American journal of physiology. Lung cellular and molecular physiology最新文献

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease marked by aberrant fibroblast activation, resulting in excessive proliferation, survival, and accumulation of extracellular matrix (ECM). A critical barrier to developing effective therapies for IPF is the limited understanding of druggable molecular regulators that control fibroblast activation. In this study, we identify the proto-oncogene MYCN as a key driver upregulated in dysregulated fibroblasts from IPF lungs. Mechanistically, we show that transforming growth factor α (TGFα) induces MYCN expression via the profibrotic transcription factor Wilms' tumor 1 (WT1). Notably, the knockdown of MYCN significantly attenuated fibroblast proliferation, survival, and ECM production. We further show that MYCN positively regulates the mitotic kinase, Polo-like kinase (PLK1), and that pharmacological inhibition of PLK1 using volasertib reduced expression of MYCN, WT1, and PLK1 and mitigated fibroblast activation. In vivo, volasertib treatment attenuated fibroblast activation and collagen deposition during TGFα-induced pulmonary fibrosis. Together, these findings identify a pathogenic role for the WT1-MYCN-PLK1 axis in fibroblast activation and provide proof-of-concept evidence supporting PLK1 inhibition with volasertib as a potential therapeutic strategy for IPF.NEW & NOTEWORTHY Excessive proliferation, impaired apoptotic clearance, and extracellular matrix (ECM) production are pathological features of fibroblast activation that together result in scar tissue formation in pulmonary fibrosis. This study identifies the previously unrecognized role for the WT1-MYCN-PLK1 axis in promoting fibroblast activation and highlights the therapeutic potential of PLK1 inhibition with volasertib as an antifibrotic strategy.

Aims and objectives: Lung implantable devices such as stents and valves are used for treatment of lung cancer and COPD. They apply continuous supraphysiological compressive stress to airway tissue, potentially triggering adverse effects such as chronic inflammation, granulation tissue hyperplasia and fibrosis at the implant site. In order to identify the biological responses underlying this process we developed an in vitro contact-compression model that applies variable compressive stress to bronchial epithelial cells. Methods: Confluent layers of bronchial epithelial cells (16HBE) were subjected to compressive stress using agarose-embedded weights (3g, 6g, 9g and 15g). After 24hrs, cell viability, inflammation, fibrosis and mechano-transduction were assessed using cell viability assays, qRT-PCR, ELISA and immunofluorescent staining. Results: Maximum compressive stress (15g) led to reduced cell viability. Compression increased the expression of inflammation, CXCL8, TNF, IL1α, GM-CSF, and remodeling-related genes, EGR1, TNC, COL1A1, CTGF, while no changes in TGFB1, TNC and FN1 expression were observed. These changes were reflected in protein levels with increased CXCL8, IL-1α and CTGF in supernatant upon compression. Compressed cells showed increased actin polymerization, mechanoreceptor re-localization, and YAP nuclear translocation, reflecting a mechanotransducive response. Conclusion: We developed a viable in vitro model to study contact-compression, showing biomechanical inflammatory and remodeling responses. With adjustable components, this model can be applied to further study tissue responses to lung implants.

As bioactive microproteins, Mitochondrial-Derived-MicroProteins (MDPs) are encoded within the small open reading frames (sORFs) of mitochondrial DNA. MDPs have been shown to be altered in a number of disease states and have mitochondrial, nuclear and extracellular actions. Most published work on MDP's has focused on MOTS-c and Humanin's actions in tissues with high mitochondrial density (heart, skeletal muscle, and brain) or in disease states of advanced age - Alzheimer's, Cancer, Cardiovascular disease. This review aims to highlight the existing gaps in knowledge related to MDPs' role in lung homeostasis and disease - including Acute Lung Injury (ALI), Chronic Obstructive Pulmonary Disease (COPD), allergic asthma (AA) and Pulmonary Fibrosis (PF). The increasingly recognized role of MDPs in non-pulmonary diseases sheds light on the importance of more investigations of MDPs, their clinical and mechanistic roles, and their therapeutic potential for pulmonary diseases.

