaling phenotype characterized by augmented IL-6 production and suppressed canonical targets. ConclusionsPathogenic AHR signaling in pulmonary fibrosis is highly cell-context dependent and driven by complex cell-cell interactions. Reductionist monocultures fail to replicate tissue- level dendritic cell phenotypes, highlighting the necessity of co-culture models and providing a cautionary note for the systemic clinical use of AHR-targeted therapeutics.

Chronic inflammation induced by cigarette smoke (CS) plays a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD), but its impact on lung epithelial progenitor function and regenerative capacity remains incompletely understood. Here, we combined in vivo and in vitro approaches to dissect how CS exposure and subsequent inflammatory insults shape epithelial repair dynamics. A 6-week whole-body CS exposure model in mice induced lung function impairment and altered gene expression profiles in alveolar epithelial cells, prominently activating interferon (IFN)-related pathways and the cGAS-STING axis. Alveolar epithelial cells from CS-exposed mice generated a similar number, but larger organoids with reduced alveolar differentiation compared to air-exposed mice. Notably, these cells obtained from CS-exposed mice displayed resistance to IFN{gamma}-induced suppression of organoid growth, contrasting with the strong inhibitory effect of IFN{gamma} observed in controls. This phenotype was recapitulated in a two-hit in vitro model using cigarette smoke extract (CSE), in which chronic CSE exposure impaired regeneration and differentiation while inducing resistance to IFN{gamma}. Gene expression and proteomic analyses revealed upregulation of Zbp1, Irf7, and other upstream IFN regulators, correlating negatively with alveolar differentiation potential. Inhibition of the cGAS-STING pathway with RU.521 partially rescued organoid formation, increased proliferation, and alveolar differentiation. Together, our data reveal that CS exposure alters the alveolar epithelial landscape, inducing a stress-adapted, IFN{gamma}-resistant state that compromises alveolar regeneration, with cGAS-STING activation as a key driver of early CS-associated alveolar type 2 cell dysfunction. These findings provide new insight into how chronic inflammation reshapes progenitor cell function during early lung injury.

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.

Proteostasis declines with lung aging, while the role of the Unfolded Protein Response (UPR) in lung aging and age-associated pulmonary diseases remains understudied. We investigated how deficiency in the UPR sensor ATF6 affects physiological and smoke exposure-accelerated lung aging. ATF6 -deficient mice exhibited accelerated alveolar simplification, a sign of lung parenchymal aging, which was exacerbated by smoking. Nevertheless, small airway vascular fibrotic remodeling, a prominent smoking induced pathology, was not evident in smoke-exposed ATF6 -deficient mice. Mechanistically, these divergent phenotypes arose from cell-type-specific ATF6 programs. In alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of the lung parenchyma, ATF6 maintained mitochondrial bioenergetics and sustained efficient re-differentiation into alveolar epithelial type 1 cells (AEC1s). In lung pericytes, ATF6 promoted extravasation, re-differentiation into myofibroblast-like cells, and production of collagens 1 and 3. These findings identify ATF6 as a cell-type-specific regulator of differentiation programs during lung aging and highlight the need to study ATF6 under defined physiological and pathological contexts before therapeutically targeting this pathway.

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.

Rationale: Fibrotic hypersensitivity pneumonitis (fHP) is an antigen-driven, life-threatening interstitial lung disease characterized by heterogeneous radiologic features, clinical outcomes, and treatment responses. Objectives: To identify blood-based fHP endotypes that inform mechanism, prognosis and therapeutic response. Methods: We performed integrative analyses of multi-compartment transcriptomic data derived from whole blood, peripheral blood mononuclear cells, bronchoalveolar lavage, and surgical lung biopsies, alongside circulating plasma proteomics. Multiple clustering algorithms were cross-compared to ensure robustness and reproducibility of endotypes identification. Immune cell composition was inferred using bulk RNA-seq deconvolution and annotated with BAL single-cell RNA-seq. Pathway activities were characterized using Gene Set Enrichment Analysis. Transplant-free survival (TFS) was evaluated for endotype and corticosteroid exposure by Kaplan-Meier methods, with hazard ratios analyzed using multivariable Cox proportional hazards models. Results: Two molecular endotypes, lymphocytic-associated (L-fHP) and non-lymphocytic-associated (N-fHP), were identified and validated. L-fHP showed enrichment of adaptive immune signaling and lymphocyte predominance, whereas N-fHP demonstrated myeloid-cell activation with neutrophil and macrophage predominance. Corticosteroid exposure was associated with worse TFS in L-fHP but not in N-fHP after adjusting for age, sex, and baseline pulmonary function. Compared to L-fHP, N-fHP had poorer baseline pulmonary function, faster 12-month FVC decline, and shorter TFS. N-fHP also exhibited elevated neutrophil-associated markers, including matrix metalloproteinase-9, across paired transcriptomic and proteomic datasets, supporting a neutrophil-driven, cross-compartment disease process. Conclusion: Multi-omic, multi-compartment analysis identifies two reproducible fHP endotypes with distinct clinical outcomes and corticosteroid responses, supporting a precision medicine approach beyond current clinical and radiologic classification.

