American Journal of Respiratory Cell and Molecular Biology

Inflammation is a key driver of cystic fibrosis (CF) lung disease, not addressed by current standard care. Improved understanding of the mechanisms leading to aberrant inflammation may assist the development of effective anti-inflammatory therapy. Single-cell RNA sequencing (scRNA-seq) allows profiling of cell composition and function at previously unprecedented resolution. Herein, we seek to use multimodal single-cell analysis to comprehensively define immune cell phenotypes, proportions and functional characteristics in preschool children with CF. We analyzed 42,658 cells from bronchoalveolar lavage of 11 preschool children with CF and a healthy control using scRNA-seq and parallel assessment of 154 cell surface proteins. Validation of cell types identified by scRNA-seq was achieved by assessment of samples by spectral flow cytometry. Analysis of transcriptome expression and cell surface protein expression, combined with functional pathway analysis, revealed 41 immune and epithelial cell populations in BAL. Spectral flow cytometry analysis of over 256,000 cells from a subset of the same patients revealed high correlation in major cell type proportions across the two technologies. Macrophages consisted of 13 functionally distinct sub populations, including previously undescribed populations enriched for markers of vesicle production and regulatory/repair functions. Other novel cell populations included CD4 T cells expressing inflammatory IFN/{beta} and NF{kappa}B signalling genes. Our work provides a comprehensive cellular analysis of the pediatric lower airway in preschool children with CF, reveals novel cell types and provides a reference for investigation of inflammation in early life CF.

BackgroundTwo molecular phenotypes of the acute respiratory distress syndrome (ARDS) with divergent clinical trajectories and responses to therapy have been identified. Classification as "hyperinflammatory" or "hypoinflammatory" depends on plasma biomarker profiling. Differences in pulmonary biology underlying these phenotypes are unknown. MethodsWe analyzed tracheal aspirate (TA) RNA sequencing (RNASeq) data from 41 ARDS patients and 5 mechanically ventilated controls to assess differences in lung inflammation and repair between ARDS phenotypes. In a subset of subjects, we also analyzed plasma proteomic data. We performed single-cell RNA sequencing (scRNASeq) on TA samples from 9 ARDS patients. We conducted differential gene expression and gene set enrichment analyses, in silico prediction of pharmacologic treatments, and compared results to experimental models of acute lung injury. FindingsIn bulk RNASeq data, 1334 genes were differentially expressed between ARDS phenotypes (false detection rate < 0.1). Hyperinflammatory ARDS was characterized by an exaggerated innate immune response, increased activation of the integrated stress response, interferon signaling, apoptosis, and T-cell activation. Gene sets from experimental models of lipopolysaccharide lung injury overlapped more strongly with hyperinflammatory than hypoinflammatory ARDS, though overlap in gene expression between experimental and clinical samples was variable. ScRNASeq demonstrated a central role for T-cells in the hyperinflammatory phenotype. Plasma proteomics confirmed a role for innate immune activation, interferon signaling, and T-cell activation in the hyperinflammatory phenotype. Predicted candidate therapeutics for the hyperinflammatory phenotype included imatinib and dexamethasone. InterpretationHyperinflammatory and hypoinflammatory ARDS phenotypes have distinct respiratory tract biology, which could inform targeted therapeutic development. FundingNational Institutes of Health; University of California San Francisco ImmunoX CoLabs; Chan Zuckerberg Foundation; Genentech

ObjectiveChronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear. DesignUsing an in vivo mouse model of cigarette smoke-induced COPD and fecal microbial transfer (FMT), we characterized the fecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology, and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in sixteen COPD patients using a randomized, double-blind, placebo-controlled pilot study of inulin supplementation. ResultsFMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in cigarette smoke-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes. ConclusionThe gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically. What is already known on this topicO_LIChanges in gut microbiota are associated with COPD but the underlying host and microbial mechanisms are unclear, limiting the therapeutic applications. C_LI What this study addsO_LIMicrobiome composition and metabolism is reproducibly correlated with lung and gastrointestinal pathology in experimental COPD. C_LIO_LIMicrobiome modifying interventions effectively alleviate disease, including protective effects supplementing smoking cessation. C_LIO_LINutritional interventions targeting the microbiome in COPD patients demonstrate efficacy in a small pilot study. C_LI How this study might affect research, practice or policyO_LIMicrobiome-targeting therapeutics and nutritional interventions may be developed for COPD, including as supplements to smoking cessation. C_LI

