American Journal of Respiratory Cell and Molecular Biology

Background Idiopathic Pulmonary Fibrosis (IPF) is a fatal chronic lung disease with limited therapeutic options. While alveolar epithelial injury and fibroblast activation are well-studied, endothelial-mesenchymal transition (EndoMT) is emerging as a critical pathogenic mechanism. The regulatory role of exosomal miRNAs in pulmonary fibrosis remains unclear. This study investigates serum exosomal miRNAs, particularly let-7a-5p, in modulating EndoMT during the onset of pulmonary fibrosis. Methods Clinical cohorts of IPF patients and healthy controls were enrolled. Serum exosomal miRNAs were profiled, followed by differential expression and functional enrichment analyses. In vitro experiments involved human pulmonary artery endothelial cells (HPAECs) transfected with let-7a-5p mimic or inhibitor. Dual-luciferase reporter assays confirmed the binding between let-7a-5p and TGFBR1. HPAECs were co-cultured with lung epithelial cells to examine paracrine signaling. In vivo studies used a bleomycin-induced mouse model with let-7a-5p agomir administration. Assessments included histopathological staining, hydroxyproline content, Western blot, qPCR, micro-CT, and pulmonary function tests. Results Let-7a-5p was significantly downregulated in serum exosomes from IPF patients, correlating with clinical indicators. Mechanistically, let-7a-5p directly bound the TGFBR1 3'UTR to inhibit its expression. Inhibition of let-7a-5p upregulated -SMA, FN1, smad2/3 phosphorylation, and collagen I, while downregulating CD31 and VE-cadherin. Therapeutically, let-7a-5p mimic reversed bleomycin-induced EndoMT and suppressed epithelial-mesenchymal transition (EMT) via paracrine signaling. Mice administered agomir showed reduced fibrosis, improved lung function, and suppressed TGF-{beta}/Smad signaling. Conclusion Serum exosomal let-7a-5p suppresses pulmonary fibrosis by targeting TGFBR1 to inhibit EndoMT. Its downregulation in IPF patients correlates with disease progression, highlighting its biomarker potential.

The mechanisms underlying the progression of pulmonary fibrosis in idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases remain unclear. Increased extracellular matrix stiffness is a hallmark of fibrotic lung diseases. Monocyte-derived macrophages can promote fibrosis progression. However, there is limited understanding of how the mechanical properties of the fibrotic microenvironment influence macrophage phenotypes. Profibrotic macrophages are apoptosis-resistant, and this phenotype is modulated by enhanced mitochondrial bioenergetics. The objective of the study was to determine how lung tissue stiffness impacts macrophage phenotypes and fibrotic progression. We demonstrate that mechanoactivated macrophages exhibit apoptosis-resistance, increased expression of the antiapoptotic protein Bcl-xL and increased mitochondrial oxidative phosphorylation. Critically, the metabolic reprogramming observed in mechanoactivated macrophages is dependent on increased glutaminolysis. Inhibition of glutaminolysis attenuated apoptosis resistance in mechanoactivated macrophages. Moreover, inhibition of Bcl-xL in vivo protected mice against experimental pulmonary fibrosis. Lastly, mechanoactivated primary IPF macrophages produce more profibrotic cytokines and promote extracellular matrix production in precision-cut lung slices. We describe a mechanism for acquired macrophage apoptosis resistance dependent on metabolic reprogramming regulated by extracellular matrix stiffness. Our results identify mechanoactivated apoptosis-resistant macrophages as pro-fibrotic mediators, suggesting a novel therapeutic target in IPF and related fibrotic disorders.

