American Journal of Physiology-Lung Cellular and Molecular Physiology

The precise mechanisms by which Covid-19 infection leads to hypoxia and respiratory failure have not yet been elucidated. Interactions between sulfated glycosaminoglycans (GAGs) and the SARS-CoV-2 spike glycoprotein have been identified as participating in viral adherence and infectivity. The spike glycoprotein binds to respiratory epithelium through the angiotensin converting enzyme 2 (ACE2) receptor, which endogenously interacts with Angiotensin (Ang) II to yield Angiotensin 1-7. In this report, we show that stimulation of human vascular smooth muscle cells by Ang II leads to increased mRNA expression of two chondroitin sulfotransferases (CHST11 and CHST15), which are required for synthesis of chondroitin 4-sulfate (C4S) and chondroitin 4,6-disulfate (CSE), respectively. Also, increased total sulfated GAGs, increased sulfotransferase activity, and increased expression of the proteoglycans biglycan, syndecan, perlecan, and versican followed treatment by Ang II. Candesartan, an Angiotensin II receptor blocker (Arb), largely, but incompletely, inhibited these increases, and the differences from baseline remained significant. These results suggest that another effect of Ang II also contributes to the increased expression of chondroitin sulfotransferases, total sulfated GAGs, and proteoglycans. We hypothesize that activation of ACE2 may contribute to these increases and suggest that the SARS-CoV-2 spike glycoprotein interaction with ACE2 may also increase chondroitin sulfotransferases, sulfated GAGs, and proteoglycans and thereby contribute to viral adherence to bronchioalveolar cells and to respiratory compromise in SARS-CoV-2 infection.

BackgroundCachexia is an extrapulmonary manifestation of Chronic Obstructive Pulmonary Disease (COPD) characterized by weight loss and muscle wasting. Transcriptomic profiling of vastus lateralis biopsies enables profiling of COPD-cachexia relevant dysregulation. As obtaining muscle biopsies is invasive and yields limited tissue, human muscle derived cultures (HMDC) may enable mechanistic research into cachexia. However, questions remain regarding the extent to which HMDC recapitulate transcriptomic signatures of bulk skeletal muscle in COPD-cachexia. To address this gap, we tested whether COPD and COPD-cachexia associated transcriptional dysregulation signatures in bulk skeletal muscle are preserved in derived myoblasts, myocytes, and myotubes. MethodsVastus lateralis biopsies were collected from 13 (6M/7F, 64{+/-}9 years) participants; COPD n=5, COPD-cachexia n=4, and 4 age-matched controls. Cachexia was defined using a composite measure of weight loss coupled with reduced muscle strength, fatigue, anorexia, low muscle mass and/or systemic inflammation. Satellite cells were isolated and differentiated into myoblasts, myocytes, and myotubes. Differential gene expression testing, generated from RNA-sequencing, identified transcripts significantly dysregulated (p>0.05) in bulk tissue. Weighted gene co-expression network analysis (WGCNA) was performed to identify modules of co-expressed genes at the whole-transcriptome and mitochondrial transcriptome levels. Bulk tissue modules were tested for preservation in HMDC (Z-summary >2) and correlated with clinical traits. Gene set enrichment analysis was performed for all modules. Results1,379 genes were significantly differentially expressed in bulk samples from all COPD participants compared to controls. The top upregulated gene was IL32 (L2FC=4.5, p=1.3x10-3) and top downregulated CGN (L2FC=-5.8, p=8.8x10-3). A total of 632 genes were significantly differentially expressed in bulk samples from COPD participants with and without cachexia. The top upregulated gene was SEMA4F (L2FC=5.0, p=6.9x10-4) and top downregulated ARC (L2FC=-4.9, p=3.1x10-2). WGCNA generated 9 modules (Modules 1 - 9) at the whole-transcriptome level and 2 modules (Modules A and B) at the mitochondrial transcriptome level. Modules 1, 4, 5, and 9 were significantly correlated with COPD-cachexia. Of these, module 1 was preserved in myoblasts and modules 4, 5 and 9 in myocytes. These modules are enriched with genes involved in metabolic and inflammatory remodeling, catabolic stress and atrophy, and chromatin-driven regeneration. ConclusionsThese results provide a foundation for using myocytes and myoblasts as in vitro models of degeneration and repair pathway dysregulation in COPD-cachexia. Several modules were preserved between bulk skeletal muscle and HMDC, suggesting HMDC have utility for studying COPD-cachexia.