1Acute respiratory distress syndrome (ARDS) and pediatric ARDS (PARDS) can be triggered by multiple pulmonary and non-pulmonary insults and are the source of substantial morbidity and mortality. The nasal and lower conducting airways have similar cell composition and nasal transcriptomes identify disease state and sub-classes in lung cancer, COPD, and asthma. We conducted an observational, prospective trial to determine whether this technique could identify PARDS endotypes in 26 control and 25 PARDS subjects <18 admitted to the pediatric ICU. RNA from inferior turbinate brushing was collected on days 1, 3, 7, and 14. Standard RNA-processing yielded 29% usable specimens by mRNA-Seq, and a low-input protocol increased yield to 95% usable specimens. 64 low-input specimens from 10 control and 15 PARDS subjects were used for model development. Control and some PARDS subjects clustered together in Group A while some day 1, 3, and 7 specimens clustered into Groups B and C with specimens from these subjects moving to Group A with PARDS resolution. In multivariate analysis, the only clinical variables associated with specimen Group B or C assignment was severity of lung injury or viral PARDS trigger. Compared to Group A, Group B had upregulation of innate immune processes and Group C had upregulation of ciliary and microtuble processes. Analysis of the 15 standard processing specimens identified the same grouping. Mortality trended higher in group B (25%) and C subjects (28.6%) compared to A (5%, p=0.1). Comparison of groups with 16 PARDS-associated serum biomarkers identified correlation of Endotype B with Tumor Necrosis Factor-, but not other inflammatory cytokines and Endotype C with Surfactant Protein D. We identified three nasal transcriptomic PARDS endotypes. A is similar to control. B is marked by an innate immune signature only weakly reflected in the serum. C may be associated with loss of epithelial barrier integrity. Nasal transcriptomics may be useful for prognostic and predictive enrichment in future PARDS trials. ClinicalTrials.gov Identifier NCT03539783

The identification of clinically predictive serum biomarkers for pulmonary fibrosis is a significant challenge and important goal. Multiple recent proteomic biomarker studies have identified latent transforming growth factor binding protein-2 (LTBP2) as a circulating factor associated with disease progression in fibrotic lung diseases in humans (including IPF), but its role in the development of fibrosis is incompletely defined. LTBP2 competes with the large latent transforming growth factor-beta (TGF{beta}) complex (LLC) for binding to the N-terminus of fibrillin and is thought to promote the release of active TGF{beta}. We hypothesized that LTBP2 deficiency would promote LLC sequestration in matrix and reduce TGF{beta} signaling. We recently reported an LTBP2 knockout (Ltbp2-/-) mouse with no baseline lung abnormalities. Here we show that Ltbp2-/- mice exposed to either bleomycin or silica have a significant reduction in fibrosis compared to wild type controls. Consistent with reduced fibrosis, after bleomycin Ltbp2-/- mouse lungs have reduced TGF{beta} signaling and isolated fibroblasts from Ltbp2-/-mice exhibit impaired migration in an in vitro wound closure assay. Transcriptomic analysis of bleomycin-treated control and Ltbp2-/- mouse lung tissue identified multiple LTBP2-regulated genes, including the lncRNA antisense of IGFR2 non-coding RNA (Airn) which has reported antifibrotic effects. Interestingly, we also observed that Ltbp2-/- mice had impaired epithelial repair after bleomycin treatment, a phenotype that also occurred in a naphthalene model of club cell injury. These findings provide evidence that LTBP2 is profibrotic and facilitates TGF{beta} signaling but is also required for normal airway epithelial repair.