Objective: Lymphatic dysfunction has been implicated in exacerbating fibrosis in numerous diseases, yet the role of the lymphatic system in laryngotracheal injury has not previously been explored. This study aims to evaluate lymphatic vascular remodeling in a murine model of laryngotracheal stenosis (LTS) and determine how pharmacologic blockade of lymphangiogenesis impacts airway healing after mucosal injury. Methods: LTS was induced in C57BL6 mice using an established chemomechanical injury model. Lymphatic density was quantified using LYVE-1 immunohistochemistry. Mice were treated with the VEGFR-3-selective tyrosine kinase inhibitor SAR131675 to block lymphangiogenesis after injury. Outcomes assessed included survival, histopathology, immunohistochemistry, and Evans blue dye vascular leakage. Results: Laryngotracheal injury induced a substantial increase in subepithelial lymphatic vessel density concomitant with fibrotic remodeling. Pharmacologic inhibition of VEGFR-3 signaling with SAR131675 abrogated this lymphangiogenic response and resulted in markedly increased mortality, impaired epithelial repair with obstructive sloughing, increased edema, and persistent histopathologic evidence of tissue injury. A qualitative increase in pathologic fibrocellular remodeling was also observed, though with no measurable difference in lamina propria thickness. Conclusion: These findings establish lymphatic remodeling as an essential component of successful airway repair following mucosal injury. Lymphatic dysfunction is a common feature of known risk factors for LTS including diabetes, obesity, and prematurity, and can be exacerbated by positive pressure ventilation. Disruption of the lymphangiogenic response to airway injury may lead to stasis of pro-inflammatory factors that result in chronic inflammation, maladaptive remodeling, and pathologic tissue changes. The lymphatic vasculature is a viable target for future mechanistic study and potential therapeutic intervention following airway injury.

Background: Although the lung is of low biomass, it harbors a diverse and dynamic microbiome that may influence disease and healing. Existing studies have used diverse sampling methods with high propensities for contamination and sampling error, leading to diverse and unclear results. Here, we characterized the lung microbiome via airway and parenchymal samples to determine variation across patients and sampling methods. Methods: We recruited adult patients undergoing lung resection for suspected or confirmed malignancy. After resection and under sterile conditions, a 1 cm cubic piece of non-cancerous lung parenchyma and a swab from the specimen's bronchus were collected and sent for microbiome analysis via 16S rRNA gene amplicon (V4) sequencing on an Illumina platform. An established bioinformatics pipeline was used to determine taxonomic identification. Baseline clinical and demographic data were compared to microbiome composition. Results: A total of 86 patients were included in the study. Beta diversity (microbial composition) varied significantly by sampling method (biopsy of lung parenchyma versus airway swabs), so all further results were analyzed within sample types. Further analyses revealed significant differences in beta diversity by lobe of the lung, indicating a different microbial composition by anatomic location. Analyses of patient demographics revealed significant differences by age and comorbidities, including chronic obstructive pulmonary disease and atrial fibrillation. Conclusions: The lung harbors a diverse microbiome that differs by anatomic location and patient characteristics. This study provides a framework for more accurate future lung microbiome sampling and characterization.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal lung disease with a median survival of 3-5 years after diagnosis. Current antifibrotic therapies slow disease progression, but do not halt or reverse fibrosis, underscoring the need for new therapies. We identified a dysregulated oxysterol-GPR183 axis as a driver of IPF. Oxidized cholesterols were elevated in lungs from IPF patients, with myofibroblasts representing the dominant source of 7,25-hydroxycholesterol (7,25-OHC), the endogenous high affinity ligand for the oxysterol-sensing receptor GPR183. IPF patients had increased GPR183 expression in interstitial and monocyte-like macrophages compared to controls. In a bleomycin-induced model of pulmonary fibrosis genetic deletion of GPR183 reduced disease severity characterized by reduced fibrosis, inflammation, and accumulation of macrophages and myofibroblasts. Pharmacological inhibition of GPR183 with the antagonist NIBR189 attenuated fibrosis when administered preventatively from day 1-7 after bleomycin exposure. Notably, therapeutic treatment with the GPR183 antagonist after commencement of fibrosis development at day 10 post-bleomycin also significantly reduced fibrotic pathology, achieving efficacy comparable to the approved antifibrotic nintedanib. However, the GPR183 antagonist was more potent in reducing inflammation and myofibroblast activation compared to nintedanib. Together, these findings identify an oxysterol-GPR183 signaling axis that contributes to pulmonary fibrogenesis and provide a strong preclinical rationale for targeting GPR183 as a novel therapeutic strategy for IPF. One Sentence SummaryTargeting GPR183 reduced lung fibrosis and inflammation in a preclinical model, supporting GPR183 as a promising new therapy.