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory airway disease in which impaired mucosal barrier function may increase susceptibility to aspirated oral microbial products. Periodontal disease has been associated with COPD development and exacerbation, but the epithelial mechanisms linking periodontal pathogens to pulmonary immune remodeling remain unclear. Here, we investigated whether gingipain-containing Porphyromonas gingivalis culture supernatant (PCS) promotes {gamma}{delta} T-cell-associated inflammation in COPD-like airways. Repeated intratracheal administration of PCS to {beta}ENaC-transgenic mice induced airway-centered immune cell accumulation and increased {gamma}{delta} TCR-positive cell accumulation, together with elevated expression of the {gamma}{delta} T-cell-associated cytokines Ifng and Il17a. PCS also increased pulmonary Ccl20 and Ccr6 expression, whereas epithelial alarmin-related genes and M2 macrophage-associated responses were not induced in parallel. In ENaC-overexpressing human airway epithelial cells, PCS induced CCL20 and F2RL1, the gene encoding protease-activated receptor 2 (PAR-2), and reduced the N-terminal PAR-2 signal, consistent with proteolytic receptor cleavage. Direct PAR-2 activation reproduced CCL20 induction, whereas pharmacological PAR-2 inhibition suppressed PCS-induced CCL20 expression. In contrast, PAR-1 inhibition or LPS neutralization with polymyxin B did not suppress this response. These findings support a mucosal epithelial protease-sensing model in which gingipain-containing P. gingivalis products activate PAR-2-dependent CCL20 production in airway epithelial cells and are associated with CCR6-linked {gamma}{delta} T-cell accumulation in COPD-like airways.

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.

Mutations in the bone morphogenetic protein receptor 2 (BMPR2) are a major genetic driver of pulmonary arterial hypertension (PAH), yet their penetrance is strikingly sex-biased: females are disproportionately affected, while males experience poorer outcomes. While hormonal and chromosomal factors have been implicated, the biological basis for this disparity remains not fully understood. Here, we investigated the role of the lung microbiome in sex-linked PAH pathogenesis. We hypothesized that increased BMPR2 mutation penetrance in females is partly driven by the accumulation of potent vasoactive molecules, such as endothelin-1 (ET-1), in response to lung microbiome dysbiosis. Using humanized Bmpr2+/R899X mice, we integrate lung metagenomics with basic functional immune profiling to show that females develop a distinct microbiome profile, characterized by increased microbial-derived lipopolysaccharide (LPS), potentially fueling the pathogenic effects of the estrogen metabolite 16-hydroxyestrone (16-OHE). These signals converge on macrophages, where co-exposure led to a hyperactivated state characterized by enhanced phagocytosis and ET-1 secretion. Tissue-level analyses confirmed immune cell infiltration and spatial association with elevated ET-1, providing evidence that these factors may contribute to the onset of sex-linked PAH. Taken together, these findings identify a previously unrecognized microbiome-estrogen-immune axis that amplifies BMPR2 dysfunction and provides a mechanistic basis for female-biased disease penetrance.

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.