Bacterial pneumonia poses a significant challenge to public health, often leading to antibiotic treatment failure. Enhancing innate immunity represents a promising adjunctive strategy to conventional antibiotic therapy. Bacterial flagellin, a Toll-like receptor 5 (TLR5) agonist, has been shown to stimulate innate immune defenses when delivered via the respiratory route, demonstrating efficacy in both preventing and treating bacterial pneumonia in murine models. This protective effect is primarily mediated through TLR5-driven activation of airway epithelial cells. This study aimed to characterize the immunomodulatory effects of flagellin on human primary respiratory epithelium. Using the MucilAir air-liquid interface model and RNA sequencing, we demonstrated that apical administration of flagellin induced robust immune responses in airway epithelium derived from healthy individuals, as well as patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). TLR5-mediated epithelial signaling triggered key immune-related pathways, including cytokine production, leukocyte chemotaxis, neutrophil recruitment, and antimicrobial defense, with strong commonalities across healthy and diseased airway epithelia. Furthermore, we demonstrated that flagellin effectively activated epithelial immune responses even in the presence of the bacteria Pseudomonas aeruginosa or Streptococcus pneumoniae. However, epithelial activation alone was insufficient to directly limit bacterial colonization or replication, highlighting the potential role of epithelial-immune cell interactions in achieving effective bacterial clearance. These findings support TLR5 activation as a promising therapeutic strategy to enhance host defense mechanisms and improve treatment outcomes for bacterial pneumonia in both healthy individuals and patients with COPD or CF.

Obstructive sleep apnea and its characteristic intermittent hypoxia (IH) are widely recognized as significant contributors to various pulmonary diseases, including asthma, pulmonary arterial hypertension, fibrosis, and chronic obstructive pulmonary disease. While single-cell RNA sequencing (scRNA-seq) has provided valuable insights into cell-type-specific responses to IH, previous studies have primarily focused on post-hypoxic recovery states, leaving immediate molecular responses during active IH exposure unexplored. To address this critical knowledge gap, we investigated real-time transcriptional responses to IH at single-cell resolution in lung tissue using male mice (n=3/group) exposed to either normoxia or IH (30 cycles/h, nadir 6% O2, 12 h/day) for 14 days, with tissue collection during active IH exposure. Our analysis revealed pronounced cell-type-specific transcriptional reprogramming, particularly in airway smooth muscle cells (ASMC), arterial endothelial cells (AEC), and lymphatic endothelial cells (LEC). These changes were characterized by enrichment in pathways related to epithelial-to-mesenchymal transition (ASMC, LEC), myogenesis (ASMC), and antioxidant defenses (AEC, LEC). Most cell types demonstrated substantial upregulation of genes encoding mitochondrial complex I-IV proteins and TCA cycle enzymes accompanied by a decreased expression of genes encoded by mitochondrial DNA that was markedly present in LEC, AEC, and cells of the alveolar-capillary unit, revealing a mito-nuclear imbalance. These findings provide novel insights into the immediate cellular responses to IH, showing previously uncharacterized metabolic reorganization that may underlie the development of IH-related pulmonary complications. This improved understanding of early molecular events during active IH exposure advances our knowledge of sleep apnea-related lung pathologies and may inform future therapeutic strategies.

The heterogeneity of fibroblasts in the murine and human lung during homeostasis and disease is increasingly recognized. It remains unclear if the different phenotypes identified to date are characteristic of unique subpopulations with unique progenitors or whether they arise by a process of differentiation from common precursors. Our understanding of this ubiquitous cell type is limited by an absence of well validated, specific, markers with which to identify each cell type and a clear consensus on the distinct populations present in the lung. Here we describe single cell RNA sequencing (scRNA-seq) analysis on mesenchymal cells from the murine lung throughout embryonic (E) development (E9.5 - 17.5), at post-natal day (P1 - 15), as well as in the adult and the aged murine lungs before and after bleomycin-induced fibrosis. We carried out complementary scRNA-seq on human lung tissue from a P1 lung, a month 21 lung and lung tissue from healthy donors and patients with idiopathic pulmonary fibrosis (IPF). The murine and human data were supplemented with publicly available scRNA-seq datasets. We consistently identified lipofibroblasts, myofibroblasts, pericytes, mesothelial cells and smooth muscle cells. In addition, we identified a novel population delineated by Ebf1 (early B-cell factor 1) expression and an intermediate subtype. Comparative analysis with human mesenchymal cells revealed homologous mesenchymal subpopulations with remarkably conserved transcriptomic signatures. Comparative analysis of changes in gene expression in the fibroblast subpopulations from age matched non-fibrotic and fibrotic lungs in the mouse and human demonstrates that many of these subsets contribute to matrix gene expression in fibrotic conditions. Subtype selective transcription factors were identified and putative divergence of the clusters during development were delineated. Prospective isolation of these fibroblast subpopulations, localization of signature gene markers, and lineage-tracing each cluster are under way in the laboratory. This analysis will enhance our understanding of fibroblast heterogeneity in homeostasis and fibrotic disease conditions.