2.Rhinoviruses (RVs) are the predominant cause of the common cold and a major trigger of acute asthma exacerbations. Yet, unlike related enteroviruses such as poliovirus (PV) and enterovirus A71 (EV-A71), no approved vaccines or antivirals exist. Because enteroviruses depend heavily on host factors for replication, cellular proteins that support the replication of multiple enteroviruses have emerged as attractive broad-spectrum antiviral targets that may offer a higher barrier to resistance than virus-targeted therapies. The 70-kDa heat shock protein (Hsp70) family, a highly conserved class of molecular chaperones, is required for the replication of several enteroviruses, including EV-A71 and coxsackievirus A16, but whether RVs share this dependency was unknown. Here, we show that two mechanistically distinct small-molecule inhibitors of the Hsp70 family abolish replication of RV-A16, a clinically relevant RV type widely used in asthma research. Using siRNA knockdown, we demonstrate a role for HSPA8, the major constitutively expressed Hsp70 isoform, in RV replication. We further demonstrate that Hsp70 activity is indispensable for viral translation, identifying this as a key Hsp70-dependent step in the RV replication cycle. Together, these findings establish Hsp70 chaperones as essential host factors for RV replication and strengthen the rationale for targeting them as a broad-spectrum antiviral strategy. 3. Impact statementRhinoviruses are the predominant cause of the common cold and major triggers of exacerbations in people with asthma and chronic obstructive pulmonary disease, yet no licensed antiviral therapies exist. Although several enteroviruses are known to require Hsp70 chaperones for replication, whether rhinoviruses share this dependence was unknown. Here, we address this gap by demonstrating that Hsp70 chaperones are essential host factors for rhinovirus replication and are required for viral translation. These findings advance our understanding of how rhinoviruses exploit the host cell machinery and broaden the evidence supporting Hsp70 chaperones as potential antiviral targets across the Enterovirus genus. As host-targeted therapies may be less vulnerable to resistance than direct-acting antivirals, these findings represent an important step towards the development of urgently needed anti-rhinoviral therapeutics and will be of interest to virologists, respiratory clinicians and antiviral drug developers.

Motile cilia demonstrate coordinated beating to propel fluids across epithelial tissues, and changes to their beating frequency are largely regulated by intracellular second messengers including Ca2+. In the airway epithelium, ciliary beating is essential to mucociliary clearance. Mucociliary clearance involves trapping inhaled pathogens and irritants in sticky mucus lining the airways for motile cilia to sweep away contaminated mucus, preventing infection and reducing general airway inflammation. Many chronic respiratory diseases, including chronic rhinosinusitis and asthma, are characterized by an acquired ciliary dysfunction. Despite the importance of Ca2+ signaling in cilia physiology, the identity and molecular mechanisms governing localized ciliary Ca2+ transport remain poorly understood. MS4A8B is an uncharacterized cilia-localized transmembrane protein. Other MS4A homologs have been indirectly linked to Ca2+ signaling via uncharacterized mechanisms. Using primary human nasal epithelial cells differentiated at air-liquid interface, we demonstrated that MS4A8B regulates motile cilia function. MS4A8B knockdown impairs ciliary beating and impacts cilia structure. Live-cell imaging combined with genetic analysis revealed that MS4A8B potentiates Orai1-mediated Ca2+ influx. Co-immunoprecipitation and FRET microscopy in ectopic expression systems demonstrated that MS4A8B interacts with Orai1 channels. Orai1 was further identified to reside in motile cilia of primary human nasal epithelial cells, allowing ciliary beat frequency to be stimulated by Orai1 agonists including arachidonic acid. MS4A8B functional coupling with Orai1 acts as an autonomous cilia signaling network. Targeting this compartmentalized signaling pathway offers a novel therapeutic approach to restore or enhance mucociliary clearance in airway diseases.