PurposeMucus abnormalities are central to the pathophysiology of several chronic airway diseases. Mucus secretion and clearance are regulated, in part, by cholinergic innervation. Prolonged cholinergic stimulation may contribute to mucus abnormalities in disease. Thus, we tested the hypothesis that prolonged cholinergic stimulation gives rise to lasting mucus abnormalities in airways. MethodsWe delivered aerosolized bethanechol, a cholinergic agonist, to pig airways. Forty-eight hours later, we measured mucus secretion and mucociliary transport in tracheal segments ex vivo. Tracheal and bronchoalveolar lavage concentrations of the major secreted mucus glycoproteins, mucin5B (MUC5B) or mucin5AC (MUC5AC), were measured with ELISA and antibody labeling. Pig airway epithelia were cultured at the air-liquid interface and treated with bethanechol for forty-eight hours. Stimulated fluid secretion was measured with reflected microscopy and Ussing chambers were used to measure ion transport. ResultsAirways from bethanechol-challenged pigs exhibited sheet-like mucus films, which were not associated with a greater abundance of MUC5AC or MUC5B. Epithelia treated with bethanechol had diminished fluid secretion and decreased Cl- transport. However, mucus and fluid alterations were not associated with impaired mucociliary transport. ConclusionsThese data suggest that cholinergic transmission induces sustained alterations in airway mucus properties. Such defects might compound and/or contribute to persistent mucus phenotypes found after the resolution of airway inflammation.

Ventilator-associated pneumonia (VAP) is the most frequent nosocomial infection in critically ill patients. Local pH variations affect bacterial growth. Whether airway acidification contributes to the pathogenesis and pathophysiology of Pseudomonas aeruginosa (PA)-induced VAP is currently unknown. This study was undertaken to investigate the role and mechanisms of airspace acidification by mechanical ventilation (MV) in PA-induced VAP. C57BL/6J mice were subjected to high (HVt: 34 mL/kg) or low (LVt: 9 mL/kg) tidal volume MV for 4 h. PA was instilled via the tracheal tube, and animals were allowed to recover from sedation and breathe spontaneously for 24 h following extubation. Fluorescence microscopy was applied to determine alveolar pH in ex vivo perfused and ventilated murine lungs. Bacterial growth and adhesion on cyclically stretched A549 and human alveolar epithelial cells was examined. Upon PA infection, HVt mice showed increased alveolo-capillary permeability, elevated lung and blood leukocyte counts, and higher bacterial load in lungs and extrapulmonary organs as compared to LVt controls. HVt MV induced acidification of alveolar lining fluid (ALF) in lungs and decreased pulmonary expression of Na+/H+ exchanger 1 (NHE1). Inhibition of NHE1 enhanced PA growth in vitro on alveolar epithelial cells and increased pulmonary bacterial loads in LVt-MV mice in vivo. In a novel murine VAP model, key characteristics of PA-VAP were replicated. HVt MV induced mild VILI with acidification of airway lining fluid, increasing susceptibility to PA pneumonia. NHE1 was identified as critical factor for MV-induced airspace acidification, and thus as potential target to combat PA-VAP.

Cysteinyl leukotriene receptor 1 (CysLTR1), a potent lipid mediator, is known for its critical role in regulating inflammatory responses, particularly in asthma and airway diseases. While its function in immune cell recruitment has been previously reported, its broader impact on pulmonary metabolism remains largely unexplored. In this study, we investigated the metabolic consequences of CysLTR1 deletion in mice using GC-TOFMS-based metabolomics analysis of bronchoalveolar lavage fluid (BALF) from both CysLTR1 knockout (KO) and wild-type (WT) mice. The BALF from CysLTR1 KO mice exhibited significantly reduced levels of glucose, gluconic acid, sedoheptulose, D-xylose, glucosamine, glyceric acid, and 1-methylinosine, indicating impaired glucose uptake and dysregulation of glycolysis and gluconeogenesis. Further disruption of glucose-associated pathways, including the pentose phosphate pathway and purine metabolism, alongside reduced 1-methylinosine levels, suggests altered RNA turnover. In addition, decreases in butanoic acid, decan-2-ol, and 1-hexadecanol point to dysregulated fatty acid metabolism, potentially as a compensatory response to glucose deficiency. Altered levels of mandelic acid, glutaric acid, tricarballylic acid, and decan-2-ol, some of which are derived from corn-based diets also indicate changes in the pulmonary microbiome. Overall, the deletion of CysLTR1 significantly disrupts pulmonary metabolic homeostasis, affecting the metabolism of carbohydrates, lipids, amino acids, nucleotides, and microbial-derived metabolites.

RationalePulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that nuclear factor kappa-B (NF{kappa}B) promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF{kappa}B activation in the pulmonary vasculature are unknown. ObjectivesTo identify mechanisms that activate pro-angiogenic NF{kappa}B signaling in the developing pulmonary vasculature. MethodsProteomic analysis of the lung secretome was performed using 2D-DIGE. NF{kappa}B activation and angiogenic function was assessed in primary pulmonary endothelial cells (PEC) and TGFBI-regulated genes identified using RNA-sequencing. Alveolarization and pulmonary angiogenesis was assessed in WT and TGFBI null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Measurements and Main ResultsSecreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PEC. Proteomic analysis identified transforming growth factor beta-induced protein (TGFBI) as a protein highly expressed by myofibroblasts in the early alveolar lung that promoted PEC migration by activating NF{kappa}B via v{beta}3 integrins. RNA-sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PEC. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. ConclusionsOur studies identify TGFBI as a developmentally-regulated protein that promotes NF{kappa}B-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.

BackgroundAcute respiratory distress syndrome (ARDS) remains a lethal inflammatory lung condition lacking reliable biomarkers that reflect lung-specific pathology. Extracellular vesicles (EVs) circulate systemically and may carry molecular signals from injured organs, but the correspondence between EV cargo and lung tissue alterations remains unclear. MethodsWe established aspiration-, lipopolysaccharide (LPS)-, and COVID-19-induced murine ARDS models and applied a tissue-guided multiomics framework integrating proteomic and metabolomic analyses of lung tissue and plasma-derived EVs to identify lung-originating circulating biomarkers. ResultsFour proteins--haptoglobin (HP), inter-alpha-trypsin inhibitor heavy chains 3 and 4 (ITIH3, ITIH4), and clusterin (CLU)--were consistently upregulated in both lung tissue and plasma EVs across all ARDS etiologies. Metabolomic integration revealed dysregulation of arachidonic acid metabolism as a unifying inflammatory axis. Multiomics network analysis further distinguished etiology-specific molecular programs, including glycolytic activation in aspiration-induced, platelet aggregation in LPS-induced, and vascular smooth muscle dysregulation in COVID-19-induced ARDS. ConclusionsThis study establishes a tissue-informed EV profiling framework that links local lung pathology to systemic molecular signatures, revealing HP, ITIH3, ITIH4, CLU, and arachidonic-acid-related metabolites as potential diagnostic markers for ARDS. These findings provide a foundation for developing clinically translatable, EV-based biomarker assays for early detection and molecular subtyping of lung injury.

RationaleThe mechanistic role of canonical transient receptor potential 6 (TRPC6) channel in chronic obstructive pulmonary disease (COPD) is poorly understood. ObjectivesThe purpose of this study is to determine the role of TRPC6 channel in COPD and its underlying signaling mechanisms in human airway smooth muscle cells (HASMCs). Methods and Main ResultsThe present study examined the effects of TRPC6 channel on nicotine and cigarette induced HASMCs proliferation, migration and mouse airway remodeling models. mRNA and protein expression of TRPC6 were increased in cultured HASMCs incubated with nicotine using real-time PCR and western blot analysis. Nicotine treatment significantly increased TRPC6 transcriptional activity through NF-{kappa}B in HASMCs with Co-IP and electrophoretic mobility shift assays (EMSA). Nicotine treatment also increased ROS level in HASMCs, this increase was attenuated by Nox inhibitor apocynin. miR-135a/b-5p down-regulated mRNA and protein level of TRPC6 in HASMCs, while luciferase reporter assay showed that miR-135a/b-5p targeted at the 3-UTR of TRPC6 mRNA. microRNA-135a/b-5p (miR-135a/b-5p), with a negative correlation to TRPC6 expression, was low in airway smooth muscle of COPD patients. Cigarette-induced airway remodeling mice model also exhibited a large increase in smooth muscle cell proliferation and smooth muscle layer mass with immunohistochemistry assay, this well-characterized airway remodeling was eliminated by lentivirus of TRPC6 knockdown or miR-135a/b-5p overexpression. ConclusionsNicotine exposure results in increased HASMCs proliferation and migration through NF-{kappa}B signaling. Inhalation of cigarette causes airway smooth muscle layer re-modeling due to altered TRPC6 elicited Ca2+ influx, miR-135a/b-5p abolishes this change both in vitro and in vivo.