Many acute and chronic lung diseases affect the distal lung alveoli. Although airway-derived human cell lines exist, alveolar epithelial cell (AEC)-derived lines are needed to better model these diseases. We have generated and characterized novel immortalized cell lines derived from human AECs. They grow as epithelial monolayers expressing lung progenitor markers SOX9 and SOX2, with little to no expression of mature AEC markers. Co-cultured in 3-dimensions (3D) with lung fibroblasts, the cells form NKX2-1+ organoids expressing mature AEC markers AQP5 and GPRC5A. Single-cell RNA sequencing of an AEC line in 2D versus 3D revealed increased cellular heterogeneity and induction of cytokine and lipoprotein signaling, consistent with organoid formation. Activating WNT and FGF pathways resulted in larger organoids. Our approach appears to yield lung progenitor lines that retain a genetic and structural memory of their alveolar cell lineage despite long-term expansion and whose differentiation may be modulated under various 3D conditions. These cell lines provide a valuable new system to model the distal lung in vitro.

RationaleExposure to humidifier disinfectants has been linked to an array of pulmonary disorders and diminished lung functionality particularly reduced Forced Vital Capacity (FVC). ObjectivesThis investigation sought to identify diagnostic biomarkers for early detection of children at elevated risk of developing chronic respiratory conditions following such exposure. MethodsOur research employed a comprehensive multi-omics strategy analyzing 70 pediatric patients alongside 10 controls, seamlessly integrating clinical assessments with transcriptomics, methylomics, proteomics, and metabolomics data. The analytical framework utilized a sophisticated combination of Non-negative Matrix Factorization (NMF), Multi-Omics Factor Analysis (MOFA), and advanced machine learning algorithms. Measurements and Main ResultsNMF clustering uncovered distinctive protein expression patterns associated with integrin-mediated signaling pathways and immune response mechanisms. Complementarily, MOFA identified latent factors correlating with lung function metrics, highlighting critical molecular pathways involved in integrin cell surface interactions and lipid metabolism regulation. Machine learning-based analysis facilitated the development of a multi-marker panel-comprising IGHV2-70, LysoPC (16:0), and hexadecyl ferulate-which achieved 81.46% accuracy in identifying pulmonary dysfunction cohort. ConclusionsThese findings suggest that alterations in integrin-related signaling networks and dysregulation of lipid metabolism play pivotal roles in mediating the long-term pulmonary consequences of humidifier disinfectant exposure. The proposed multi-marker panel offers significant potential for enhanced risk stratification and timely therapeutic intervention. At a Glance CommentaryO_ST_ABSScientific Knowledge on the SubjectC_ST_ABSExtensive epidemiological evidence has established the causal relationship between humidifier disinfectant exposure and pulmonary dysfunction; however, clinically validated biomarkers for predicting chronic lung disease progression remain limited. Pediatric populations demonstrate unique pathophysiological mechanisms distinct from adults, highlighting the critical necessity for biomarker identification grounded in comprehensive molecular understanding. Despite advances in omics technologies, recent investigations have encountered significant obstacles in achieving deeper mechanistic insights, predominantly attributable to methodological constraints in harmonizing clinical phenotypes with high-dimensional molecular datasets. What This Study Adds to the FieldThis investigation elucidates the fundamental contributions of integrin-mediated signaling cascades and lipid metabolic networks to persistent pulmonary dysfunction following humidifier disinfectant exposure. Our analyses revealed coordinated regulation of integrin signaling pathways and immune response networks through NMF clustering, indicating dynamic temporal evolution of inflammatory responses during chronic disease progression, with temporally distinct molecular signatures identified across discrete observation intervals. Multi-omics factor analysis (MOFA) corroborated integrin pathway dysregulation while additionally uncovering systematic suppression of lipid metabolic processes. Furthermore, machine learning algorithms enabled development of a robust three-component biomarker panel--encompassing IGHV2-70, LysoPC (16:0), and hexadecyl ferulate--demonstrating 81.46% classification accuracy for pulmonary dysfunction phenotypes. Collectively, these findings substantially advance mechanistic understanding of chronic lung injury in vulnerable pediatric cohorts and identify clinically relevant biomarkers with translational potential for risk stratification and therapeutic targeting in clinical practice.

