American Journal of Respiratory and Critical Care Medicine

BackgroundTracheostomies in children are associated with significant morbidity, poor quality of life, excess healthcare costs, and excess mortality. The underlying mechanisms facilitating adverse outcomes in tracheostomised children are poorly understood. We aimed to characterise airway host defence in tracheostomised children using serial molecular analyses. MethodsTracheal aspirates, tracheal cytology brushings, nasal swabs and stool samples were prospectively collected from children with a tracheostomy and controls. Transcriptomic, proteomic, and metabolomic methods were applied to characterise the impact of tracheostomy on host immune response and the airway microbiome. ResultsChildren followed up serially from the time of tracheostomy up to three months post-procedure (n=9) were studied. A validation cohort of children with a long-term tracheostomy was also enrolled (n=24). Controls (n=13) comprised children without a tracheostomy undergoing bronchoscopy. Tracheostomy was associated with new, rapidly emergent and sustained airway neutrophilic inflammation, superoxide production and evidence of proteolysis when compared with controls. In contrast, reduced airway microbial diversity was established pre-tracheostomy and sustained thereafter. ConclusionsChildhood tracheostomy is associated with rapidly emergent and persistent airway neutrophil recruitment and activation, with sustained proteolysis and superoxide generation. These findings suggest neutrophil recruitment and activation as potential exploratory targets in seeking to prevent recurrent airway complications in this vulnerable group of patients. Key messageThe effect tracheostomy has on children is not described. Tracheostomy in children results in persistent local airway neutrophilic inflammation, proteolysis, superoxide production and dysbiosis.

Purpose: Airway remodeling is a convergent feature across respiratory diseases, yet current CT tools provide limited characterization of the airway tree. We present Radiomics of the Airway (RadAr), an automated framework for multi-scale airway phenotyping from routine chest CT. Methods: RadAr extracts multi-scale, interpretable airway measurements capturing luminal dimensions, tapering, architectural distortion, and global morphology and provides an interactive web portal for analysis and visualization. It was evaluated across four settings: 63-week mortality prediction in fibrotic interstitial lung disease (fILD; N=147), COVID-19 severity prediction (N=1164), structure-function association in progressive pulmonary fibrosis (PPF; N=9) and structure-inflammation markers in pediatric cystic fibrosis (CF; N=11). Unsupervised clustering identified airway phenotypes across the fILD and COVID-19 cohorts. Results: In fILD, lower-lobe architectural distortion was associated with mortality (balanced accuracy 0.654). In COVID-19, severe disease was independently associated with luminal dilation (AUC 0.719, odds ratio 2.32, p=0.017). In PPF, airway phenotypes correlated with forced vital capacity ({rho}=0.83), mid-expiratory flow ({rho}=0.87), and 129Xe MRI alveolar gas exchange impairment ({rho}=0.70). In pediatric CF, reduced tapering and increased cylindricity were associated with prior exacerbations and bronchoalveolar lavage neutrophilia ({rho}=-0.64 to -0.78). Five phenotypes were identified from extensive, tapered airway trees to sparse, dilated, thick-walled, tortuous trees, with increasing COVID-19 severity and fILD mortality across this spectrum. Conclusions: RadAr identified interpretable, disease-specific airway signatures associated with function and outcomes across restrictive, obstructive, and mixed lung diseases in adult and pediatric settings. It provides a scalable framework that may support diagnosis, risk stratification, and longitudinal monitoring across pulmonary diseases.

In this study, we provide a comprehensive characterization of the alveolar immune landscape in patients suffering from severe acute respiratory failure, predominantly caused by pneumonia or acute respiratory distress syndrome, conditions defined by intense pulmonary inflammation and immune dysregulation. Despite diverse underlying causes, the overall composition of alveolar immune cells was largely consistent, with neutrophils and macrophages comprising the majority of cells. However, the maturation and activation states of immune cell subsets varied significantly, not only between patients with and without pneumonia, but also among pneumonia cases stratified by pathogen type. We also observed dynamic shifts in immune cell subsets over the disease course and found that an increased proportion of CD123bright immature neutrophils and a reduction in alveolar resident macrophages were associated with increased 28-day mortality. Integration with alveolar cytokine profiles revealed strong correlations between immune cell populations and the local cytokine milieu. These findings highlight the importance of assessing immune cell function, not merely abundance, through broad and longitudinal investigation to better understand the pathophysiology of acute respiratory failure and to guide precision immunomodulatory therapy.

