American Journal of Respiratory and Critical Care Medicine

BackgroundChronic lung allograft dysfunction (CLAD) is the major cause of late mortality after lung transplantation and includes two principal phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). RAS and other phenotypes with RAS-like opacities (RLO) on chest imaging have a poorer prognosis. Despite clear clinical and pathological differences, molecular distinctions between phenotypes remain poorly defined. We aimed to explore gene transcriptional profiles across CLAD phenotypes and relevant controls. MethodsWe performed bulk RNA sequencing on explanted lung tissue from 45 lung transplant recipients with end-stage CLAD (20 with RLO and 25 without RLO). Samples from twenty-seven control donor and lobectomy lungs and sixteen idiopathic pulmonary fibrosis (IPF) lungs served as comparators. Non-negative matrix factorization (NMF) was used to identify latent transcriptomic signatures, which were correlated with clinical, radiologic, and histopathologic features. ResultsNMF identified seven distinct gene signatures that segregated CLAD phenotypes. RLO-CLAD lungs were enriched for extracellular matrix remodeling and B-cell/plasma cell-associated signatures, overlapping partly with IPF, whereas non-RLO-CLAD showed relative enrichment of epithelial injury and surfactant-response pathways. Signatures related to epithelial homeostasis and ciliary/microtubule function were progressively reduced from control lungs to non-RLO-CLAD and were most suppressed in RLO-CLAD. ConclusionsRLO-CLAD and non-RLO-CLAD, aligning with RAS and BOS phenotypes, show distinct transcriptomic signatures. RLO-CLAD is characterized by profibrotic and humoral immune signatures with profound epithelial dysfunction, whereas non-RLO-CLAD shows relative enrichment of epithelial injury responses. These data provide molecular stratification of CLAD and support the development of phenotype-specific biomarkers and targeted therapies.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease (ILD) characterized by progressive fibrosis, irreversible loss of lung elasticity, and chronic respiratory failure, with a mean survival of 3-5 years. The disease is believed to result from repeated alveolar epithelial injury that sustains transforming growth factor-beta (TGF-{beta}) signaling, driving fibroblast-to-myofibroblast differentiation and excessive collagen deposition. Although current IPF models--including animal studies, 2D cultures, and basic 3D systems--have enhanced understanding of disease mechanisms, they inadequately replicate epithelial-fibroblast interactions, extracellular matrix (ECM) remodeling, and epithelial barrier dysfunction. To address this limitation, we engineered a 3D lung co-culture model that simulates the physiological epithelial-fibroblast crosstalk and ECM remodeling characteristic of IPF. Our model embeds fibroblasts within a collagen-hyaluronic acid matrix overlaid with an epithelial monolayer cultured at an air-liquid interface. Basolateral TGF-{beta} exposure generated a profibrotic microenvironment that weakened epithelial barrier integrity and drove myofibroblast differentiation marked by elevated -SMA and vimentin. Elevated pro-inflammatory cytokine secretion and increased collagen disorganization further demonstrated active fibrogenesis. Together, these features show that our model captures key early events in IPF pathogenesis and offers a versatile platform for next-generation lung-on-a-chip studies in fibrotic disease.

IntroductionClonal hematopoiesis of indeterminate potential (CHIP) is a recognized risk factor for hematologic malignancies, but its contribution to different types of solid cancers remains incompletely defined. MethodsHere, we performed a systematic, gene-specific analysis of CHIP across 19 common solid cancer types using two large population-based cohorts, the UK Biobank and All of Us with Cox proportional hazards models and nested case-control logistic models. ResultsWe demonstrate that the relationship between CHIP and solid tumors is highly cancer-type specific, with lung cancer exhibiting the strongest association. In lung cancer, this association is largely driven by ASXL1-mutant clones. Specifically, high variant allele fraction (high-VAF) ASXL1 conferring a significantly increased risk (hazard ratio = 3.2), and the associations remained robust after adjustment for age, sex, body mass index (BMI), smoking status, and genetic ancestry. Notably, ASXL1 CHIP was substantially enriched among smokers, and its association with lung cancer risk was restricted to ever-smokers, highlighting a key interaction between CHIP and environmental exposure. The enrichment of ASXL1 CHIP in lung cancer was further validated in two independent cancer-only cohorts, including MSK-IMPACT and TCGA. In addition, rare germline variant association analysis revealed that germline variation in ASXL1 had the strongest association with lung cancer susceptibility among all solid tumors. ConclusionsCollectively, our findings support a model in which smoking-associated expansion of ASXL1-mutant clones contributes to lung cancer development and suggest that gene-specific CHIP metrics may enhance risk stratification and early detection strategies.