Neutrophil extracellular traps contribute to lung injury in cystic fibrosis and asthma, but the mechanisms are poorly understood. We sought to understand the impact of human NETs on barrier function in primary human bronchial epithelial and a human airway epithelial cell line. We demonstrate that NETs disrupt airway epithelial barrier function by decreasing transepithelial electrical resistance and increasing paracellular flux, partially by NET-induced airway cell apoptosis. NETs selectively impact the expression of tight junction genes claudins 4, 8 and 11. Bronchial epithelia exposed to NETs demonstrate visible gaps in E-cadherin staining, a decrease in full-length E-cadherin protein and the appearance of cleaved E-cadherin peptides. Pretreatment of NETs with alpha-1 antitrypsin (A1AT) inhibits NET serine protease activity, limits E-cadherin cleavage, decreases bronchial cell apoptosis and preserves epithelial integrity. In conclusion, NETs disrupt human airway epithelial barrier function through bronchial cell death and degradation of E-cadherin, which are limited by exogenous A1AT. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/484920v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@841dbforg.highwire.dtl.DTLVardef@1bd0188org.highwire.dtl.DTLVardef@1afaf97org.highwire.dtl.DTLVardef@130fd03_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundExtensive comorbidity between cardiovascular (CVD) and respiratory (RT) diseases is well-documented, yet the shared genetic mechanisms remain elusive. Genetic pleiotropy may play a pivotal role in understanding the intricate comorbidity patterns associated with cardiovascular and respiratory conditions. MethodsOur comprehensive analysis encompasses the largest available GWAS dataset of European ancestry covering six major CVDs (atrial fibrillation, coronary artery disease, venous thromboembolism, heart failure, peripheral arterial disease, and stroke) and four prevalent RTs (asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and sleep apnea). Initially, we aimed to unveil the common genetic basis of major CVDs, through genome-wide and local genetic correlations and polygenic overlap. Subsequently, the shared genetic mechanisms between RTs and CVDs was investigated in terms of both horizontal and vertical pleiotropy. From a horizontal pleiotropy perspective, cross-trait analysis was utilized to identify pleiotropic genetic determinants including genomic loci, single nucleotide polymorphisms (SNPs), genes, biological pathways, and protein targets. From a vertical pleiotropic perspective, Mendelian randomization was employed to evaluate potential causal relationships between CVDs and RTs. ResultsOur study confirmed the significant existence of genetic correlations and overlaps between CVDs and RTs. Pleiotropy analysis under the composite null hypothesis identified 17,964 significant potential pleiotropic SNPs in 24 trait pairs, with 73 pleiotropic loci and 69 colocalized loci detected. Gene-based analysis revealed 59 candidate pleiotropic genes, highly enriched in unsaturated fatty acid biosynthetic processes and MHC class I-mediated antigen processing and presentation. Mendelian randomization analysis demonstrated a positive causal relationship only between chronic obstructive pulmonary disease and heart failure. Overall, the genetic basis between CVDs and RTs was inconsistent with vertical pleiotropy, suggesting the dramatic impact of horizontal pleiotropy. ConclusionsOur findings indicate widely distributed pleiotropic genetic determinants between RTs and CVDs across the genome. These results support a common genetic basis for RTs and CVDs and are important for intervention and therapeutic targets in comorbidities. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIA common genetic underpinning for CVDs and RTs has been identified using a variety of approaches and further explained as a shared genetic mechanism mediated by pleiotropy. C_LIO_LIThe systematic atlas of horizontal pleiotropy addressed key questions about pleiotropic SNPs, genomic loci, genes, functional features, and protein targets contributing to comorbidity between CVDs and RTs. C_LIO_LIThe systematic atlas of vertical pleiotropy highlighted causal associations between CVDs and RTs beyond the observed correlations. C_LI What Are the Clinical Implications?This study may help to elucidate the shared genetic mechanism between respiratory and cardiovascular diseases and further prioritize shared drug targets between RTs and CVDs.