Cell confluency is a major determinant of cell-cell communication, protein interactions, access to nutrients, and signalling dynamics, thereby significantly impacting biological outcomes. Lung cancer cells like A549 are widely used as screening models for scientific studies wherein their growth in vitro progress from non-confluent to confluent growth. In this study, we investigated the transcriptomic adaptations associated with the transition of A549 cells from baseline non-confluent to confluent growth. Comparative transcriptomic analysis between confluent and cells at baseline identified 815 upregulated and 671 downregulated transcripts. Pathway enrichment analysis of deregulated transcripts in confluent cells revealed enhanced cholesterol and sterol biosynthetic pathways, along with suppression of chromosomal segregation and mitotic pathways. At confluency, an increased expression of glucose transporters (SLC2, SLC60, and SL37 families) and glycolytic pathways, and a decrease in amino acid transporters (SLC1, SLC7, SLC38, and SLC36) and amino acid metabolic pathways is observed. A reduced one-carbon metabolic signature (SHMT2, DHFR, and MTHFD2) and enhanced fatty acid precursor synthesis (HMGCLL1, ALDH6A1, and AASS) were also observed at confluency. 1H NMR profiling of culture media revealed higher glucose and glutamine utilisation with lactate accumulation during culture maturation. Collectively, the data suggest transcriptome-level rewiring in A549 cells with preferential biosynthesis of lipids and sterols at confluency and underscore the importance of considering culture maturity in cancer biology, metabolism, and therapeutic studies.

Background: The systemic immune responses associated with persistent respiratory symptoms (PRS) after exposure to airborne environmental pollutants remain poorly understood. Objective: To identify immune disturbances associated with PRS, defined as persistent wheeze, cough, or breathlessness, we examined systemic immune responses and airway function in a cross-sectional cohort with detailed histories of airborne pollutant exposure. Methods: Never-smoking post-deployment Veterans with PRS (n=16) or without PRS (n=24) underwent chest computed tomography, pulmonary function testing, and oscillometry to assess structural and functional airway abnormalities. Peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3/CD28 antibodies, lipopolysaccharide, or {beta}-glucan, and cytokine production was measured. Correlation analyses evaluated associations between cytokine responses and physiological measures of airway function. Results: Oscillometry, but not conventional pulmonary function testing or chest computed tomography, detected small-airway abnormalities in participants with PRS, including significantly greater frequency dependence of resistance and higher resonant frequency. Baseline PBMC cytokine concentrations were similar between groups. After stimulation, however, PBMCs from participants with PRS showed increased IL-17A production consistent with a type 17 (T17) response; innate stimulation also increased the type 2 (T2) cytokines IL-33 and IL-4. T2/T17 cytokine responses correlated positively with oscillometric measures of small-airway dysfunction. Conclusion: Individuals with PRS exhibited a stimulus-dependent systemic T2/T17 immune signature that was associated with early small-airway dysfunction. Clinical Implication: Stimulus-dependent systemic immune profiling, combined with oscillometry, may help identify early respiratory abnormalities in pollutant-exposed individuals whose conventional pulmonary tests remain normal.

Inadequate autophagy promotes smoking-induced cellular senescence involved in chronic obstructive pulmonary disease (COPD) pathogenesis. Transcription factor EB (TFEB) is a master regulator of the autophagy-lysosome axis. For the first time, we investigated the therapeutic potential of pemafibrate, a putative TFEB inducer. COPD lung epithelial cells showed reduced TFEB expression. Pemafibrate enhanced autophagy/mitophagy flux and restored lysosomal acidification observed during cigarette smoke (CS) extract exposure in human bronchial epithelial cells, resulting in reduced cellular senescence. TFEB knockdown demonstrated involvement of pemafibrate-induced TFEB in these effects. Pemafibrate induced TFEB expression, mitigated alveolar enlargement and airflow obstruction, and attenuated the CS-induced increase in static lung compliance in a long-term CS-exposed mouse model. It reduced the CS exposure-induced cellular senescence, possibly through autophagy/mitophagy, as suggested by bulk RNA sequencing of mouse lungs. A retrospective cohort study showed that patients given pemafibrate displayed attenuated FEV1.0 decline compared with those given bezafibrate or fenofibrate. In conclusion, pemafibrate is a promising therapeutic agent for COPD, potentially exerting its effects through the regulation of the TFEB-autophagy/mitophagy-lysosome axis.