Alveolar capillary endothelial cells are positioned adjacent to the alveolar epithelium and contribute to lung homeostasis and injury responses. Single-cell studies have identified aerocyte capillary endothelial cells (aCap), which are specialized for gas exchange, and general capillary endothelial cells (gCap), which contribute to endothelial maintenance and inflammatory signaling. Apelin and its receptor are differentially enriched across these endothelial compartments, but their roles in emphysema development remain incompletely understood. Using an elastase-induced emphysema model in male C57BL/6J mice, we combined bulk RNA sequencing, CIBERSORTx-based cell-type deconvolution, histology, inflammatory assays, pulmonary function testing, and pharmacologic activation of the apelin receptor with [Pyr1]-Apelin-13. At 24 hours after elastase exposure, the inferred fraction of gCap was reduced, and lung expression of apelin and the apelin receptor was decreased. Early [Pyr1]-Apelin-13 administration reduced lung inflammatory mediator expression, Ly6G-positive neutrophil accumulation, bronchoalveolar lavage neutrophil counts, and matrix metalloproteinase-9 activity. Early treatment also attenuated subsequent airspace enlargement, whereas treatment initiated after emphysema was established did not improve physiological or histological outcomes. In a chronic {beta}ENaC-transgenic mouse model, the inferred gCap fraction was maintained, the aCap fraction was reduced, and apelin receptor activation did not improve disease phenotypes. These findings suggest that early activation of the apelin receptor modifies acute inflammatory and endothelium-associated responses following elastase injury and limits emphysematous remodeling in mice. Together, these results support a time-sensitive role for apelin-APJ signaling during the early phase of emphysema development.

Genome-wide association studies have identified rare and common mutations associated with increased risk of pulmonary arterial hypertension (PAH), but the mechanism by which impaired SOX17 expression increases PAH risk is not known. Notably, SOX17 plays a critical role in endothelial identity during development by suppressing RUNX1 through binding to its promoter and directing stem and progenitor cells toward an endothelial rather than a hematopoietic cell fate. RUNX1 functions as a key regulator of myeloid differentiation, aberrant angiogenesis and adverse cardiac remodeling. Previously, we found that RUNX1 inhibition reverses pulmonary hypertension (PH) in multiple animal models. Here, we hypothesize that impaired expression of SOX17 in PAH leads to endothelial cell (EC) dysfunction by failing to suppress RUNX1. METHODSHuman pulmonary artery endothelial cells (HPAECs) with stable SOX17 CRISPR/Cas9 knockout or RUNX1 overexpression were generated and examined for endothelial and hematopoietic gene expression, proliferation, migration, apoptosis, and angiogenesis. Immortalized lymphoblastoid cell lines (LCLs) from PAH patients with SOX17 mutations and healthy controls were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into ECs. The effect of RUNX1 inhibition on Sugen/hypoxia-PH was examined in rats, SOX17 enhancer knockout (SOX17enhKO) mice, and Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic mice. SOX17 and RUNX1 expression were analyzed in peripheral blood samples from PAH patients (n=359). RESULTSHPAECs with SOX17 deletion or RUNX1 overexpression exhibited decreased expression of EC markers, enhanced proliferation and migration, defective angiogenesis, and decreased apoptosis. RUNX1 siRNA knockdown or RUNX1 inhibition by Ro5-3335 partially restored the endothelial properties in SOX17 KO HPAECs. ECs differentiated from SOX17 mutant PAH patient iPSCs exhibited upregulated RUNX1 expression and loss of endothelial identity, which was also partially restored by RUNX1 siRNA or Ro5-3335. In addition, SOX17enhKO mice had increased RUNX1 expression and susceptibility to Sugen/hypoxia-induced PH (SuHx-PH). Treatment with RUNX1 inhibitors or inducible endothelial-specific deletion of RUNX1 rescued SuHx-PH susceptibility in SOX17enhKO mice. RUNX1 inhibitors Ro5-3335 and Ro24-7429 also reversed SuHx-PH in wild-type rats. In addition, plasma RUNX1 expression was higher in PAH patients lacking detectable SOX17 expression than in patients with detectable SOX17 expression. CONCLUSIONSImpaired SOX17 expression increases the risk of PAH through insufficient suppression of RUNX1, leading to pulmonary endothelial dysfunction. RUNX1 inhibition mitigates PH associated with SOX17 deficiency and may represent a novel therapeutic strategy for PAH, especially those with rare or common SOX17 mutations.