RationaleChronic obstructive pulmonary disease (COPD) is characterized by pathologic changes in the airways, lung parenchyma, and persistent inflammation, but the links between lung structural changes and patterns of systemic inflammation have not been fully described. ObjectivesTo identify novel relationships between lung structural changes measured by chest computed tomography (CT) and systemic inflammation measured by blood RNA sequencing. MethodsCT scan images and blood RNA-seq gene expression from 1,223 subjects in the COPDGene study were jointly analyzed using deep learning to identify shared aspects of inflammation and lung structural changes that we refer to as Image-Expression Axes (IEAs). We related IEAs to COPD-related measurements and prospective health outcomes through regression and Cox proportional hazards models and tested them for biological pathway enrichment. Measurements and Main ResultsWe identified two distinct IEAs: IEAemph captures an emphysema-predominant process with a strong positive correlation to CT emphysema and a negative correlation to FEV1 and Body Mass Index (BMI); IEAairway captures an airway-predominant process with a positive correlation to BMI and airway wall thickness and a negative correlation to emphysema. Pathway enrichment analysis identified 29 and 13 pathways significantly associated with IEAemph and IEAairway, respectively (adjusted p<0.001). ConclusionsIntegration of CT scans and gene expression data identified two IEAs that capture distinct inflammatory processes associated with emphysema and airway-predominant COPD. At a Glance CommentaryO_ST_ABSScientific Knowledge on the SubjectC_ST_ABSChronic obstructive pulmonary disease (COPD) is characterized by lung structural changes and has a prominent systemic inflammatory component, but the links between lung structural changes and patterns of systemic inflammation in COPD have not been fully described. What This Study Adds to the FieldWe identified novel relationships between lung structural changes and systemic inflammation by simultaneously analyzing CT scans and blood RNA-sequencing gene expression using deep learning models. We identified two distinct Image-Expression Axes (IEAs) that characterize different inflammatory processes associated with emphysema and airway predominant COPD. This article has an online data supplement, which is accessible from this issues table of content online at www.atsjournals.org.

RationaleChronic obstructive pulmonary disease (COPD) is a leading cause of mortality in the United States. Electronic health records provide large-scale healthcare data for clinical research, but have been underutilized in COPD research due to challenges identifying these individuals, especially in the absence of pulmonary function testing data.\n\nObjectivesTo develop an algorithm to electronically phenotype individuals with COPD at a large tertiary care center.\n\nMethodsWe identified individuals over 45 years of age at last clinic visit within Vanderbilt University Medical Center electronic health records. We tested phenotyping algorithms using combinations of both structured and unstructured text and examined the clinical characteristics of the resulting case sets.\n\nMeasurement and Main ResultsA simple algorithm consisting of 3 International Classification of Disease codes for COPD achieved a sensitivity of 97.6%, a specificity of 76.0%, a positive predictive value of 57.1%, and a negative predictive value of 99.0%. A more complex algorithm consisting of both billing codes and a mention of oxygen on the problem list that achieved a positive predictive value of 86.5%. However, the association of known risk factors with chronic obstructive pulmonary disease was consistent in both algorithm sets, suggesting a simple code-only algorithm may suffice for many research applications.\n\nConclusionsSimple code-only phenotyping algorithms for chronic obstructive pulmonary disease can identify case populations with epidemiologic and genetic profiles consistent with published literature. Implementation of this phenotyping algorithm will expand opportunities for clinical research and pragmatic trials for COPD.