Objective White smoke inhalation injury (WSI) causes severe acute lung damage with no specific therapy currently available. Sphingolipid metabolism is implicated in pulmonary inflammation, but its transcriptional regulatory landscape in WSI remains unexplored. This study aimed to identify key sphingolipid metabolism related genes and evaluate their regulatory roles and therapeutic potential in WSI. Methods We established a rat model of WSI and performed integrated bulk RNA sequencing, weighted gene coexpression network analysis (WGCNA), and single-cell RNA sequencing (scRNAseq) to screen for differentially expressed sphingolipid metabolism-related genes (DESRGs). Protein-protein interaction (PPI) network with four centrality algorithms was used to prioritize hub genes. In silico gene knockout and molecular docking were conducted to assess regulatory functions and identify potential drug candidates. Results We identified 22 DESRGs that were predominantly enriched in DNA replication and cell cycle pathways rather than canonical sphingolipid metabolic processes. PPI consensus prioritized three hub genes--Top2a, Ttk, and Ccna2--with Top2a exhibiting the highest expression in epithelial cells and significant downregulation after smoke exposure. ScRNAseq revealed immune cell infiltration and epithelial differentiation trajectories. Virtual knockout showed that Top2a depletion affected the largest transcriptomic fraction (~0.4%) and was enriched in lysosome biogenesis, innate immunity, phagocytosis, and lipid catabolism. Molecular docking identified thalidomide as a high affinity ligand for Top2a (Vina score: -8.5 kcal/mol). Conclusion Our multiomics integrative framework identifies Top2a as a central regulatory hub linking sphingolipid associated inflammation to epithelial responses in WSI, and nominates thalidomide as a potential drug repurposing candidate. These findings provide prioritized targets for future translational investigation.

Interstitial lung diseases (ILDs) are heterogeneous pulmonary disorders characterized by chronic inflammation and/or fibrosis. 30-40% of ILD patients develop fibrotic disease that is associated with progressive respiratory decline and poor prognosis, particularly in idiopathic pulmonary fibrosis. Current antifibrotic therapies slow disease progression but do not reverse fibrosis, highlighting the need for improved therapeutic strategies. Robust histopathological evaluation in preclinical models is essential for drug development; however, conventional scoring systems are semi-quantitative, labor-intensive, subject to inter-observer variability, and rely on limited field sampling. Here, we introduce FibroSight, a standalone platform for compartment-resolved quantification of lung remodeling in Sirius Red-stained sections. By integrating deep learning- based structural segmentation with color-based feature extraction, FibroSight enables highly automated whole-lobe analysis without requiring complex computational setup. The platform quantifies complementary remodeling parameters, including parenchymal collagen fraction, parenchymal tissue density, nuclear area fraction, parenchymal airspace fraction, and airway- and vascular-associated remodeling. Validated in the bleomycin-induced fibrosis model, FibroSight-derived metrics strongly correlated with expert Ashcroft scoring and showed stronger associations with histological severity than corresponding outputs from a semi-automated ImageJ-based workflow. The platform further distinguished inflammatory from fibrotic remodeling in influenza-induced lung injury and demonstrated translational proof-of-concept applicability in human ILD biopsy specimens. By enabling scalable, reproducible, and multi-compartment histological quantification, FibroSight provides a practical framework for objective assessment of lung remodeling. This approach expands conventional fibrosis evaluation by integrating fibrotic, inflammatory, airway, and vascular-associated readouts, supporting more precise analysis of disease mechanisms and therapeutic responses in preclinical and translational ILD research.

Background: Chronic lung allograft dysfunction (CLAD) is the leading cause of death beyond the first year after lung transplantation, and its most frequent phenotype is bronchiolitis obliterans syndrome (BOS), a fibrotic small-airway disease. Mechanistic work has focused on the immune compartment, yet intensified immunosuppression does not alter established disease. Aim: To resolve which structural cell states populate the BOS graft and how they are spatially organized during airway obliteration. Methods: We profiled explanted lungs from 33 BOS patients undergoing re-transplantation and 33 controls, combining single-nucleus RNA sequencing (14 BOS, 13 controls) with targeted spatial transcriptomics of 108 regions (27 BOS, 24 controls) and multiplex immunofluorescence validation. Single-nucleus data were integrated with a published restrictive allograft syndrome (RAS) atlas. Results: Across 175,128 nuclei and 1.67 million spatially resolved cells, BOS lungs harbored a profibrotic circuit of Aberrant Basaloid cells and CTHRC1+ fibrotic fibroblasts previously described in fibrotic lung diseases, including RAS. Spatial mapping identified a CXCL14+TNC+ injury-associated basal cell state arising early in the obliterative cascade, identifying basal cells as their major reservoir. CTHRC1+ fibroblasts expanded subepithelially replacing resident peribronchial fibroblasts, alongside a peribronchial vascular shift toward systemic venous endothelium. The circuit extended beyond the airway wall to the alveolar interface, defining two convergent remodeling fronts. Conclusion: BOS engages structural-cell circuits largely shared with RAS and fibrotic lung diseases, but along an airway-centered rather than parenchyma-centered axis. CLAD thus emerges as a spatial rather than cellular spectrum, defined by anatomical distribution more than cell identity. Shared structural programs may therefore be targetable across CLAD phenotypes.