Background Genetic studies to date are yet to define the major portion of the genetic risk for adult-onset pulmonary fibrosis (PF). Further the dearth of knowledge of clinically actionable variants for PF is hampering efforts to implement genetic testing to aid early diagnosis and improve disease management. Here we evaluated the contribution of rare and common variants to PF in cohorts with and without a family history of PF. Method Whole genome sequencing (WGS) was performed in a familial cohort comprising PF cases and their family members (85 individuals representing 55 families); and 122 cases from the Australian IPF Registry (AIPFR) with and without a self-reported family history of PF. WGS data were interrogated for rare potentially PF-causing variants in 33 genes previously associated with PF. Variants that were rare and predicted to be likely causative were formally curated using the American College of Medical Genetics and Association for Molecular Pathology (ACMG-AMP) guidelines. Additionally, to examine the common risk variant contribution, a weighted polygenic risk score (PRS) was generated using 16 previously IPF-associated common SNPs. PRS were generated from WGS for the 85 clinically confirmed familial cases and 122 AIPFR cases. In the remaining 202 AIPFR cases, PRS were generated from TaqMan genotyping data. Results Interrogation of WGS generated from 207 individuals with PF revealed multiple rare putative pathogenic variants in both familial and AIPFR cohorts. Formal curation revealed pathogenic (P) or likely pathogenic (LP) variants confirmed in TERT or RTEL1 in four families (7.3%) with the majority of remaining variants classified as variants of uncertain significance (VUS; 12.7%) in seven additional families. Amongst AIPFR participants, four variants met the threshold for classification as P/LP variants (3.3%), with a further six individuals found to harbour VUS following curation (4.9%). Overall weighted PRS did not differ significantly between individuals with familial PF or with no reported family history. However, PRS in all patient groups were significantly elevated compared with population controls. Conclusion VUS remain the major portion of rare variants identified in known PF -related genes. For ~80% individuals with a confirmed family history no potentially causative variants were identified in known PF related genes nor was there evidence that a high burden of common variants contributed to risk in these families. Similarly, we found no evidence that a high burden of common variants contributes to a significant proportion of risk PF in those individuals with no reported family history.

Background: The chromosome 1q31 Th2 pathway-associated interval has been linked to asthma, but its phenotype specificity and cross-ancestry architecture remain unclear. Methods: We analyzed African (AFR) and European (EU) ancestry datasets, including 9,965 asthma cases and 37,391 controls of AFR, and 6,074 cases and 116,255 controls of EU ancestry. Imputed dosage-based association analyses were performed for asthma, steroid-dependent asthma (SDA), and non-steroid-dependent asthma, followed by QC-filtered SDA remapping, leave-one-batch-out analysis, cross-ancestry comparison, and functional enrichment. Results: Strong regional association was observed only for SDA. After quality-control (QC) filtering, the SDA signal remained significant in both ancestries, with 2,280 genome-wide significant variants in AFR and 859 in EU. Cross-ancestry comparison identified 3,129 significant variants: 10 shared, 2,270 AFR-specific, and 849 EU-specific. Shared variants showed concordant effects, whereas 237 variants showed nominal heterogeneity. AFR-specific signals included PTPRC variants with larger effects in AFR. Functional enrichment suggested different biological emphases within the same interval: immune and contractile airway-wall biology in AFR, and additional neuroaxonal components in EU. Conclusions: The 1q31 interval is strongly associated with SDA in both AFR and EU populations, and its fine-scale architecture differs by ancestry. These findings highlight population-specific effects within a shared SDA susceptibility interval, with potential implications for population-informed precision medicine in steroid responsiveness and asthma management.