Background Chronic rhinosinusitis (CRS) and nasal polyps (NP) are closely related inflammatory airway diseases, and their co-occurrence is often associated with more persistent symptoms, frequent recurrence, and substantial respiratory morbidity. However, the extent to which CRS without and with NP (CRSsNP and CRSwNP) share genetic susceptibility-and which genetic mechanisms are disease-specific-remains poorly characterized. Methods We conducted cross-population genome-wide association meta-analyses of overall CRS (including both CRSwNP and CRSsNP) and NP (a proxy for CRSwNP) using data from six biobanks. We estimated genome-wide genetic correlations between overall CRS, CRSwNP, and a spectrum of respiratory diseases. We applied five complementary gene-prioritization strategies to nominate CRS- and CRSwNP-associated genes and performed pathway enrichment analyses to infer implicated biological processes. For CRSwNP, we integrated single-cell transcriptomic data to characterize cell-type-specific expression of prioritized genes and used stratified LD score regression to quantify heritability enrichment across immune and epithelial annotations. To delineate shared versus disease-specific genetic signals, we performed three comparative analyses-local genetic correlation, CRSwNP-CRS colocalization, and genomic structural equation modeling. Finally, we performed proteome-wide Mendelian randomization to identify circulating proteins with putative causal effects on CRS and CRSwNP. Results This GWAS meta-analysis identified 96 genome-wide significant loci for CRSwNP and 41 for overall CRS, prioritizing 92 and 39 candidate genes, respectively. CRSwNP and overall CRS showed shared genetic susceptibility (rg = 0.59; P = 6.8e-16), while CRS exhibited broader genetic correlations across multiple respiratory disorders. Pathway analyses consistently implicated immune signaling albeit with disease-specific emphases and lipid-metabolism networks. Single-cell analyses demonstrated distinct expression of CRSwNP-prioritized genes across nasal epithelial and immune cell clusters, and immune annotations explained more CRSwNP heritability (enrichment score = 4.1; P = 0.010) than epithelial annotations (2.5; P = 0.072). Comparative genetic analyses highlighted multiple shared loci-including BACH2, CD247, FADS2, FOXP1, FUT2, GPX4, IL7R, NDFIP1, RAB5B, RORA, SMAD3, TSLP - as well as 3 CRSwNP-specific and 6 CRS-specific loci. Proteome-wide MR identified 10 and 8 putatively causal circulating proteins for CRSwNP and overall CRS, respectively, with protein TNFSF11, IL2RB, and STX4 associated with both conditions. Conclusions This multi-population GWAS meta-analysis expanded genetic discovery for CRS and CRSwNP and showed substantial shared liability with distinct disease-specific components. Immune components explained a larger proportion of CRSwNP heritability than epithelial annotations, reinforcing the primacy of immune-driven mechanisms in polyp disease.

ObjectiveAs our understanding of autoimmune laryngeal stenosis evolves, distinguishing patients who may benefit from systemic immunosuppression versus those needing only local treatment is increasingly important. In this study, we identify a distinct subset of autoimmune laryngeal stenosis characterized by edema of the inferior true vocal folds that extends to the superior aspect of the cricoid cartilage, termed "subcordal stenosis." The objective of this study is to characterize the clinical presentation and treatment outcomes of subcordal stenosis and compare it to typical autoimmune-related subglottic stenosis (AI-SGS). MethodsWe conducted a retrospective review of patients with laryngeal stenosis evaluated by both rheumatology and otolaryngology at our institution to identify two groups: patients with subcordal stenosis and those with typical AI-SGS. Data on immunosuppressive treatments and airway dilation procedures were collected. Time to first dilation was compared between groups. ResultsAmong 49 patients with laryngeal stenosis, 11 had subcordal involvement. Five of these also had subglottic disease, while six had isolated subcordal stenosis. Kaplan-Meier analysis showed significantly longer time to first dilation in patients with subcordal involvement (median 792 vs. 44 days; p = 0.048). They also underwent fewer dilations within two years (median 0 vs. 1; p = 0.05). ConclusionAmong laryngeal stenosis patients referred to rheumatology, those with subcordal involvement experienced fewer dilations and longer intervals before first dilation compared to those with typical AI-SGS. These findings suggest that subcordal stenosis may represent a distinct, glucocorticoid-responsive phenotype within autoimmune laryngeal stenosis, with implications for treatment selection and multidisciplinary care.