Chronic obstructive pulmonary disease (COPD) is characterized by continuous and irreversible inflammation frequently caused by persistent exposure to toxic inhalants such as cigarette smoke (CS). CS may trigger mitochondrial DNA (mtDNA) extrusion into the cytosol, extracellular space, or foster its transfer by extracellular vesicles (EVs). The present study aimed to elucidate whether mtDNA is released upon CS exposure and in COPD. We measured cell-free mtDNA (cf-mtDNA) in the plasma of former smokers affected by COPD, in the serum of mice that developed CS-induced emphysema, and in the extracellular milieu of human bronchial epithelial cells exposed to cigarette smoke extract (CSE). Further, we characterized cells exposed to sublethal and lethal doses of CSE by measuring mitochondrial membrane potential and dynamics, superoxide production and oxidative stress, cell cycle progression, and cytokine expression. Patients with COPD and mice that developed emphysema showed increased levels of cf-mtDNA. In cell culture, exposure to a sublethal dose of CSE decreased mitochondrial membrane potential, increased superoxide production and oxidative damage, dysregulated mitochondrial dynamics, and triggered mtDNA release in extracellular vesicles. The release of mtDNA into the extracellular milieu occurred concomitantly with increased expression of DNase III, DNA-sensing receptors (cGAS, NLRP3), proinflammatory cytokines (IL-1{beta}, IL-6, IL-8, IL-18, CXCL2), and markers of senescence (p16, p21). Exposure to a lethal dose of CSE preferentially induced mtDNA and nuclear DNA release in cell debris. Our findings demonstrate that CS-induced stress triggers mtDNA release and is associated with COPD, supporting cf-mtDNA as a novel signaling response to CS exposure.

Acute lung injury (ALI) is associated with cytokine release, pulmonary edema and in the longer term, fibrosis. A severe cytokine storm and pulmonary pathology can cause respiratory failure due to acute respiratory distress syndrome (ARDS), which is one of the major causes of mortality associated with ALI. In this study, we aimed to determine a novel neural component through cardiopulmonary spinal afferents that mediates lung pathology during ALI/ARDS. We ablated cardiopulmonary spinal afferents through either epidural T1-T4 dorsal root ganglia (DRG) application or intra-stellate ganglia delivery of a selective afferent neurotoxin, resiniferatoxin (RTX) in rats 3 days post bleomycin-induced lung injury. Our data showed that both epidural and intra-stellate ganglia injection of RTX significantly reduced plasma extravasation and reduced the level of lung pro-inflammatory cytokines providing proof of principle that cardiopulmonary spinal afferents are involved in lung pathology post ALI. Considering the translational potential of stellate ganglia delivery of RTX, we further examined the effects of stellate RTX on blood gas exchange and lung edema in the ALI rat model. Our data suggest that intra-stellate ganglia injection of RTX improved pO2 and blood acidosis 7 days post ALI. It also reduced wet lung weight in bleomycin treated rats, indicating a reduction in lung edema. Taken together, this study suggests that cardiopulmonary spinal afferents play a critical role in lung inflammation and edema post ALI. This study shows the translational potential for ganglionic administration of RTX in ARDS.