Biological sex has systemic effects on gene expression, cell behavior, and disease etiology. Despite these widespread effects, sex as a biological variable is understudied, particularly in chronic lung diseases. In idiopathic pulmonary fibrosis (IPF), 70% of patients are male, and male patients have overall worse survival post-diagnosis. While behavioral differences between sexes might account for some of the epidemiological differences, the contribution of underlying biology is not known. In this study, we performed regional proteomic analysis via laser-captured microdissection-coupled mass spectrometry and analyzed the data for sex-biased protein expression. We discovered that even in control lung, sex differences existed in both airway and alveolar regions. Sex differences became more pronounced in diseased regions, with sex-biased expression of diverse proteins including those involved in extracellular vesicle secretion, cellular metabolism, and extracellular matrix remodeling. These data suggest that baseline sex differences in lung proteome may contribute to sex-specific susceptibility, progression, and clinical outcomes in IPF, underscoring the need for future mechanistic and clinical studies to account for sex as a biological variable.

RationaleFibrotic lung diseases, such as idiopathic pulmonary fibrosis (IPF) and fibroproliferative remodeling in acute respiratory distress syndrome (ARDS), are characterized by increased extracellular matrix (ECM) deposition. However, measuring collagen accumulation alone does not capture differences in ECM organization or biochemical maturation that may distinguish persistent fibrosis from potentially reversible remodeling. ObjectivesTo examine collagen organization characteristics and mature (pyridinoline) collagen crosslinking amount in established end stage fibrotic lung disease (IPF) and fibroproliferation following an acutely damaged lung (non-resolving (NR) ARDS) and to investigate any relationships in these parameters and temporal tissue remodeling. MethodsHuman lung tissue samples from control subjects, patients with IPF, and NR-ARDS were analyzed. Collagen amount and fiber organization were digitally quantified using picrosirius red staining. Mature collagen crosslinking was assessed by quantification of pyridinoline crosslinks. Measurements and Main ResultsLung tissue from both IPF and NR-ARDS lungs had higher collagen content compared with controls. Collagen fiber organization differed between groups. IPF lungs exhibited collagen architectures consistent with established fibrosis, whereas NR-ARDS lungs showed altered but less stabilized collagen organization despite similarly elevated collagen levels. Mature collagen crosslinks were significantly higher in IPF lungs but not in NR-ARDS lungs compared to controls. Integrated analyses identified distinct disease-associated ECM phenotypes, indicating that higher collagen abundance in NR-ARDS, unlike IPF, is not accompanied by more mature and persistent collagen crosslinking. ConclusionsDespite shared increases in collagen content, IPF and NR-ARDS lungs differ fundamentally in collagen organization and crosslinking maturity, suggesting differences in the reversibility of these conditions.

ABSTRACT Background: Corticosteroids reduce mortality in severe COVID-19 requiring oxygen or invasive mechanical ventilation, yet emerging data suggest that SARS-CoV-2-associated acute lung injury is biologically heterogeneous and that treatment response may vary across molecularly defined disease states. Lung-derived molecular endotypes of severe COVID-19-associated acute lung injury have been described, but direct molecular profiling is not routinely available at the bedside. We evaluated whether a clinical predictor of previously defined lung molecular endotype identifies heterogeneity in corticosteroid treatment effect among mechanically ventilated patients with COVID-19. Methods: We utilized a single-center cohort of 5,000 patients with COVID-19 treated at the University of North Carolina Hospital between January 1, 2020, and December 31, 2022, to emulate a target trial assessing the effect of corticosteroid receipt on mortality, length of stay, and incident organ support. Confounding was addressed through inverse probability of treatment weighting (IPTW). Outcomes for severely ill patients requiring mechanical ventilation were compared to the RECOVERY trial results, with subsequent moderation analysis and stratified analysis by clinically predicted lung molecular endotype and vaccination status. The primary outcome was 28-day mortality. Secondary Outcomes were time to discharge alive and progression to additional organ support. Results: This emulated target trial showed a directionally favorable but non-statistically significant association between corticosteroid treatment and reduced 28-day mortality in patients requiring mechanical ventilation for SARS-CoV-2 infection. A clinical predictor of lung molecular endotype moderated the effect of corticosteroids on 28-day mortality (p-value for interaction 0.038) and identified distinct predicted endotype-specific treatment effect. Corticosteroid treatment was associated with lower 28-day mortality in the predicted Hyper-Inflammatory endotype (OR 0.62, 95% CI 0.39, 0.99) but not in the predicted Metabolic Dysregulation endotype (OR 1.15, 95% CI 0.82, 1.61). We did not detect significant effect modification by vaccination status (p-value for interaction 0.65), although inference was limited by the small, vaccinated subgroup (28-mortality OR 0.78, 95% CI 0.37, 1.65 in vaccinated vs 0.94, 95% CI 0.70, 1.26 in unvaccinated). Conclusions: In this target trial emulation of mechanically ventilated patients with severe COVID-19, corticosteroid treatment showed a directionally favorable but non-statistically significant association with reduced 28-day mortality in the overall cohort. However, a clinical predictor of lung molecular endotype identified significant heterogeneity in treatment effect, with benefit concentrated in the predicted Hyper-Inflammatory endotype and no apparent benefit in the predicted Metabolic Dysregulation endotype. These findings support prospective validation of clinically deployable endotype-guided corticosteroid treatment strategies in acute lung injury and ARDS.