RationaleHuman lung development has been mainly described in morphologic studies and the potential underlying molecular mechanisms were extrapolated from animal models. Therefore, there is a need to gather knowledge from native human lung tissue. In this study we describe changes at a single-cell level in human fetal lungs during the pseudoglandular stage. MethodsWe report the cellular composition, cell trajectories and cell-to-cell communication in developing human lungs with single-nuclei RNA sequencing (snRNA-seq) on 23,251 nuclei isolated from nine human fetuses with gestational ages between 14 to 19 weeks of gestation. ResultsWe identified nine different cell types, including a rare pulmonary neuroendocrine cells population. For each cell type, marker genes are reported, and selected marker genes are used for spatial validation with fluorescent RNA in situ hybridization. Enrichment and developmental trajectory analysis provide insight into molecular mechanisms and signaling pathways within individual cell clusters according to gestational age. Lastly, ligand-receptor analysis highlights determinants of cell-to-cell communication among the different cell types through the pseudoglandular stage, including general developmental pathways (NOTCH and TGFB), as well as more specific pathways involved in vasculogenesis, neurogenesis, and immune system regulation. ConclusionThese findings provide a clinically relevant background for research hypotheses generation in projects studying normal or impaired lung development and help to develop and validate surrogate models to study human lung development, such as human lung organoids. TAKE HOME MESSAGEUsing a single-cell transcriptomic approach (single-nuclei RNA sequencing), we describe here, for the first time, the cellular landscape, cell developmental trajectories, and cell-to-cell communication in the developing human lung during the pseudoglandular stage.

Lung tissue from fetal and neonatal lung samples is tough to obtain, and capturing cells from a living patient with evolving or established disease is very challenging. We hypothesized that airway organoids derived from bronchoalveolar lavage (BAL) samples obtained from intubated preterm infants with bronchopulmonary dysplasia (BPD) will recapitulate the epithelial heterogeneity seen in human airways and can be used to study lung injury and therapeutic response in vitro. Here, we demonstrate that BAL sample-derived airway organoids from ventilator-dependent patients with established BPD exhibited cellular heterogeneity consistent with that observed in the human airway. Developed organoids contain basal cell progenitors and a spectrum of differentiated epithelial subtypes, including secretory, ciliated, PNECs, and hillock cells. Hyperoxia exposure and treatment with dexamethasone caused significant cellular transcriptional changes and highlighted biological pathways, both known and novel, with distinct findings based on sex as a biological variable. Findings were validated in an independent dataset from human BPD lung samples. Infant BAL-derived human lung organoids represent a cutting-edge model that bridges a critical gap in BPD research. They combine the advantages of being patient-specific and capturing developmental lung biology, with the experimental flexibility of an in vitro system.

There is mounting evidence that macrophage-fibroblast communication is key to the understanding of disease processes. To gain insights into these relationships in the context of progressive lung damage, we measured changes in protein and RNA expression of pulmonary macrophages and fibroblasts upon exposure to IL-33, IL-13, and IL-17A, which are three cytokines often implicated in pathways driving chronic lung remodeling and severe disease like emphysema. Applying an in vitro culture system, bulk-RNA sequencing, and protein assays, it was determined that IL-33, IL-13, and IL-17A used alone or in combination activated mouse alveolar macrophages to a modest extent with IL-13 inducing the most vigorous response. While lung fibroblasts also responded modestly to single and paired treatments with IL-33, IL-13, and IL-17A, simultaneous exposure to all three cytokines induced significant activation that was characterized by expression of genes associated with immune cell trafficking and activation, tissue remodeling, and maintenance of the extracellular matrix. Importantly, factors secreted by triple-treated lung fibroblasts resulted in the activation of macrophages in vitro. In addition to being the first report describing the cooperative interactions of IL-33, IL-13, and IL-17A on lung fibroblasts, these findings provide additional evidence that fibroblast-macrophage communication is a key component to repair and remodeling in the lung, as well as mechanisms that drive progression of emphysema.