Cigarette smoke-induced lung inflammation is a central driver of pulmonary diseases. The limited efficacy of current anti-inflammatory agents underscores the need for structurally novel therapeutics with distinct mechanisms. We recently demonstrated that AB-free kava, a flavokavains A/B-depleted formulation from Piper methysticum containing six major kavalactones, effectively suppresses cigarette smoke-induced lung inflammation in mice. This study aims to identify the bioactive constituent(s) and elucidate underlying mechanisms. These kavalactones revealed a clear structure-activity relationship in suppressing lipopolysaccharide (LPS)-stimulated prostaglandin E2 (PGE2) production in macrophages with desmethoxyyangonin (DMY) as the most potent kavalactone whereas dihydrokavain (DHK, a structurally similar analog) with minimal activity. DMY also effectively reduced LPS-induced interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-) production while DHK was ineffective. Mechanistically, DMY, but not DHK, attenuated COX-2 induction and reduced phosphorylation of cAMP response element-binding protein (CREB). Pharmacological inhibition of protein kinase A (PKA) similarly reduced p-CREB, COX-2 and PGE2, supporting a PKA-dependent CREB/COX-2 signaling in mediating PGE2 suppression while these effects were independent of nuclear factor kappa B (NF-{kappa}B) and activator protein 1 (AP-1) signaling. Similar results were observed for DMY and DHK in attenuating cigarette smoke condensate-induced proinflammatory pathways and PGE2 production. Consistently, DMY demonstrated significant in vivo efficacy in suppressing cigarette smoke-induced lung inflammation while DHK was not effective. Interestingly, dihydromethysticin (DHM) demonstrated the greatest in vivo anti-inflammatory efficacy, although it only exhibited moderate in vitro potency, likely due to its superior bioavailability over DMY. Concordantly, cigarette smoke exposure elevated p-CREB and COX-2 expressions in mouse lungs, which were attenuated by AB-free kava and its bioactive kavalactones with the extent of suppression correlating with their in vivo anti-inflammatory efficacy. DHM effectively suppressed LPS-induced neutrophil accumulation in mouse lungs as well. Collectively, these studies identify bioactive kavalactones in AB-free kava that suppress cigarette smoke- and LPS-induced lung inflammation through the modulation of the PKA/CREB/COX-2 signaling axis, providing a foundation for developing structurally distinct anti-inflammatory agents, particularly targeting smoke-induced inflammation and associated pulmonary diseases.

Chronic Pseudomonas aeruginosa lung infections are becoming harder to treat due to global escalation of antimicrobial resistance (AMR). Bacteriophage (phage) therapy has emerged as a promising adjunct to conventional antibiotics, especially in chronic lung infections such as those seen in cystic fibrosis (CF). However, phage monotherapy may be limited by the emergence of phage-resistant bacterial populations and there remains limited preclinical evidence evaluating both antimicrobial efficacy and host safety in physiologically relevant human airway models. Here, we evaluated the safety and antimicrobial activity of Kara-mokiny 3, a myovirus bacteriophage, alone and in combination with subinhibitory concentrations of tobramycin using fully differentiated paediatric primary airway epithelial cells (pAECs) cultured at the air-liquid interface (ALI). Kara-mokiny 3 rapidly reduced P. aeruginosa viability and exhibited synergistic activity with tobramycin, resulting in significantly greater bacterial killing than either treatment alone. Importantly, phage treatment replicated efficiently in the presence of its bacterial host while preserving epithelial morphology, mucin production and epithelial barrier architecture., without inducing cytotoxicity or excessive IL-6 and IL-8 inflammatory responses. These findings demonstrate that phage-antibiotic combination therapy can enhance antimicrobial activity while maintaining epithelial safety in a physiologically relevant human airway model. This study represents one of the first comprehensive evaluations of phage-antibiotic combination therapy in differentiated primary airway epithelial cultures, providing important preclinical evidence supporting the development of personalised phage-based therapies for the treatment of MDR pulmonary infections. ImportanceThe rise of MDR P. aeruginosa has created an urgent need for alternative treatment strategies for chronic lung infections. Although phage therapy is receiving increasing clinical attention, there is limited evidence evaluating its safety and efficacy in physiologically relevant human airway models. Using differentiated primary airway epithelial cultures, we demonstrate that a phage-antibiotic combination reduces bacterial burden without compromising epithelial integrity and toxicity or excessive inflammatory responses. These findings provide translational evidence supporting phage-antibiotic combination therapy and highlight the value of primary airway epithelial models for the preclinical assessment of emerging antimicrobial interventions, supporting the translation of personalised phage therapies.