IntroductionPulmonary arterial hypertension (PAH) involves progressive cellular and molecular change within the pulmonary vasculature, leading to increased vascular resistance. Current therapies targeting nitric oxide (NO), endothelin, and prostacyclin pathways yield variable treatment responses. Patients with systemic sclerosis-associated PAH (SSc-PAH) often experience worse outcomes than those with idiopathic PAH (IPAH). MethodsLung tissue samples from four SSc-PAH, four IPAH, and four failed donor specimens were obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI) lung tissue bank. Single-cell RNA sequencing (scRNAseq) was performed using the 10X Genomics Chromium Flex platform. Data normalization, clustering, and differential expression analysis were conducted using Seurat. Additional analyses included gene set enrichment analysis (GSEA), transcription factor activity analysis, and ligand-receptor signaling. Pharmacotranscriptomic screening was performed using the Connectivity Map. ResultsSSc-PAH samples showed a higher proportion of fibroblasts and dendritic cells/macrophages compared to IPAH and donor samples. GSEA revealed enriched pathways related to epithelial-to-mesenchymal transition (EMT), apoptosis, and vascular remodeling in SSc-PAH samples. There was pronounced differential gene expression across diverse pulmonary vascular cell types and in various epithelial cell types in both IPAH and SSc-PAH, with epithelial to endothelial cell signaling observed. Macrophage to endothelial cell signaling was particularly pronounced in SSc-PAH. Pharmacotranscriptomic screening identified TIE2, GSK-3, and PKC inhibitors, among other compounds, as potential drug candidates for reversing SSc-PAH gene expression signatures. DiscussionOverlapping and distinct gene expression patterns exist in SSc-PAH versus IPAH, with significant molecular differences suggesting unique pathogenic mechanisms in SSc-PAH. These findings highlight the potential for precision-targeted therapies to improve SSc-PAH patient outcomes. Future studies should validate these targets clinically and explore their therapeutic efficacy.

BackgroundMitochondrial dysfunction, characterized by impaired lipid metabolism and heightened reactive oxygen species (ROS) generation, results in lipid peroxidation and ferroptosis. Ferroptosis is an inflammatory mode of cell death that promotes complement activation and macrophage recruitment. In pulmonary arterial hypertension (PAH), pulmonary arterial endothelial cells (PAEC) exhibit cellular phenotypes that promote ferroptosis. Moreover, there is ectopic complement deposition and inflammatory macrophage accumulation in the pulmonary vasculature. However, the effects of ferroptosis inhibition on these pathogenic mechanisms and the cellular landscape of the pulmonary vasculature are incompletely defined. MethodsMulti-omics and physiological analyses evaluated how ferroptosis inhibition modulated preclinical PAH. The impact of AAV1-mediated expression of the pro-ferroptotic protein ACSL4 on PAH was determined, and a genetic association study in humans further probed the relationship between ferroptosis and pulmonary hypertension (PH). ResultsFerrostatin-1, a small-molecule ferroptosis inhibitor, mitigated PAH severity in monocrotaline rats. RNA-seq and proteomics analyses demonstrated ferroptosis was associated with PAH severity. RNA-seq, proteomics, and confocal microscopy revealed complement activation and pro-inflammatory cytokines/chemokines were suppressed by ferrostatin-1. Additionally, ferrostatin-1 combatted changes in endothelial, smooth muscle, and interstitial macrophage abundance and gene activation patterns as revealed by deconvolution RNA-seq. Ferroptotic PAEC damage associated molecular patterns restructured the transcriptomic signature, mitochondrial morphology, and promoted proliferation of pulmonary artery smooth muscle cells, and created a pro-inflammatory phenotype in monocytes in vitro. AAV1-Acsl4 induced an inflammatory PAH phenotype in rats. Finally, single-nucleotide polymorphisms in six ferroptosis genes identified a potential link between ferroptosis and PH severity in the Vanderbilt BioVU repository. ConclusionsFerroptosis promotes PAH through metabolic and inflammatory mechanisms in the pulmonary vasculature.