Stem cell dysfunction and loss of renewal capacity are primary characteristics of tissue aging and decremental regeneration in response to injury. Alveolar type 2 cells (AT2) are key progenitor cells responsible for lung repair and are thought to be dysfunctional in diseases such as chronic obstructive pulmonary disease (COPD). AT2 cells are highly metabolic and rely on mitochondria, but how mitochondrial mechanisms influence their maintenance and cell fate is unclear. This gap is critical as no current therapies target lung repair or mitochondrial function in COPD. Here, we report that adenine nucleotide translocase 2 (ANT2), a key ATP/ADP transporter, is reduced in AT2 cells from COPD lungs, and that ANT2 loss impairs bioenergetics (ATP). We also identify, for the first time, ferroptotic susceptibility as a consequence of ANT2 loss in AT2 cells, leading to impaired self-renewal and progenitor capacity in alveolar organoids. Together, loss of ANT2 and the associated cellular dysfunction resulted in worsened lung damage or emphysema due to cigarette smoke in mice. Therapeutic restoration of ANT2 expression resulted in renewed AT2 stem cell function and prevention of emphysema by reducing oxidative stress and ferroptosis. These findings highlight the importance of ANT2 in metabolic regulation, plasticity, and cell resiliency of AT2 cells in the lung and that ANT2 is a potential target for lung repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/737954v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@96d0caorg.highwire.dtl.DTLVardef@165b15dorg.highwire.dtl.DTLVardef@15f86baorg.highwire.dtl.DTLVardef@862745_HPS_FORMAT_FIGEXP M_FIG C_FIG

Single-cell transcriptomic profiling of chronic obstructive pulmonary disease (COPD) lungs identified QKI, an RNA-binding protein, as a candidate emphysema-associated gene, but its epithelial role in COPD pathobiology remains unclear. We show that QKI expression is reduced in human COPD lungs and that alveolar type 2 epithelial (AT2) cell QKI protein levels correlate strongly with spirometric indices and diffusing capacity (DLCO). Lung epithelium-specific QKI knockout mice (QKI{Delta}/{Delta}) developed spontaneous airspace enlargement with emphysema-like mechanics, and QKI-deficient AT2 cells showed impaired spheroid colony formation and increased apoptosis. Integrated transcriptomic and proteomic analyses of primary AT2 cells revealed a selective reduction in functional mitochondrial (respiratory-chain and metabolic) protein abundance despite relatively preserved transcript levels, consistent with mitochondrial transcriptome-proteome discordance. QKI loss increased mtDNA abundance and TOMM20 staining but decreased ATP5A, indicating accumulation of structurally increased but functionally dysfunctional mitochondria. In human epithelial cells, CRISPR-mediated QKI deficiency reduced oxidative respiration, increased glycolytic reliance, elevated mitochondrial ROS and membrane potential, and increased apoptosis; these phenotypes were partially rescued by QKI re-expression. These findings identify epithelial QKI as a regulator of mitochondrial integrity and stress tolerance in COPD.

Background: Alveolar macrophage (AM) dysfunction contributes to Acute Respiratory Distress Syndrome (ARDS) pathogenesis. We investigated the role of extracellular vesicles (EVs) in mediating this dysfunction. Methods: Pulmonary EVs were isolated from broncho-alveolar lavage and non-directed bronchial lavage samples of ventilated sepsis patients with and without ARDS, and post-operative control patients via ultracentrifugation. AMs were isolated from lung tissue resections of lobectomy patients. AMs were treated with pooled EVs for 24 hours prior to functional, metabolic and autophagy profiling. EV cargo was profiled via small RNA transcriptomics and proteomics. Mechanistic role of EV microRNAs was assessed via mimic / antagomir transfection. Results: Pulmonary EVs from sepsis patients with ARDS impaired AM efferocytosis, and control EVs had no effect. ARDS EV treatment enhanced AM mitochondrial-linked respiration, but not glycolysis. ARDS EV treatment impaired LC3B-II and LAMP1 expression, indicating dysregulated AM autophagy-lysosomal machinery. Proteomics revealed downregulation of innate immune pathways in ARDS EVs. Transcriptomics revealed enrichment of 24 microRNAs in ARDS EVs; miR-652-3p was the most enriched, validated by RT-qPCR. EV miR-652-3p was associated with 90-day mortality (9.20 vs 0.59 RQ, p=0.0295) and inversely correlated with oxygenation (PaO2/FiO2). AM transfection with miR-652-3p mimic induced similar dysregulation of function and autophagy as ARDS EVs. Transfection of ARDS EVs with antagomirs to miR-652-3p prior to AM treatment partially rescued efferocytosis and autophagy. Conclusions: Targeting EV miR-652-3p may restore alveolar macrophage function and reduce excessive inflammation, thus offering a novel therapeutic strategy for patients with ARDS.