Current approaches for imaging fibrotic remodeling have sensitivity, specificity and cost drawbacks that limit both preclinical research and clinical diagnosis. Here, we show that fast green FCF, a small molecule that binds to fibrillar collagen, enables highly sensitive and specific imaging of fibrosis in lung samples from mice and humans using fluorescence microscopy. We report strategies for using fast green FCF staining to assess fibrotic remodeling using precision-cut lung slice and whole-biopsy preparations. Our findings demonstrate that fluorescence imaging of fast green FCF-stained collagen will be useful for fibrosis research and may help to improve detection of fibrosis in clinical pathology.

Immune adaptation after birth requires coordinated remodeling across the airway-blood axis, yet how these compartments communicate during early postnatal life remains poorly understood. In preterms, dysregulation of this immune response determines mortality and morbidity. We performed paired single-cell RNA sequencing (scRNA-seq) and mass spectrometry-based proteomic profiling of airway samples (deep pharyngeal aspirates, DPA) and matched whole blood from 19 neonates spanning extreme preterm (<28 weeks) to term gestation, sampled at two postnatal timepoints (1-3 days and 4-10 days). This integrated multiomic atlas revealed coordinated and compartment-specific immune adaptation across the airway-blood axis during the first week of life. We observed gestational age-dependent shifts in cell composition in both compartments, including expansion of immature hematopoietic and myeloid populations in blood and distinct myeloid and epithelial programs in DPA, accompanied by compartment-specific inflammatory and innate immune gene expression that evolved during the first week of life. Unexpectedly, we identified a circulating respiratory epithelial-like cell population in neonatal blood whose abundance correlated with prematurity and lung disease and which we validated by flow cytometry as well as in independent datasets. Matched plasma proteomics revealed a gestational age axis and a disease-associated axis; an Organ-to-System Score derived from lung-restricted plasma proteins tracked lung injury severity and distinguished trajectories toward chronic lung disease as early as 72 hours after birth. Together, these multiomic data describe coordinated and divergent immune programs across mucosal and systemic compartments in early preterm life, identify circulating respiratory epithelial-like cells as a candidate blood-accessible signal of airway-blood interface perturbation, and prioritize ciliated-myeloid signaling (LAMA5-ITGB1) as a candidate axis underlying neonatal lung disease. This systems-level framework provides a platform for biomarker discovery and mechanistic studies in larger cohorts.

Research on Pseudomonas aeruginosa adaptation in cystic fibrosis (CF) has historically relied on comparing chronic isolates to laboratory reference strains, or evolving reference strains in environments simulating chronic CF. This work has established a small set of genes, including lasR, mucA, and mexZ, as canonical markers of CF patho-adaptation. However, without broad non-CF comparators, it remains unclear how specific these signatures are to CF. We used a structured literature review to define 20 historically emphasized "canonical CF genes", then evaluated their mutational patterns across 4,475 genetically distinct P. aeruginosa genomes from seven defined clinical and environmental contexts. We tested four competing hypotheses: (1) enrichment in adult CF alone, (2) in adult and pediatric CF combined, (3) in chronic lung infections broadly (including non-CF bronchiectasis), or (4) no strong environment-specific enrichment. We found little evidence that canonical gene mutations were specifically enriched in adult CF or CF more broadly. Instead, loss-of-function and individual mutations in genes including mucA, mexB, and mexZ were enriched across chronic lung infections, while most canonical genes (including lasR) showed no strong environment-specific enrichment. These results demonstrate that a canon of genes believed to drive patho-adaptation in CF instead largely reflects the narrow comparative framework of past studies rather than CF-exclusive selection. Our findings emphasize shared evolutionary pressures between CF and non-CF bronchiectasis, highlighting opportunities to exchange research and therapeutic insights across chronic infection clinical contexts.