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

RationaleWe showed that levels of a murine mitochondrial noncoding RNA, mito-ncR-LDL805, increase in alveolar epithelial type 2 cells exposed to extracts from cigarette smoke. The transcripts translocate to the nucleus, upregulating nucleus-encoded mitochondrial genes and mitochondrial bioenergetics. This response is lost after chronic exposure to smoke in a mouse model of chronic obstructive pulmonary disease. ObjectivesTo determine if mito-ncR-LDL805 plays a role in human disease, this study aimed to (i) identify the human homologue, (ii) test if the smoke-induced response occurs in human cells, (ii) determine causality between the subcellular localization of the transcript and increased mitochondrial bioenergetics, and (iii) analyze mito-ncR-LDL805 transcript levels in samples from patients with chronic obstructive pulmonary disease. MethodsLevels and subcellular localization of the human homologue identified from an RNA transcript library were assessed in human alveolar epithelial type 2 cells exposed to smoke extract. Lipid nanoparticles were used for nucleus-targeted delivery of mito-ncR-LDL805 transcripts. Analyses included in situ hybridization, quantitative PCR, cell growth, and Seahorse mitochondrial bioenergetics assays. Measurements and Main ResultsThe levels of human homologue transiently increased and the transcripts translocated to the nuclei in human cells exposed to smoke extract. Targeted nuclear delivery of transcripts increased mitochondrial bioenergetics. Alveolar cells from humans with chronic obstructive pulmonary disease had reduced levels of the mito-ncR-LDL805. Conclusionsmito-ncR-LDL805 mediates mitochondrial bioenergetics in murine and human alveolar epithelial type 2 cells in response to cigarette smoke exposure, but this response is likely lost in diseases associated with chronic smoking, such as chronic obstructive pulmonary disease, due to its diminished levels. ImpactThis study describes a novel mechanism by which epithelial cells in the lungs adapt to the mitochondrial stress triggered by exposure to cigarette smoke. We show that a noncoding RNA in mitochondria is upregulated and translocated to the nuclei of alveolar epithelial type 2 cells to trigger expression of genes that restore mitochondrial bioenergetics. Mitochondria function and levels of the noncoding RNA decrease under conditions that lead to chronic obstructive pulmonary disease, suggesting that the mitochondrial noncoding RNA can serve as potential therapeutic target to restore function to halt disease progression.

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, characterized by a progressive decline in lung function, alveolar loss (emphysema), and airflow limitation due to excessive mucus secretion (chronic bronchitis), that can occur even after the injurious agent is removed. It is slated to rise to the 3rd leading cause of death due to chronic disease by 2030 globally, and the 4th leading cause of death due to chronic disease in the USA. While there is substantial evidence indicating loss of E-cadherin in the lung epithelium of patients with COPD, it is not known if this is causal to the disease. We investigated if loss of E-cadherin can result in lung disease using in both in vitro models of primary, differentiated human cells and in mouse models. Using a cell type-specific promoter using Cre/LoxP mice system to knock-out E- cadherin in ciliated and alveolar epithelial cell (Type 1 and Type 2) populations in adult mouse models, we determined that loss of E-cadherin caused airspace enlargement, as well as increased airway hyperresponsiveness indicating that it does have a causative role in causing COPD. Strategies to upregulate CDH1 (encodes for E-cadherin) in CHBEs and cigarette-smoke injured NHBEs can rescue the dysfunctional epithelium.

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

Goblet cell metaplasia and mucus hyper-production are key features of chronic muco-obstructive lung diseases such as asthma, chronic bronchitis, and cystic fibrosis. Various mechanisms lead to goblet cell metaplasia in the airways; the driving mechanism for goblet cell metaplasia in a specific patient may be unknown. We recently found that heat shock protein 90 (HSP90) is important for both IL-13- and IL-17- induced airway goblet cell metaplasia. HSP90 interacts with multiple clients that are important in goblet cell metaplasia including Akt, Jak/STAT, IRS, Notch, and various kinases involved in NF{kappa}B signaling. Here, we used a targeted phospho-proteomic approach to identify candidate HSP90 clients modulated by the HSP90-inhibitor geldanamycin. NF{kappa}B family members were enriched amongst the top candidate targets of HSP90 inhibition in IL-13 an organotypic model of human airway epithelia. We hypothesized that HSP90 inhibition modulated goblet cell metaplasia by interfering with NF{kappa}B signaling. We used transcription factor activation, nuclear translocation, and phospho-specific immunofluorescence assays to investigate how IL-13 exposure and HSP90 inhibition modulated NF{kappa}B. We found that HSP90 inhibition prevented goblet cell metaplasia by non-canonically blocking NF{kappa}B p100/p52 function in human airway epithelia. NF{kappa}B modulation via its interaction with HSP90 is a pharmaceutically feasible therapeutic target for goblet cell metaplasia; this approach may enable treatment of patients with chronic muco-inflammatory lung diseases with both known or unidentified disease-driving mechanisms.

