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

Pulmonary fibrosis (PF) is a major cause of morbidity and mortality. Although increased oxidative stress and altered metabolism are implicated in PF pathobiology, our knowledge regarding the contribution of the glucose metabolism to the synthesis of extracellular matrix (ECM) is still incomplete. Therefore, our objective was to determine altered metabolic pathways that contribute to bleomycin (BLM; 5 mg/kg) sulfate-induced PF in rats. We determined the effects of nebulized BLM on PF in CRISPR-edited rats expressing glucose-6-phosphate dehydrogenase (G6PD) variant (S188F; G6PD^S188F) and their wild-type (WT) littermates. Unexpectedly, application of BLM increased lung tissue volume in G6PD^S188F rats as compared with WT littermates. Masson's Trichrome staining and Ashcroft scoring revealed increased collagen in perivascular regions and around the airways and hydroxyproline within the lungs of G6PD^S188F + BLM as compared with WT + BLM rats. In addition, mass spectrometry-based proteomics and spatial proteomics confirmed increased expression of profibrotic proteins, including collagen1a1 and baculoviral IAP repeat containing 5, in the lungs of G6PD^S188F + BLM rats compared with WT + BLM rats. Since BLM increased expression of KEAP1, we suggest that BLM inactivated NRF2 and increased oxidized glutathione, an indicator of oxidative stress that increases ECM, in lungs of G6PD^S188F rats. Finally, unbiased metabolomics revealed downregulated spermidine, a polyamine pathway metabolite that decreases BLM-induced collagen deposition, in the lungs of G6PD^S188F + BLM rats. Therefore, we propose that dysregulated polyamine pathway and antioxidant state exacerbated BLM-induced synthesis of ECM-related proteins in G6PD^S188F variant rats as compared with their WT littermates.NEW & NOTEWORTHY This study reports that a loss-of-function G6PD variant exacerbates BLM-induced lung fibrosis in rats by suppressing polyamine pathway and increasing oxidative stress that oxidized the key ECM-related proteins and antioxidants.

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.

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.

Chronic obstructive pulmonary disease (COPD) is characterized by a deficiency within the lungs of regulatory T cells and an excess of Th17 cells, termed T17/Treg imbalance. Conventional dendritic cells type-1 (cDC1) and type-2 (cDC2) are known to drive Treg and Th17 polarization, respectively, but their roles in COPD are incompletely understood. Using a murine cigarette smoke (CS)-exposure model, we found that after 8 wk of CS exposure, the percentage of lung cDC1, but not cDC2, was significantly decreased (P < 0.05) relative to air-exposed controls. Following intranasal adoptive transfer into naïve recipients, cDC1s were less likely to be retained in the lungs and instead were enriched in mediastinal lymph nodes. Coculture with DCs from cDC1-deficient BATF3^-/- mice induced greater expression of intracellular IL-17 protein by naïve lung CD4⁺ T cells compared with DCs from BATF3^+/+ mice (P < 0.001), which instead more greatly induced intracellular IFN-gamma. LPS-stimulated lung DCs from CS-exposed wild-type mice produced significantly higher amounts of the Th17-polarizing cytokines IL-6 and IL-23. Congruently, whole lung homogenates of CS-exposed mice had increased IL-17 protein expression compared with air-exposed mice (P < 0.05). Naïve CD4⁺ T cells cocultured with lung DCs from CS-exposed mice produced more IL-17 (P < 0.01) than coculture with lung DC from air-exposed mice. Thus, loss of cDC1 and predominance of cDC2 in the lungs of CS-exposed mice drive naïve CD4⁺ T cells toward IL-17 production and could play a role in the Th17/Treg imbalance seen in COPD.NEW & NOTEWORTHY Dendritic cell subsets play unique roles in chronic obstructive pulmonary disease (COPD) pathogenesis. We demonstrated that conventional DC type 1 (cDC1) are decreased, leading to a predominant cDC2 population in the lungs of a clinically relevant murine model of cigarette smoke exposure. This drives naive CD4⁺ T cells toward IL-17 production and could play a role in the Th17/Treg imbalance seen in COPD.

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.