Background Acute respiratory distress syndrome (ARDS) is characterised by substantial physiological heterogeneity, which contribute to a very variable clinical outcomes and therefore inconsistent responses to ventilatory strategies. We aimed to externally validate physiological ARDS subphenotypes previously identified using routine ventilatory and gas-exchange variables, assess their prognostic relevance across independent cohorts, and examine heterogeneity of treatment effect according to PEEP strategy. Methods Unsupervised Gaussian Mixture Modelling was used to identify physiological subphenotypes based on ventilatory mechanics and gas-exchange parameters. Labels were subsequently used to train and validate supervised classifiers using XGBoost. Prognostic relevance was assessed across three independent cohorts, including two randomised controlled trials (ALVEOLI and LOVS). Predictive enrichment for PEEP strategy was evaluated using individual patient data from ALVEOLI and LOVS (n = 1,532) using intention-to-treat analyses, applying both one-stage and two-stage fixed-effects IPD meta-analytic approaches to test for interaction between physiological subphenotype and PEEP strategy. Results Two distinct physiological subphenotypes, termed Efficient and Restrictive, were replicated across independent cohorts. Across each cohort, patients classified as Restrictive consistently exhibited higher all-cause 28-day mortality compared to Efficient patients. When pooled across studies, the Restrictive subphenotype was associated with a significantly increased risk of death (pooled odds ratio 1.75, 95% CI 1.36-2.24), with no evidence of between-study heterogeneity. Predictive analyses showed a statistically significant interaction between physiological subphenotype and PEEP strategy in the one-stage IPD model (p for interaction = 0.037), with concordant findings in the two-stage fixed-effects IPD meta-analysis (interaction OR 1.91, 95% CI 1.00-3.66; I2 = 0%). Higher PEEP was associated with increased mortality in Efficient patients and reduced mortality in Restrictive patients, indicating effect modification by physiological subphenotype. Interpretation Physiological ARDS subphenotypes derived from routinely collected bedside data provide robust and externally validated prognostic stratification across observational and randomised trial cohorts. The observed interaction with PEEP strategy suggests that underlying physiological profiles may influence treatment response, supporting the concept that physiology-based be a starting point for personalized medicine and therefore better ventilatory strategies in future clinical trials.

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.

BackgroundAnimal models are used widely to study pulmonary hypertension (PH). The cell populations that respond to disease-inducing stimuli in these models and their relationship to human disease remain incompletely defined. Materials and methodThis study analyzed the relationship between several rodent models of PH and human disease at single-cell resolution. scRNA-seq was performed on lungs from mice exposed to hypoxia or Sugen/hypoxia, rats exposed to monocrotaline, and controls. A cross-species single-cell dataset was integrated with human lung cell atlas (HLCA) and single-cell dataset from idiopathic pulmonary arterial hypertension (IPAH) to identify overlapping cell subsets between experimental and human disease and species. ResultsHigh levels of overlap were found between species and models of PH, HLCA, and IPAH datasets. Cell subsets perturbed in rat and mouse PH were similar to those found in human disease, with macrophages and endothelial cells being most affected. A novel Dhcr24high macrophage subset harboring both tissue-remodeling and pro- inflammatory features was consistently increased across models. Several functionally diverse endothelial subtypes were found, including novel Ednrb+ and Nox2+ subpopulations, reflecting enhanced apoptosis, dysregulated angiogenesis and proliferation, and reactive oxygen species-mediated stress. These macrophage and endothelial subtypes expressed numerous PH drug target genes, and exhibited potential disease-specific intercellular interactions involving Angptl4, Cxcl12, and Sema3 signaling. Disease-associated changes in these populations were confirmed by immunofluorescence in lung tissues from animals and patients. ConclusionsWe established a comprehensive cross-species single-cell atlas of mainstream rodent PH models, highlighting several novel macrophage and endothelial subtypes and signaling motifs potentially contributing to human disease.