Adjunctive therapies that enhance the efficacy of existing antitubercular drugs are needed for drug-resistant tuberculosis. We evaluated the efficacy of intranasally administered recombinant D29 LysB, a mycobacteriophage-derived mycolylarabinogalactan esterase, in murine and guinea pig models of pulmonary tuberculosis. BALB/c mice and guinea pigs were aerosol-infected with Mycobacterium tuberculosis H37Rv and treated for 4 weeks with LysB alone or with standard antitubercular therapy (ATT: rifampicin, isoniazid, pyrazinamide). Outcomes included pulmonary and extrapulmonary bacterial burden (CFU), lung and spleen histopathology, cytokine profiling, and humoral immune responses. LysB monotherapy produced modest pulmonary CFU reductions. When given adjunctively with ATT, LysB produced an additional 0.6-0.7 log10 reduction in lung CFU compared with ATT alone and decreased splenic dissemination in both species. Combination therapy improved tissue pathology, reducing granulomatous involvement and preserving pulmonary architecture. LysB treatment increased TNF- with a moderate rise in IL-10, a profile consistent with enhanced antibacterial immunity without excessive inflammatory damage. Repeated intranasal administration was well tolerated; no IgE-mediated hypersensitivity was detected. LysB-specific IgG developed but did not diminish therapeutic efficacy. These results show that intranasal D29 LysB augments the bactericidal and histopathological effects of standard ATT in vivo and support further development of inhaled phage-derived lysins as adjunctive therapies for drug-resistant tuberculosis. ImportanceTuberculosis remains a major cause of infectious mortality worldwide, and the increasing burden of multidrug-resistant and extensively drug-resistant disease continues to challenge effective treatment. New therapeutic approaches that complement conventional antibiotics are urgently needed. In this study, intranasally delivered recombinant mycobacteriophage-derived LysB was well tolerated and enhanced treatment efficacy in experimental pulmonary tuberculosis. Adjunctive LysB improved bacterial clearance, reduced tissue pathology, and modulated host immune responses in both murine and guinea pig models. These findings highlight phage-derived endolysins as promising inhalable adjunctive therapeutics for drug-resistant tuberculosis.

RationaleHeterogeneous alveolar collapse is prevalent in inflammatory lung conditions such as chronic obstructive pulmonary disease, acute respiratory distress syndrome, and pneumonia. Although neutrophil-released proteases contribute to the tissue remodeling that leads to alveolar collapse, how this altered mechanical environment in turn affects neutrophil migration remains largely unexplored. ObjectivesIn this study, we investigate how alveolar collapse and stretch influence neutrophil migration and identify the mechanical and biochemical factors that govern regional migration differences. MethodsWe developed a novel precision-cut lung slice platform that generates collapsed vs non-collapsed regions within the same slice. Neutrophils in both regions were longitudinally imaged for up to 5 hours to quantify motility behavior. Migration mechanisms were probed using migration-related inhibitors, collagenase, and cigarette smoke extract. A crystal ribcage system, which preserves intact alveolar shape and the air-liquid interface, was also used to assess the effects of ventilation on neutrophil migration. ResultsNeutrophil migration was faster in the collapsed region compared to not-collapsed regions. This regional difference was eliminated by Rho-associated protein kinase (ROCK) inhibition, which selectively increased migration speed in the non-collapsed region. The regional difference persisted with the addition of collagenase and cigarette smoke extract, both of which significantly increased the migration speed in both regions. In the crystal ribcage, the preserved air-liquid interface and ventilation together enhanced neutrophil migration compared with a collapsed lung. ConclusionsAlveolar collapse and stretch facilitate neutrophil migration, indicating the role of localized tissue remodeling in driving neutrophil activity and further disease progression.