Background: Early airflow changes associated with tobacco smoking often occur without observable obstruction or symptoms. Spirometry, the gold standard, has limitations in detecting early disease highlighting the need for sensitive diagnostic methods. We aimed to evaluate the utility of the forced oscillation technique (FOT) and biomarkers in detecting early airway abnormalities in smokers and patients with COPD, and to explore the correlation between FOT parameters, spirometry measures, and biomarkers of airway inflammation. Methods: A cross-sectional study was conducted on 71 participants divided into three groups: patients with COPD (CP, n=27), normal lung function smokers (NS, n=22), and healthy controls (HC, n=22). Lung function was assessed using spirometry and FOT, while biomarkers of inflammation (MMP-9, TIMP-1, TIMP-2) were measured. Statistical analyses included group comparisons and correlation between lung function parameters and biomarker levels. Results: Patients with COPD had significantly lower spirometry and higher FOT values compared to NS and HC (p<0.01). In contrast, NS participants had similar spirometry values to HC, except for FEF25-75% and PEF. The NS group exhibited significantly higher values for R5 compared to HC (p<0.05). FOT parameters, particularly R5, demonstrated comparable diagnostic accuracy to spirometry in smokers, and all other parameters showed excellent discriminatory ability in COPD patients. MMP-9 correlated positively with percentage predicted FOT parameters, R5-R20 and AX, and X5 (r'=0.29,0.30, 0.31; p=0.02,0.04,0.02 respectively) in the combined group of smokers and COPD patients and positively with percentage predicted Fres (r'=0.30; p=0.01) when all groups were analysed together. Conclusion: FOT may be a sensitive and complementary measure to detect early airway changes in smokers and patients with COPD. MMP-9 correlating with FOT further supports the role of FOT combined with biomarkers in detecting early airway abnormalities in smokers and earlier stages of COPD.

The airway epithelium, a primary target for viral infection, plays a critical role in disease response-particularly in individuals with pre-existing airway conditions such as cystic fibrosis (CF). At the onset of the SARS-CoV-2 pandemic, CF individuals were expected to have severe outcomes based on prior viral outbreaks; however, those on effective CFTR modulators showed milder disease. CF patients on the CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ETI) combination therapy showed attenuated viral infection and reduced airway epithelial damage. To investigate how this is accomplished, we used an iPSC-derived airway epithelium model of CF and syngeneic CFTR-corrected cells to examine responses to SARS-CoV-2 infection. CF iPSC-airways were significantly more susceptible to viral infection and epithelial injury compared to their corrected counterparts, despite comparable expression of viral entry factors. Strikingly, pretreatment with ETI conferred significant protection in CFTR-corrected and non-CF, wildtype (WT) airway epithelia, as well as in iPSC-derived and primary epithelia. Single-cell RNA sequencing analysis confirmed a heightened infection and pro-inflammatory response in CF iPSC-airways, while ETI treatment significantly reduced these responses in both CF and CFTR-corrected iPSC-airways. Mechanistically, ETI treatment led to increased type I interferon signaling and induction of antiviral genes, while expression of many other pro-inflammatory genes were suppressed in both CF and non-CF iPSC-airways. These results underscore the therapeutic promise of CFTR-modulators like ETI in mitigating SARS-CoV-2 infection and inflammation, not only in CF airways but also in non-CF airways, highlighting the broad applicability of CFTR-modulators as a therapeutic strategy in viral pneumonia and inflammatory lung disease.

Pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) induce airway smooth muscle (ASM) cell proliferation. Previously we showed that TNFα induces an endoplasmic reticulum (ER) stress response involving autophosphorylation of inositol requiring enzyme 1α at serine 724 (pIRE1α^S724) and alternative splicing of X-box binding protein 1 (XBP1s). XBP1s transcriptionally activates expression of Cyclin dependent kinases 1 and 5 (CDK1 and CDK5). In the present study, we hypothesized that TNFα induced activation of the pIRE1α^S724/XBP1s ER stress pathway mediates transcriptional activation of Cyclin B1 and ASM cell proliferation. Human ASM (hASM) cells were dissociated from bronchiolar tissue samples obtained from female and male patients with no history of respiratory disease. Isolated hASM cells were either treated or untreated with TNFα (20 ng/ mL) for 6 h. For loss of function experiments, hASM cells were either treated with 4μ8C, a pharmacological inhibitor of IRE1α endoribonuclease activity or transfected with a non-spliceable XBP1 mutant (δXBP1). The binding site sequences of XBP1s to the CCNB1 (Cyclin B1 gene) promoter was identified by bioinformatic analysis and confirmed by chromatin immunoprecipitation (ChIP) assay. hASM cell proliferation was measured using a CyQuant cell proliferation assay. TNFα induced pIRE1α^S724 phosphorylation and XBP1s splicing in hASM cells. XBP1 transcriptionally activates expression of Cyclin B1 mRNA and protein. Nuclear localization of Cyclin B1 increased significantly in TNFα treated hASM cells consistent with the formation of Cyclin B1/CDKs complexes, and increased cell proliferation. Inhibition of pIRE1α^S724/XBP1s pathway mitigated TNFα induced Cyclin B1 and CDKs expression and hASM cell proliferation.

Alveolar macrophages (AMs), a highly plastic immune cell population, are among the first responders to inhaled ozone (O₃) and its ozonated products in the lung airspaces. However, the comprehensive understanding of how AMs respond to O₃, particularly across different concentrations, remains incomplete. To address this knowledge gap, we exposed adult male C57BL/6J mice to filtered air (FA), 1 ppm O₃, or 1.5 ppm O₃ for 3 hours. Compared with FA-exposed mice, O₃-exposed mice exhibited increased recruitment of alveolar macrophages and neutrophils into the lung airspaces, consistent with elevated levels of the macrophage- and neutrophil-specific chemokines, i.e., MCP-3, MCP-5, and MIP-2. To delineate the transcriptomic landscape of AMs following O³-exposure and determine how these alterations relate to AM heterogeneity and function states, we subjected AMs to single-cell RNA sequencing (scRNA-seq) analyses. Differentially expressed genes (DEGs) analysis of the AM population revealed significant transcriptional changes in both the 1 ppm and 1.5 ppm O₃-exposed groups. Compared with AMs from FA-exposed group, AMs from both O₃-exposed groups exhibited enrichment of pathways such as oxidative phosphorylation, EIF2 signaling, and non-canonical NF-kB signaling. Furthermore, AMs from 1 ppm O₃-exposed mice exhibited enrichment of IL-10 signaling pathway, whereas AMs from 1.5 ppm O₃-exposed mice were uniquely enriched for DNA damage bypass and repair pathways. Interestingly, uniform manifold approximation and projection (UMAP) analysis of annotated AMs identified five distinct subclusters. DEGs and ingenuity pathways (IP) analyses of these subcluster revealed O₃ concentration-dependent enrichment of pathways associated with protein translation, cholesterol biosynthesis and mitochondrial biogenesis. Further analyses revealed that O₃ exposure induces cluster-specific alterations in the expression of gene signatures associated with macrophage activation. Additionally, AMs from 1.5 ppm O₃-exposed mice displayed increased expression of proliferation-associated gene signatures. Taken together, these findings identify O₃ concentration-dependent transcriptomic alterations in AMs and associated functional modulations at single-cell resolution.

Idiopathic pulmonary fibrosis (IPF) is a serious respiratory disease with a poor prognosis and limited treatments. IPF is characterized by accumulation of extracellular matrix, destruction of gas exchange units, and decline in lung function. The pathogenesis and underlying mechanisms are poorly understood although a combination of environmental and genetic factors is implicated. Epigenetic modifications represent a facet of gene regulation likely important to key pathways including transforming growth factor (TGF-β1) signaling, fibroblast-to-myofibroblast differentiation, epithelial-to-mesenchymal transition, cellular aging, and apoptosis. Methylation, acetylation, noncoding RNAs, telomere length, and G-quadruplexes have all been investigated with varying levels of progress but are certainly potential targets for drug development. This review discusses the underlying epigenetic biology of IPF, associated clinical applications, and potential novel treatments.