RationaleRestrictive allograft syndrome (RAS) is a major cause of mortality in patients following lung transplantation due to rapid progressive fibrosis in the pulmonary graft. We have only limited knowledge of the cellular and molecular mechanisms that characterize the fibrosis in the RAS lung. ObjectiveTo elucidate cellularly-resolved transcriptomic and histologic characteristics of the structural cells in human RAS lungs. MethodsSingle-nuclei RNA-sequencing was performed in peripheral lung tissues from 15 RAS patients undergoing lung re-transplantation, and from 9 healthy control lungs. Findings were validated and complemented by various histologic techniques, including immunofluorescence, RNAscope, combined Elastica van Gieson-immunohistochemistry stains, and micro-CT scans. Measurement and Main resultsDifferential gene expression analysis of our single-nuclei RNA-sequencing data revealed in human RAS lungs previously undescribed and uniquely distributed aberrant basaloid cells, ectopic COL15A1+ vascular endothelial cells, and CTHRC1+ fibrotic fibroblasts, all first characterized in idiopathic pulmonary fibrosis (IPF). In contrast to IPF, RAS lacks the cellular equivalent of bronchiolization. Histologic stains confirmed our transcriptomic discoveries and disclosed distinctive distribution patterns: Aberrant basaloid cells are primarily localized at the edge of the fibrotic pushing border, forming together with the juxtaposed CTHRC1+ fibrotic fibroblasts the fibrotic niche of alveolar fibroelastosis (AFE), the histopathological hallmark in RAS lungs. On the endothelial side, PRX+ alveolar microvasculature is lost in AFE areas. Micro-CT scans revealed that blood supply, now facilitated by expanded and ectopic COL15A1+ VE cells, changes from pulmonary to systemic perfusion. Last, our data reveals potential therapeutically-modifiable expression patterns in RAS, including genes coding for the integrin subunits v{beta}6, activators of TGF{beta}. ConclusionConsidering the marked clinical, histologic and etiologic dissimilarities of RAS and IPF, our snRNAseq study revealed a surprising general principle of cellular and molecular pathogenesis in the fibrosing lung: the entity-spanning composition of the fibrotic niche by a) aberrant basaloid cells localized at the fibrotic pushing border, b) ectopic COL15A1+ vascular ECs and c) effector CTHRC1+ fibrotic fibroblasts. This general principle justifies a flexible but cellular pathogenesis-guided transferability of potential therapeutic approaches between progressive fibrotic lung diseases.

Congenital diaphragmatic hernia (CDH) is a devastating condition characterized by incomplete closure of the diaphragm and herniation of abdominal organs into the chest. As a result, fetuses have pulmonary hypoplasia, whose severity is the main determinant of poor outcome. The pathogenesis of pulmonary hypoplasia secondary to CDH is at least in part explained by lack or dysregulation of miRNAs that are known to regulate lung developmental processes. Herein, we report that intra-amniotic administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs) rescues lung growth and maturation in a fetal rat model of CDH. To understand which fetal lung cells and biological pathways are affected by AFSC-EVs, we conducted whole lung single nucleus RNA-sequencing. We discovered that CDH lungs have a multilineage inflammatory signature with macrophage enrichment, and confirmed these findings in autopsy samples of lungs from human fetuses with CDH. Transcriptomic analysis of CDH fetal rat lungs also showed that AFSC-EV treatment reduced macrophage density and inflammation to normal levels. Analyzing the miRNAs contained in the AFSC-EV cargo with validated mRNA targets, we found that the downregulated genes in AFSC-EV treated CDH lungs were involved in inflammatory response and immune system processes. This study reports a single cell atlas of normal and hypoplastic CDH fetal rat lungs and provides evidence that AFSC-EVs restore lung development by addressing multiple pathophysiological aspects of CDH. One Sentence SummaryAmniotic fluid stem cell extracellular vesicle treatment for fetal lung macrophage modulation