RationaleTranscriptionally-defined populations of interstitial macrophages (IMs) and airspace macrophages (AMs) have recently been identified in the human lung. However, the anatomic locations occupied by these populations (i.e. alveoli, pleura, airways, or arteries) have not been fully defined. ObjectivesTo determine the distribution of transcriptionally-defined human macrophages in the major anatomical lung structures and to identify alterations in their distribution and programming induced by cigarette smoking. MethodsSingle-cell RNA sequencing was performed on lung tissue from eight human donors without pulmonary disease (four smokers and four nonsmokers). Microdissection was used to isolate distinct pulmonary anatomical structures from each lung: alveoli, pleura, airways, and arteries. Transcriptional profiles of subpopulations of interstitial macrophages (IMs) and alveolar macrophages (AMs) were analyzed based on their anatomical structure of origin and smoking status. Measurements and Main ResultsFive major IM and five AM subpopulations in human lungs are identified. We demonstrate significant differences in the accumulation patterns of each macrophage subset within anatomical structures, though each subset was detected in each. Immunofluorescent microscopy confirmed anatomical structure-specific accumulation patterns of IMs. ConclusionsIn this study, we highlight key differences in the accumulation of lung macrophage subpopulations in anatomical structures but find programming within macrophage subpopulations is largely conserved, regardless of structure of origin or smoking status. We also detect populations of inflammatory AMs and IMs which accumulate within the airways, but not the alveolar parenchyma, of human cigarette smokers. We introduce a novel three-tiered hierarchy nomenclature to distinguish transcriptionally defined human lung IM subsets as 1{degrees}) Monocyte-like vs Antigen Presenting, 2{degrees}) Quiescent vs Inflammatory, and 3{degrees}) FOLR2high vs FOLR2low. This study is the first to report the fractional accumulation of human lung macrophage subsets by lung anatomical structure. SummaryLung anatomical structure-specific single cell RNA sequencing is introduced to identify and determine the local composition of human lung leukocytes, including 5 populations of human interstitial macrophages.

Although inhaled tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of P. aeruginosa in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is unclear. Here, we demonstrate that tobramycin increases the abundance of two 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by P. aeruginosa and that the 5' tRNA-fMet halves reduce IL-8 secretion by CF bronchial epithelial cells (CF-HBECs). In mouse lung, the 5' tRNA-fMet halves attenuate KC secretion and neutrophil recruitment. We also report that the 5' tRNA-fMet halves suppress pro-inflammatory network gene expression by an Argonaut 2 (AGO2)-mediated gene silencing mechanism, thereby reducing IL-8 secretion in CF-HBECs. Moreover, tobramycin reduces the IL-8 concentration and neutrophil content in bronchoalveolar lavage fluid of pwCF. Thus, we conclude that tobramycin improves lung function in part by reducing chronic inflammation and neutrophil-mediated lung damage in pwCF.