Tissue hypoperfusion is common in clinical settings, but how tissues respond to hypoperfusion on a microphysiological scale is not clear. We used real-time confocal microscopy of live, perfused lungs to gain insights into the effects of hypoperfusion on the microcirculation and microphysiology of lung alveoli, where gas exchange occurs. We focused on effects of hypoperfusion on alveolar liquid secretion, since alveolar liquid secretion is important for alveolar homeostatic functions. Our findings show lung hypoperfusion stimulated a reversal of alveolar liquid transport, from homeostatic liquid secretion to absorption. Specifically, lung perfusion at or near physiological perfusion pressure led to alveolar liquid secretion that depended on the alveolar epithelial cystic fibrosis transmembrane conductance regulator (CFTR), Na+-K+-Cl- cotransporters, and the Na+/K+-ATPase. Within minutes of halting lung perfusion or majorly reducing it, alveoli stopped secreting liquid and instead absorbed it via the epithelial Na+ channel, CFTR, and K+-Cl- cotransporters. We provide evidence that hypoperfusion caused alveolar microvessel lumens to shrink and airspaces to expand, leading to epithelial stretch that stimulated liquid absorption. These findings show lung hypoperfusion initiates mechanical signals that stimulate the alveolar epithelium to absorb liquid, and they may inform the pathogenesis of lung diseases characterized by acute microvascular hypoperfusion.

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.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by irreversible destruction of the alveolar epithelium and impaired regeneration. Although current therapies slow disease progression, they do not restore functional alveoli, highlighting the need for regenerative approaches that promote endogenous lung repair. Here, we performed the first unbiased functional screen of 2,042 human microRNA mimics in primary mouse alveolar type II (ATII) cells to identify regulators of ATII-to-alveolar type I (ATI) cell transdifferentiation. The screen identified miR-124-3p as the most effective promoter of ATI differentiation. In vitro, miR-124-3p promoted ATII-to-ATI transdifferentiation in healthy and bleomycin-injured ATII cells while also increasing the ATII cell pool, consistent with activity on epithelial progenitors. Using the engineered AAV6.2FF capsid, we generated a vector encoding miR-124-3p, which efficiently transduced ATII cells, MHC-II club distal progenitor cells, and injury-induced KRT8 epithelial intermediates. Therapeutic administration after fibrosis establishment reduced lung fibrosis, restored alveolar architecture, and showed greater efficacy than nintedanib in the bleomycin mouse model. Mechanistically, we propose a context-dependent model whereby miR-124-3p regulates epithelial cell states through the EZH2-C/EBP axis while attenuating epithelial transcriptional programs associated with IPF. Together, these findings support AAV-mediated delivery of miR-124 to promote alveolar repair in pulmonary fibrosis.

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.

Background: Plasma proteomics may identify host-response signatures in sepsis, but it is unclear whether extracellular vesicle (EV)-enriched plasma provides distinct or redundant information compared with plasma. We compared paired plasma and EV-enriched plasma proteomes in critically ill patients with sepsis and critically ill non-sepsis controls (CINS). Methods: In this prospective observational study, paired plasma and EV-enriched plasma samples were analyzed from 56 critically ill adults, including 40 patients with sepsis and 16 CINS patients. Protein abundance was quantified using liquid chromatography-tandem mass spectrometry. Analyses compared proteomic depth, protein overlap, global concordance between compartments, and differential protein abundance between CINS and sepsis. Exploratory Gene Ontology enrichment was performed as a supplementary analysis. Results: EV-enriched plasma expanded proteomic detection, identifying 2,476 filtered proteins compared with 506 in plasma. Only 386 proteins were detected in both compartments, while 2,090 were unique to EV-enriched plasma and 120 were unique to plasma. Among shared proteins, plasma and EV-enriched plasma showed modest global concordance across critically ill patients (Spearman coeff = 0.322, p = 9.19 x 10^-11), with similar findings in sepsis alone. Differential abundance analysis identified 11 sepsis-associated proteins in plasma and 22 in EV-enriched plasma. Only SAA1, SAA2, and IGFBP6 were significant in both compartments. Exploratory pathway analysis supported acute-phase and inflammatory enrichment in plasma sepsis-associated proteins, while EV-enriched signals were directionally plausible but did not meet prespecified FDR thresholds. Conclusion: Plasma and EV-enriched plasma proteomics capture related but nonredundant sepsis-associated host-response information in critically ill patients.