Pulmonary arterial hypertension (PAH) remains an incurable and often fatal disease despite currently available therapies. Multiomics systems biology analysis can shed new light on PAH pathobiology and inform translational research efforts. Using RNA sequencing on the largest PAH lung biobank to date (96 disease and 52 control), we aim to identify gene co-expression network modules associated with PAH and potential therapeutic targets. Co-expression network analysis was performed to identify modules of co-expressed genes which were then assessed for and prioritized by importance in PAH, regulatory role, and therapeutic potential via integration with clinicopathologic data, human genome-wide association studies (GWAS) of PAH, lung Bayesian regulatory networks, single-cell RNA-sequencing data, and pharmacotranscriptomic profiles. We identified a co-expression module of 266 genes, called the pink module, which may be a response to the underlying disease process to counteract disease progression in PAH. This module was associated not only with PAH severity such as increased PVR and intimal thickness, but also with compensated PAH such as lower number of hospitalizations, WHO functional class and NT-proBNP. GWAS integration demonstrated the pink module is enriched for PAH-associated genetic variation in multiple cohorts. Regulatory network analysis revealed that BMPR2 regulates the main target of FDA-approved riociguat, GUCY1A2, in the pink module. Analysis of pathway enrichment and pink hub genes (i.e. ANTXR1 and SFRP4) suggests the pink module inhibits Wnt signaling and epithelial-mesenchymal transition. Cell type deconvolution showed the pink module correlates with higher vascular cell fractions (i.e. myofibroblasts). A pharmacotranscriptomic screen discovered ubiquitin-specific peptidases (USPs) as potential therapeutic targets to mimic the pink module signature. Our multiomics integrative study uncovered a novel gene subnetwork associated with clinicopathologic severity, genetic risk, specific vascular cell types, and new therapeutic targets in PAH. Future studies are warranted to investigate the role and therapeutic potential of the pink module and targeting USPs in PAH.

RationaleDisruption of respiratory bacterial communities predicts poor clinical outcomes in critical illness; however, the role of respiratory fungal communities (mycobiome) is poorly understood. ObjectivesWe investigated whether mycobiota variation in the respiratory tract is associated with host-response and clinical outcomes in critically ill patients. MethodsTo characterize the upper and lower respiratory tract mycobiota, we performed rRNA gene sequencing (internal transcribed spacer) of oral swabs and endotracheal aspirates (ETA) from 316 mechanically-ventilated patients. We examined associations of mycobiome profiles (diversity and composition) with clinical variables, host-response biomarkers, and outcomes. Measurements and Main ResultsETA samples with >50% relative abundance for C. albicans (51%) were associated with elevated plasma IL-8 and pentraxin-3 (p=0.05), longer time-to-liberation from mechanical ventilation (p=0.04) and worse 30-day survival (adjusted hazards ratio (adjHR): 1.96 [1.04-3.81], p=0.05). Using unsupervised clustering, we derived two clusters in ETA samples, with Cluster 2 (39%) showing lower alpha diversity (p<0.001) and higher abundance of C. albicans (p<0.001). Cluster 2 was significantly associated with the prognostically adverse hyperinflammatory subphenotype (odds ratio 2.07 [1.03-4.18], p=0.04) and predicted worse survival (adjHR: 1.81 [1.03-3.19], p=0.03). C. albicans abundance in oral swabs was also associated with the hyper-inflammatory subphenotype and mortality. ConclusionsVariation in respiratory mycobiota was significantly associated with systemic inflammation and clinical outcomes. C. albicans abundance emerged as a negative predictor in both the upper and lower respiratory tract. The lung mycobiome may play an important role in the biological and clinical heterogeneity among critically ill patients and represent a potential therapeutic target for lung injury in critical illness.

RationalePulse oximeter performance may vary by skin pigmentation, but most data are retrospective with key limitations. ObjectiveTo quantify pulse oximeter bias [mean difference between pulse oximeter oxygen saturation (SpO2) and arterial blood functional oxygen saturation (SaO2)], and average root mean square error (ARMS) and estimate adjusted effects of skin pigment on bias or ARMS in critically-ill adults MethodsProspective single-center study of 631 ICU patients (2022-2024) directly observed SpO and SaO pairs. Skin pigment was assessed using the subjective Monk Skin Tone Scale and objective spectrophotometry measurement (Individual Typology Angle [ITA]). Adjusted effects were estimated with targeted maximum likelihood estimation. Main ResultsAmong 1,760 paired measurements from 631 critically-ill adults, median SaO2 was 98% (IQR 96%, 99%) with 40 episodes of stable hypoxemia (SpO2<90%). SpO2 systematically underestimated SaO2 [median bias= -1.70 IQR (-2.84, -0.50)]. Bias was less negative in patients with darker skin (ITA <-30{degrees}) [-1.05 (-2.44, -0.10)] vs lighter skin (ITA >30{degrees}) [-2.01 (-3.34, -1.00)] and remained significantly different in adjusted analyses. ARMS was 3.87 (95% CI 3.25, 4.53) overall and 4.49 (95% CI 2.63, 7.07) in patients with darker skin. Ear probes performed worse than finger probes [bias: -2.20 vs. -1.60; ARMS: 4.90 vs. 2.70]. ConclusionsIn this large ICU cohort, hypoxemia was rare, pulse oximeters systematically underestimated SaO, performance varied by skin pigment and probe site. Bias was less negative in patients with darker skin. Pulse oximeter inaccuracies in ICU patients may be more substantial than clinicians recognize. Key PointsO_ST_ABSQuestionC_ST_ABSDoes pulse oximeter bias in critically-ill adults differ by skin pigmentation? FindingsIn this prospective, single-center study of 631 critically-ill adults, oximeter mean bias was negative for all patients but relatively less negative for patients with darker pigment. Bias variation by pigment was nonlinear and larger in ear than finger probes. MeaningPulse oximeter bias varies with skin pigmentation and may not always be positive in patients with darker pigmentation. Pulse oximeter inaccuracy may be larger than clinicians appreciate. Additional studies with multiple oximeter brands and more stable hypoxemia are needed to further understand bias variation with skin pigment.