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

RationaleAirflow obstruction refractory to {beta}2 adrenergic receptor ({beta}2AR) agonists is an important clinical feature of infant respiratory syncytial virus (RSV) bronchiolitis, with limited treatment options. This resistance is often linked to poor drug delivery and potential viral infection of airway smooth muscle cells (ASMCs). Whether RSV inflammation causes {beta}2AR desensitization in infant ASMCs is unknown. ObjectivesTo investigate the interaction of RSV inflammation with the {beta}2AR signaling pathway in infant ASMCs MethodsInfant precision-cut lung slices (PCLSs) and mouse pup models of RSV infection were subjected to airway physiological assays. Virus-free, conditioned media from RSV-infected infant bronchial epithelial cells in air-liquid interface (ALI) culture and nasopharyngeal aspirates (NPA) from infants with severe RSV bronchiolitis were collected and applied to infant PCLSs and ASMCs. Cytokines in these samples were profiled and assessed for the effects on {beta}2AR expression, cell surface distribution, and relaxant function in ASMCs. Measurements and Main ResultsConditioned media and NPA induced similar resistance to {beta}2AR agonists in ASMCs as RSV infection. Cytokine profiling identified CXCL11 as one of the most elevated signals following RSV infection. CXCL11 activated its receptor CXCR7 in a complex with {beta}2AR in ASMCs to promote {beta}2AR phosphorylation, internalization, and degradation. Blockade of CXCR7 partially restored airway relaxation in response to {beta}2AR agonists in infant PCLSs and mouse pup models of RSV infection. ConclusionsThe CXCL11-CXCR7 pathway plays a critical role in {beta}2AR desensitization in ASMCs during RSV infection and represents a potential therapeutic target in alleviating airflow obstruction in infant RSV bronchiolitis.

Dampening neutrophil-driven inflammation in the airways remains a challenge in treating cystic fibrosis (CF) lung disease. Myeloperoxidase (MPO) is a neutrophilic enzyme that produces reactive oxygen species and is highly concentrated in CF sputum samples. Greater MPO concentrations have been previously correlated with increased mucus plugging in bronchiectasis, suggesting that the enzyme could impair mucociliary transport. MPO reacts competitively with either thiocyanate (SCN-) or chloride (Cl-) in the airways to catalyze the production of hypothiocyanous acid (HOSCN) or hypochlorous acid (HOCl), respectively. HOCl has proved in prior studies to be extremely cytotoxic, while HOSCN can drastically reduce cytotoxicity. The concentration of SCN- in the airways is largely dependent on transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which is dysfunctional in individuals with CF and causes low SCN- concentrations. CFTR modulator therapies likely raise the concentration of SCN- and enhance the production of HOSCN in the airways. We found that MPO inhibits mucociliary transport in vitro in regardless of SCN- concentrations primarily due to increasing the macromolecular components and effective viscosity of airway surface liquid. The impairment of mucus clearance by MPO was similar to neutrophil elastase (NE), another neutrophilic granular enzyme that damages the host tissues and induces the secretion of mucin proteins by the airway epithelium. Overall, these findings identify MPO as a therapeutic target to resolve deficits in airway clearance function in CF and other related muco-obstructive lung diseases.

The lungs mechanosensitive immune response, which occurs when pulmonary alveoli are overstretched, is a major impediment to ventilation therapy for hypoxemic respiratory failure. The cause is not known. We tested the hypothesis that alveolar stretch causes stretch of alveolar macrophages (AMs), leading to the immune response. In lungs viewed by optical imaging, sessile AMs expressed gap junctional protein connexin-43 (Cx43), and they communicated with the alveolar epithelium through gap junctions. Alveolar hyperinflation increased Ca2+ in the AMs but did not stretch the AMs. The Ca2+ response, and concomitant TNF secretion by AMs were blocked in mice with AM-specific deletion of Cx43. The AM responses, as also lung injury due to mechanical ventilation at high tidal volume, were inhibited by AM-specific delivery of lipid nanoparticles containing Xestospongin C, which blocked the induced Ca2+ increases. We conclude, Cx43- and Ca2+-dependent AM-epithelial interactions determine the lungs mechanosensitive immunity, providing a basis for therapy for ventilator- induced lung injury.

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