BackgroundPulmonary arterial hypertension (PAH) is a rare but severe and life- threatening condition that primarily affects the pulmonary blood vessels and the right ventricle of the heart. The limited availability of human tissue for research--most of which represents only end-stage disease--has led to a reliance on preclinical animal models. However, these models often fail to capture the heterogeneity and complexity of the human condition. Analyzing the molecular signatures in patient plasma provides a unique opportunity to gain insights into PAH pathobiology, explore disease heterogeneity absent in animal models, and identify potential therapeutic targets. ObjectiveThis study aims to characterize the circulating peptides, metabolites, and lipids most relevant to PAH by leveraging unbiased mass spectrometry and advanced computational tools. Building on prior research that identified individual circulating factors, this work seeks to integrate these molecular layers to better understand their interactions and collective contribution to PAH pathobiology. MethodsPeripheral blood samples were collected from 402 patients with PAH and 76 healthy individuals. Various types of molecules in the blood - peptides, metabolites, and lipids- were measured. Statistical and machine learning methods were used to identify differences between PAH patients and healthy individuals, and further to understand how these molecules might interact with each other. A survival model was also trained to examine the association between the blood molecular signature and patient outcomes. ResultsDifferential abundance analysis revealed 832 peptides (from 291 proteins), 45 metabolites, and 222 lipids significantly altered in PAH compared to controls. Machine learning- based feature selection identified 11 key molecules, including 2-Hydroxyglutarate, that together achieved a classification accuracy of 98.6% for PAH in a multivariate model tested on a withheld cohort. Latent network discovery uncovered 7 distinct networks, highlighting interacting molecules from pathways--such as hypoxia, glycolysis, fatty acid metabolism, and complement activation--that we and others have previously linked to vascular lesions in PAH patients. A survival model incorporating 155 molecular features predicted outcomes in PAH patients with a c-index of 0.762, independent of traditional clinical parameters. This model stratified patients into risk categories consistent with established markers of cardiac function, exercise tolerance, and the REVEAL 2.0 risk score. ConclusionThis study underscores the utility of integrated omics in unraveling PAH pathobiology in human subjects. Our findings highlight the central role of hypoxia signaling pathways interacting with disrupted fatty acid metabolism, complement activation, inflammation, and mitochondrial dysfunction. These interactions, revealed through latent network analysis, emphasize the metabolic and immune dysregulation underlying PAH. Furthermore, many of the molecules identified in the circulation were consistent with pathways enriched in pulmonary vascular lesions, reinforcing their biological relevance. Circulating plasma molecules from these networks demonstrated strong prognostic capabilities, comparable to current clinical risk scores, offering insights into disease progression and potential for future clinical application.

The gain-of-function MUC5B promoter variant is the dominant risk factor for the development of idiopathic pulmonary fibrosis (IPF). However, its impact on protein expression in both non-fibrotic control and IPF lung specimens have not been well characterized. Utilizing laser capture microdissection coupled to mass spectrometry (LCM-MS), we investigated the proteomic profiles of airway and alveolar epithelium in non-fibrotic controls (n = 12) and IPF specimens (n = 12), stratified by the presence of the MUC5B promoter variant. Through qualitative and quantitative analyses, as well as pathway analysis and immunohistological validation, we have identified a distinct MUC5B-associated protein profile. Notably, the non-fibrotic control alveoli exhibited substantial MUC5B-associated protein changes, with an increase of IL-3 signaling. Additionally, we found that the epithelial cells overlying IPF fibroblastic foci cluster closely to alveolar epithelia and express proteins associated with cellular stress pathways. In conclusion, our findings suggest that the MUC5B promoter variant leads to protein changes in alveolar and airway epithelium that appears to be associated with the initiation and progression of lung fibrosis.

Acute respiratory distress syndrome (ARDS) is a clinically defined syndrome of acute hypoxaemic respiratory failure secondary to non-cardiogenic pulmonary oedema. It arises from a diverse set of triggers and encompasses marked biological heterogeneity, complicating efforts to develop effective therapies. An extensive body of recent work (including transcriptomics, proteomics, and genome-wide association studies) has sought to identify proteins/genes implicated in ARDS pathogenesis. These diverse studies have not been systematically collated and interpreted. To solve this, we performed a systematic review and computational integration of existing omics data implicating host response pathways in ARDS pathogenesis. We identified 40 unbiased studies reporting associations, correlations, and other links with genes and single nucleotide polymorphisms (SNPs), from 6,856 ARDS patients. We used meta-analysis by information content (MAIC) to integrate and evaluate these data, ranking over 7,000 genes and SNPs and weighting cumulative evidence for association. Functional enrichment of strongly-supported genes revealed cholesterol metabolism, endothelial dysfunction, innate immune activation and neutrophil degranulation as key processes. We identify 51 hub genes, most of which are potential therapeutic targets. To explore biological heterogeneity, we conducted a separate analysis of ARDS severity/outcomes, revealing distinct gene associations and tissue specificity. Our large-scale integration of existing omics data in ARDS enhances understanding of the genomic landscape by synthesising decades of data from diverse sources. The findings will help researchers refine hypotheses, select candidate genes for functional validation, and identify potential therapeutic targets and repurposing opportunities. Our study and the publicly available computational framework represent an open, evolving platform for interpretation of ARDS genomic data.