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.

Early genetic studies hinted the role of airway epithelial cells in the development of idiopathic pulmonary fibrosis (IPF), while recent single-cell RNA sequencing (scRNA-seq) atlases utilized explant IPF lungs and therefore represent late-stage disease. Here, we used air liquid interface (ALI) cultures of primary cells taken from the subsegmental bronchi of newly diagnosed IPF patients, reflecting early-stage fibrosis, to interrogate the transcriptional landscape of the airway mucosa. Profiling of 129,986 cells identified a shared proinflammatory state in epithelial cells and an early activation state of fibroblasts. Moreover, IPF basal cells initiated awry repair mechanisms and primed the airway mucosa for TGF-{beta} activation. Treatment with nintedanib, pirfenidone, both established antifibrotic drugs, and saracatinib, an Src kinase inhibitor that can limit IPF progression, only significantly affected certain IPF signatures. This study provides insight into the early disease mechanisms of IPF and may serve as a resource to further investigate pharmacological inhibition effects.

INTRODUCTIONChest imaging is necessary for diagnosis of COVID-19 pneumonia, but current risk stratification tools do not consider radiographic severity. We quantified radiographic heterogeneity among inpatients with COVID-19 with the Radiographic Assessment of Lung Edema (RALE) score on Chest X-rays (CXRs). METHODSWe performed independent RALE scoring by [&ge;]2 reviewers on baseline CXRs from 425 inpatients with COVID-19 (discovery dataset), we recorded clinical variables and outcomes, and measured plasma host-response biomarkers and SARS-CoV-2 RNA load from subjects with available biospecimens. RESULTSWe found excellent inter-rater agreement for RALE scores (intraclass correlation co-efficient=0.93). The required level of respiratory support at the time of baseline CXRs (supplemental oxygen or non-invasive ventilation [n=178]; invasive-mechanical ventilation [n=234], extracorporeal membrane oxygenation [n=13]) was significantly associated with RALE scores (median [interquartile range]: 20.0[14.1-26.7], 26.0[20.5-34.0] and 44.5[34.5-48.0], respectively, p<0.0001). Among invasively-ventilated patients, RALE scores were significantly associated with worse respiratory mechanics (plateau and driving pressure) and gas exchange metrics (PaO2/FiO2 and ventilatory ratio), as well as higher plasma levels of IL-6, sRAGE and TNFR1 levels (p<0.05). RALE scores were independently associated with 90-day survival in a multivariate Cox proportional hazards model (adjusted hazard ratio 1.04[1.02-1.07], p=0.002). We validated significant associations of RALE scores with baseline severity and mortality in an independent dataset of 415 COVID-19 inpatients. CONCLUSIONReproducible assessment of radiographic severity revealed significant associations with clinical and physiologic severity, host-response biomarkers and clinical outcome in COVID-19 pneumonia. Incorporation of radiographic severity assessments may provide prognostic and treatment allocation guidance in patients hospitalized with COVID-19.