The codevelopment and interactions between structural and immune cells in the human fetal lung can be studied by using transcriptomic and proteomic approaches. Here, we used imaging mass cytometry with a 31-antibody panel to visualize immune and structural cell development in human fetal lung tissue from elective abortions across the pseudoglandular and canalicular stages, spanning postconception weeks (pcw) 6 (n = 1), 8 (n = 1), 10 (n = 1), 13 (n = 2), and 18 (n = 3). This approach allows us to map the developing structural components of the human fetal lung. During the early pseudoglandular stage, keratin-8 (KRT8⁺) epithelial structures appeared, gradually developing into KRT8⁺EpCAM⁺ budding tips and elongated luminal structures. By the late pseudoglandular and canalicular stages, these luminal structures were lined by KRT8⁺D2-40⁺ cells, with D2-40 (Podoplanin) known to be expressed in alveolar epithelial and lymphatic endothelial cells. Surrounding these structures were layers of α-smooth muscle actin⁺ cells. The immune compartment was predominantly myeloid in origin. CD206⁺CD68⁺ macrophages were present as early as the pseudoglandular stage, whereas HLA-DR⁺ myeloid cells appeared later around 13 pcw. Cellular interaction analysis revealed an accumulation of HLA-DR⁺ cells near the KRT8⁺EpCAM⁺ structural regions, suggestive of interactions between these cells during lung development. Our findings illustrate the dynamic development of structural and immune cell components in the human fetal lung throughout the pseudoglandular and canalicular stages. Furthermore, the observed interactions between structural and HLA-DR⁺ immune cells support the notion that immune-structural cross talk plays a role in human fetal lung development.NEW & NOTEWORTHY This study provides a detailed protocol to visualize the codevelopment of structural and immune cells in the human fetal lung through imaging mass cytometry (IMC). Using IMC, spatial relationships between lung epithelial cells and myeloid cells can be visualized, which may contribute to unraveling cellular interactions important for lung maturation.

Newborn infants, especially those born preterm, often require supplemental oxygen (O₂) therapy; however, exposure to supraphysiological oxygen (hyperoxia) can disrupt normal lung development and contribute to neonatal lung injury, including bronchopulmonary dysplasia (BPD). Mitochondrial dysfunction is increasingly recognized as a contributor to oxidative lung diseases, including BPD. However, the effects of hyperoxia on mitochondrial function and mucociliary differentiation in the developing airway epithelium remain poorly understood. This study tested the hypothesis that hyperoxia impairs neonatal airway mucociliary differentiation by disrupting mitochondrial bioenergetic function. Neonatal tracheal airway epithelial cells from term infants (n = 5) were cultured in a three-dimensional air-liquid interface (ALI) model and exposed to 60% O₂ during the mid-phase of differentiation (ALI days 7-14). Cellular phenotype was assessed using immunofluorescence staining and gene expression analyses. Mitochondrial function was evaluated through Seahorse metabolic flux analysis, and global protein changes were characterized by quantitative proteomics. Hyperoxia significantly impaired terminal differentiation with reduced ciliated and goblet cells. Seahorse assay revealed a decrease in baseline oxygen consumption and mitochondrial ATP production, accompanied by a compensatory increase in glycolytic ATP production. Quantitative proteomics identified disruption of mitochondrial complex I as a central feature of the hyperoxic response. Downstream proteomic pathway analyses further confirmed the metabolic shift from mitochondrial to glycolytic ATP production and demonstrated altered epithelial differentiation pathways, including NOTCH and TGF-β signaling. These findings reveal that hyperoxia impairs mitochondrial bioenergetics and alters metabolic programming, leading to disrupted mucociliary differentiation. Future studies should evaluate mitochondrial oxidative fitness as a therapeutic target in neonatal lung disease.NEW & NOTEWORTHY We report that moderate hyperoxia during a critical window of mucociliary differentiation disrupts terminal maturation in neonatal airway epithelial cells cultured in a three-dimensional model. Hyperoxia induces mitochondrial bioenergetic dysfunction and metabolic reprogramming, with proteomic analysis identifying complex I disruption as a key driver of impaired differentiation. Overall, these findings reveal a previously underrecognized link between mitochondrial bioenergetics and airway epithelial development, positioning metabolic dysfunction as an early trigger of hyperoxia-induced neonatal airway injury.