BackgroundMicrobial colonisation of the human body begins immediately after birth, with each body site forming a distinct ecological niche. While early gut microbial dynamics and their links to paediatric health have been considerably studied, the bacterial colonisation of the early infant respiratory system remains poorly understood, particularly at the species and strain level. ResultsHere, we generated and analysed whole genome sequencing of 925 isolates from six dominant genera in nasopharyngeal samples from 58 healthy infants enrolled in the Childhood Asthma Study (CAS) in Western Australia, collected longitudinally from birth (2, 6, and 12 months old). Our results expanded genomic reference catalogues and uncovered substantial strain-level diversity in dominant infant airway taxa. Plate-sweep metagenomics identified four microbiome profile groups (MPGs) with age-dependent membership, confirming prior observations while enabling high-resolution species and strain analyses. Community maturation was characterised by a shift from early Staphylococcus aureus dominance to increased Moraxella catarrhalis dominance by 12 months of age, alongside marked temporal changes in prevalence and cohort-level strain diversity. ConclusionsThese findings resolve infant nasopharyngeal microbiota composition at the species and strain level, revealing taxon-specific colonisation patterns. By substantially expanding publicly available reference genomes for underrepresented airway taxa, this work also provides a foundation for functional follow-up studies of the early respiratory microbiota and respiratory outcomes.

Background: Without tuberculosis preventive therapy (TPT), approximately 5% of individuals infected with M. tuberculosis progress to active tuberculosis (TB) disease. Recent studies have identified body mass index (BMI) < 25 kg/m2 as a predictor of TB progression, but additional markers are needed to better identify persons at increased risk. Methods: Close contacts of patients with culture-confirmed pulmonary TB were enrolled in the Regional Prospective Observational Research in Tuberculosis (RePORT)-Brazil cohort from 2015 to 2019 and followed for up to 24 months. Analyses were restricted to interferon-{gamma} release assay (IGRA)-positive contacts who did not receive TPT or received <30 days of isoniazid. Prediction models to identify close contacts at increased TB risk were constructed using two complementary approaches: incremental models used BMI as the base predictor and evaluated whether baseline whole-blood transcriptomic signatures, human genetic polymorphism risk scores derived from low-pass whole-genome sequencing, and BMI-related plasma biomarkers improved model discrimination. Agnostic models did not impose BMI in the model and used penalized regression for predictor selection. Results: Among 285 close contacts, 15 (5%) progressed to TB. The model with BMI as unique predictor had a C-index of 0.66 (95% confidence interval [CI] 0.55; 0.77). Adding Rajan5 or Duffy9 transcriptomic signature scores to BMI improved discrimination compared with BMI alone, with C-indices of 0.78 (95% CI 0.62; 0.99) and 0.75 (95% CI 0.61; 0.89), respectively, but did not further improve discrimination after accounting for adiponectin. Adding adiponectin to BMI increased the C-index to 0.80 (95% CI 0.68; 0.91), while adiponectin alone captured most of the discriminatory performance in agnostic models (C-index, 0.80, 95% CI 0.69; 0.91). Genetic risk scores, leptin, and the adiponectin:leptin ratio did not improve model discrimination compared with the BMI-only model. In exploratory post hoc analyses, higher adiponectin was associated with increased risk of progression to TB, with each two-fold increase associated with a higher hazard of TB (HR 2.91, 95% CI 1.73; 4.91, p < 0.001). Conclusions: Baseline adiponectin strongly predicted progression to TB among close contacts and captured most of the discriminatory information contained in epidemiological and transcriptomic variables. Its consistent selection across modelling approaches supports adiponectin as a promising biomarker for TB risk stratification.