Acute Respiratory Distress Syndrome (ARDS) remains highly morbid and lacks approved disease-modifying pharmacotherapies. Direct (pulmonary) and indirect (extrapulmonary) insults may initiate biologically distinct early injury programs, but human tissue-level evidence from the first hours is scarce. Here we establish a paired, acellular ex vivo lung perfusion (EVLP) platform using human donor lungs unsuitable for transplantation to model direct (endobronchial) and indirect (perfusate) lipopolysaccharide (LPS) injury within the same donor. We profiled lung tissue proteomes at 4 h post-insult and performed therapeutic nomination by querying proteomics-derived injury signatures against the CLUE L1000 perturbational compendium with independent cross-platform validation. Both models developed histological injury and robust cytokine release. Direct injury preferentially enriched neutrophil degranulation, extracellular matrix remodelling and metabolic reprogramming modules, whereas indirect injury showed prominent complement/coagulation perturbation with greater endothelial activation markers in perfusate. Cross-platform prioritisation converged on tractable signalling and epigenetic axes, including JAK/STAT, PI3K/AKT/mTOR, SYK, CDK and HDAC inhibitor classes - yielding a tiered shortlist for EVLP intervention testing. This intact human lung perturbation platform enables injury-stratified mechanistic inference and therapeutic prioritisation in early lung injury relevant to ARDS.

Air-liquid interface (ALI) cultures consisting of well-differentiated primary respiratory epithelial cells (PRECs) provide a versatile in vitro model for pharmacological studies and to investigate host-pathogen interactions. Proliferation and differentiation of PRECs require complex media containing several growth factors, hormones, and nutrients. Usually, some of these essential components are provided by the addition of fetal calf serum (FCS). However, several disadvantages of FCS and, most importantly, ethical concerns regarding the method of serum collection have encouraged researchers to find alternatives. Human platelet lysate (hPL) has emerged as a promising alternative to FCS for supporting cell expansion in vitro. In the present study, we investigated the effects of different concentrations of hPL on the proliferation of porcine PRECs and their subsequent differentiation under ALI conditions. Cell morphology was assessed by phase-contrast microscopy, while cell proliferation was evaluated using the ClickTech EdU Cell Proliferation Kit and visualization of proliferating cells by fluorescence microscopy. Differentiation under ALI conditions was monitored by immunofluorescence staining of ciliated cells and the establishment of an intact epithelial barrier was confirmed by measuring transepithelial electrical resistance (TEER). We found that 5% hPL supported efficient cell growth and the subsequent formation of a functional, well-differentiated airway epithelium comparable to or even better than 10% FCS. Thus, hPL offers a reproducible, ethically sound, and scalable alternative to FCS for complex cell culture models in respiratory research, drug development, and host-pathogen interaction studies. LO_SCPLOWAYC_SCPLOW SO_SCPLOWUMMARYC_SCPLOWRespiratory epithelial cells from the lungs of slaughtered animals, such as pigs, can be used for cell culture models to study respiratory diseases and drug development. Air-liquid interface (ALI) cultures closely mimic the natural environment of the airways by exposing the cells to air, making them a valuable alternative to animal experiments. To grow and mature properly, these cells require nutrients and growth factors that are commonly supplied by serum from unborn calves (FCS). However, for ethical and scientific reasons, the use of FCS should be avoided. Therefore, we evaluated whether human platelet lysate (hPL) derived from expired blood donations could replace FCS in ALI cultures. We found that adding 5% hPL to the medium supported efficient cell growth and the development of a well-differentiated airway epithelium. This approach enables the use of an improved and ethically superior model of the (porcine) respiratory tract in accordance with the 3Rs principle.