Background: Diabetes mellitus (DM) worsens pulmonary tuberculosis (TB) and drives systemic hyper-inflammation, but the underlying mechanisms remain unknown. Neutrophils have key roles in TB immunopathology and lung cavitation. Here, we determine the role of neutrophils in DMTB patients and in driving TB immunopathology. Methods: Sputum and plasma from 30 TB and 30 DMTB patients were analysed for proteases and cytokines using Luminex bead array. Whole blood transcriptomics identified transcriptional differences. Single-cell RNA sequencing characterised neutrophil subsets and dysregulated pathways. Neutrophil function of poorly-controlled DM patients (HbA1c>8%) and healthy controls (HC) were examined following Mycobacterium tuberculosis stimulation, including reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and phagocytosis. Pathways were interrogated using chemical inhibitors, protein array and western blot. Results: Compared to non-diabetic TB patients, poorly-controlled DMTB patients showed up-regulated sputum MMP-8 and MMP-9, associated with increased collagen-destruction and lung cavity formation. Circulating neutrophil count and neutrophil-derived plasma MMP-8 were up-regulated, alongside transcriptional enrichment of extracellular matrix degradation and inflammatory pathways including TNF and RAGE. Single-cell profiling identified reduced cycling neutrophil subset and myelocytes in DMTB, with overall reduced antibacterial and cell-killing signatures. Ex vivo mycobacterial stimulation of DM neutrophils increased ROS and MMP-9 with impaired NETs and delayed phagocytosis. TNFR1, TNFR2, and RAGE were up-regulated. RAGE inhibition with rosiglitazone mitigated Mtb-induced ROS and MMP-8 release. Conclusion: DM worsens neutrophil-driven tissue destruction and inflammation in TB via dysregulated TNF and RAGE-signalling, priming neutrophils towards immunopathology. Targeting RAGE alongside tight glycaemic control may dampen neutrophil hyper-inflammatory responses to limit tissue destruction.

BackgroundCystic fibrosis (CF) is an autosomal recessive genetic disorder that leads to chronic infection and mucus retention in the lungs, with lung function gradually deteriorating through recurrent pulmonary exacerbations (PEx). Virulence factors (VFs) of Pseudomonas aeruginosa and Staphylococcus aureus are thought to contribute to pulmonary exacerbations. Our study objective was to identify VF genes related to PEx, high Pseudomonas abundance, and high Staphylococcus abundance in persons with CF (pwCF). MethodsThis was an ancillary study of pwCF treated with IV antibiotics for PEx between 2016-2020 at Childrens National Hospital. Using shotgun metagenomics and ShortBRED, we identified bacterial VF genes and used DESeq2 to determine differential expression of VF genes across comparators. ResultsTwenty-two PwCF experienced 43 PEx. The study cohort had a mean age of 14.6 years, 41% female, 59% white, 36% Hispanic, and 45% had an F508del homozygous CFTR mutation. Minimal differences in VF gene abundance were identified across clinical state. The most differentially increased VF genes found in Pseudomonas high samples were associated with an aminotransferase (log2FC 25.9), flagellar biosynthesis (log2FC 8.3), and type VI secretion systems (log2FC 8.2). The most differentially increased VF genes found in Staphylococcus high samples were an exotoxin (log2FC 26.7), macrolide phosphotransferase (log2FC 25.8), pathogenicity island proteins (log2FC 25.2 and 24.7), and VOC family proteins (log2FC 24.8). ConclusionsThese findings demonstrate that specific VFs associated with immune modulation, motility secretion systems, bacterial motility, and antibiotic resistance are related to P. aeruginosa and S. aureus abundance, providing potential targets for more personalized antimicrobial interventions.

ObjectiveSystemic sclerosis (SSc) predominantly affects females but exhibits greater disease severity in males, suggesting sex differences underlying SSc pathogenesis. We sought to define sex-associated alterations in the peripheral immune landscape of patients with SSc. MethodsWe performed high-dimensional immune profiling of PBMCs from 37 healthy donors (68% female) and 37 patients with SSc (11 limited, 26 diffuse; 68% female) using 30-color spectral flow cytometry, quantifying 56 immune cell subsets per donor. We conducted sex-stratified comparisons and correlation analysis, and used principal component analysis followed by linear discriminant analysis to derive a sex-discriminant immune cellular module. ResultsDiffuse cutaneous SSc (dcSSc) was associated with a distinct immune landscape characterized by increased monocyte and decreased natural killer-like and B cell frequencies, suggesting a myeloid-skewed peripheral immunophenotype. Males exhibited greater enrichment of innate immune subsets, including monocyte and dendritic cell subsets, while females exhibited greater enrichment of adaptive immune subsets. Among T cells, dcSSc was associated with coordinated remodeling across CD4+ and CD8+ subsets, including expansion of stem cell memory T cells (Tscm), and increased regulatory T cells, Th17 skewing, and decreased effector-memory CD8+ subsets. Females exhibited greater proportions of naive- and Tscm, and males exhibited higher proportions of effector-memory subsets. Integrating these data, we identified a sex-discriminant immune module comprised of 20 cell types that distinguishes males and females with dcSSc. ConclusionsSSc is associated with sex-specific differences in the peripheral immune landscape. A sex-associated immune program, further amplified in disease, may contribute to the paradox of female-biased susceptibility and male-biased severity in SSc.

Lung neuroendocrine tumours (lung NETs) are rare neoplasms comprising approximately 2% of lung cancers. Recent studies have identified distinct molecular groups based on transcriptome and methylome data, but genomic and morphological features remain underexplored due to limited whole-genome and imaging data. We have generated the largest multi-omic dataset of lung NETs to date (201 participants, for a total of n = 294 tumours), including RNA sequencing, EPIC 850K methylation arrays, and whole-genome sequencing. This multiomic dataset also include multi-regional whole-genome sequencing for 41 participants, allowing for the quantification of intra-tumoural heterogeneity. We additionally generated spatial proteomics (64 participants), spatial transcriptomics (4 participants) and whole-slide histopathology images for 212 cases. This dataset enables a comprehensive characterization of lung NET molecular groups and the identification of group-specific morphological features using deep learning algorithms. All quality control analyses, processed data, and scripts are provided to ensure reproducibility. This dataset is available as a basis for further molecular and morphological analysis of lung NETs, and for future research on multi-scale integration.

BackgroundCalcitonin gene related peptide (CGRP) hyperpolarizes pulmonary arterial smooth muscle cells (SMCs) and endothelial cells (ECs) through PKA-dependent activation of KATP channels. CGRP can diminish the severity of pulmonary fibrosis (PF), however, the effects on vascular signaling were poorly defined. We hypothesized that hyperpolarization to CGRP would be augmented in a mouse model of PF. MethodsPF was induced in male and female C57BL/6 mice by intratracheal delivery of bleomycin (3 wk), with saline used as control (sham). Pulmonary arteries (PAs; 100-150 {micro}m diameter) were cannulated and pressurized to 16 cmH2O, and endothelial tubes were studied in complementary experiments to eliminate the influence of SMCs. Membrane potential (Vm) was recorded continuously using intracellular microelectrodes. Responses were also evaluated in isolated lungs preconstricted with U46619 ([~]10 mmHg). ResultsPF led to greater indices of PH in males vs. females. Isolated lungs and PAs from male PF mice had enhanced vasodilation and hyperpolarization of Vm to CGRP, although no effect was observed in females. The greater vasodilation and hyperpolarization of SMCs to CGRP in males persisted in endothelium-disrupted PAs and during treatment with L-NAME indicating that ECs are not required for greater responsiveness to CGRP. With no effect on resting Vm, inhibition of KATP channels or PKA significantly attenuated hyperpolarization of SMCs and ECs, attenuated vasodilation to CGRP in PAs, and eliminated differences between groups in males. Direct activation of PKA, but not KATP, evoked greater Vm hyperpolarization and vasodilation in PF vs. sham PAs and lungs. Although no difference in sensory nerves was observed in fibrotic mice, perivascular nerve stimulation evoked greater vasodilation in PAs. ConclusionsIn a mouse model of PF, CGRP-dependent hyperpolarization of pulmonary arterial SMCs and ECs is augmented through increased PKA-dependent activation of KATP channels leading to increased vasodilator sensitivity.

Rationale: Air trapping in functional areas of the lung is common in chronic obstructive pulmonary disease (COPD). We developed a novel music-based intervention, Engagement of Music for Pulmonary Obstruction With Expiratory Restoration (EMPOWER) aimed at reducing air trapping and functional small airways disease (fSAD) in patients with COPD. Objectives: We conducted a pilot study to assess if air trapping and fSAD in COPD patients are reduced by our targeted EMPOWER music-based singing intervention. Methods: Participants completed four weeks of singing and vocalizing with a board-certified music therapist. Pre- and post-intervention assessments of standard pulmonary function tests (PFTs), and quantitative computed tomography (qCT) lung imaging documented changes in air trapping. Pre- and post-intervention change in psychological and patient-reported outcomes of hope, emotional wellbeing, agency and COPD symptom burden were also obtained. Main Results: All five adult participants with COPD who enrolled completed the study and reported strong interest in continuing with a similar program. Additionally, we observed trends toward improvement in qCT-measured fSAD, six-minute walk distance, and patient-reported symptoms on the COPD Assessment Test. Conclusion: Results of this preliminary study showed improvements in both patient-reported and imaging-indicated respiratory outcomes, suggesting that targeted singing components in music-based interventions such as the EMPOWER intervention may support physiological lung function changes in COPD patients.