Survivors of coronavirus disease 2019 (COVID-19) can experience long-term lung complications (pulmonary sequelae), but the underlying mechanisms remain unclear. Although most patients with COVID-19 lung injury eventually recover essentially completely, some experience significant residual damage. To investigate the underlying differences, we analyzed, using bronchoalveolar lavage fluid (BALF), the alveolar immune cell compartments of a group of patients with post-COVID-19 interstitial lung disease (ILD) 6 mo after acute COVID-19. Patients were categorized into two groups, based on high-resolution computed tomography (HRCT) evaluation a year later: those with persistent HRCT abnormalities compatible with fibrosis (persistent post-COVID-19 ILD, n = 6) and those with resolved lung lesions (resolved post-COVID-19 ILD, n = 13). In addition, six patients with preexisting ILD were included in the study, after recovery from COVID-19. Bulk RNA transcriptomics analyses of BALF cells revealed innate immunity and inflammation pathways of neutrophil and monocyte chemotaxis to be enriched in patients with persistent HRCT abnormalities post-COVID-19, consistent with an increase in monocyte-like cell recruitment in the lungs. Profibrotic secreted phosphoprotein 1 (SPP1) gene expression was significantly upregulated similar to other fibrotic lung diseases. Conversely, patients with resolved post-COVID-19 ILD showed enhanced BALF cell gene expression signatures, indicative of adaptive immune response activation. BALF gene expression patterns of low T-cell activation, high profibrotic macrophage activation, and neutrophil chemotaxis were similarly observed in patients with preexisting fibrotic ILD following COVID-19. These findings suggest that immune response imbalance leading to prolonged activation of innate immunity and subdued adaptive immune responses may be associated with persistent post-COVID-19 ILD and the development of pulmonary fibrosis.NEW & NOTEWORTHY Survivors of COVID-19 can experience long-term lung complications. We compared bronchoalveolar lavage fluid (BALF) cells from patients who recovered from COVID-19 lung injury and those who experienced significant long term residual damage. A prominent gene expression profile of increased monocyte chemotaxis coupled to decreased T cell activation was observed in persistent fibrotic post-COVID-19 ILD. These findings suggest that prolonged activation of innate immunity and subdued adaptive immune responses may be associated with persistent post-COVID-19 ILD and pulmonary fibrosis.

Hyperoxia (HO) and mechanical ventilation (MV) are the mainstay of treatment for patients with acute respiratory failure, but both interventions can also accelerate further lung injury, highlighting the need for better therapeutic approaches. We previously found that HO decreases epithelial TREK-1 expression and promotes epithelial inflammation, but the consequences of TREK-1 deficiency in a clinically relevant system of combined HO + ST (stretch) exposure remain unknown. We found that in both mouse lung tissue and primary human alveolar epithelial cells, HO + ST downregulates TREK-1 protein levels. The injurious consequences of TREK-1 downregulation are evidenced in alveolar epithelial cells following pharmacological and genetic TREK-1 inhibition and in lungs of TREK-1 KO mice by potentiation of HO + ST-induced cytosolic ROS production, caspase-8 and caspase-1 activation, IL-1β production, and MIP-1α, and CXCL-10/IP-10 secretion. In addition, HO + MV-exposed TREK-1 KO mice show increased histological lung injury scores, total cell, macrophage, and neutrophil counts in the bronchoalveolar lavage fluid (BALF). Mechanistically, HO + ST depolarized the epithelial electrical membrane potential (Em) and raised iCa²⁺ levels, which was potentiated after pharmacological and genetic TREK-1 inhibition. Both Ca²⁺ influx through voltage-gated Ca²⁺ channels and Ca²⁺ release from intracellular stores increased iCa²⁺ levels following TREK-1 inhibition. Intratracheal administration of two structurally different pharmacological TREK-1 activators (ML335, BL1249) improved HO + ST-induced BALF total and differential cell counts, total protein levels, ROS production, caspase-8 and capase-1 production, and cytokine concentrations. Therefore, these findings highlight TREK-1 as new potential target for intervention against HO + ST/MV-induced lung and epithelial injury and lay the groundwork for future rational drug development.NEW & NOTEWORTHY No targeted interventions exist that improve the outcomes of patients with acute lung injury/ARDS. A few studies investigated Na⁺ and Ca²⁺ channels/transporters for potential therapeutic intervention but with limited translational success. This study highlights the regulatory role of TREK-1 K⁺ channels during HO+stretch/mechanical ventilation-induced lung injury in ROS production, caspase activation, cytokine secretion, and explores the underlying TREK-1-mediated signaling mechanisms. These preclinical findings lay the groundwork for future rational drug design targeting TREK-1 channels.

Particulate matter (PM) < 2.5 µm (PM_2.5) contributes to many chronic respiratory disorders, but the mechanisms for this are not fully understood. The actions of PM_2.5 on lung epithelial cells have been well studied, but their effect on lung fibroblasts has not been as extensively reported. Bone morphogenetic protein 2 (BMP2), part of the transforming growth factor cytokine family, plays crucial roles in the development, morphogenesis, repair, and functions as a critical mediator in the pathogenesis of lung diseases such as pulmonary fibrosis and chronic obstructive lung disease. Here, we investigate the impact of PM_2.5 on fibroblast BMP2 production and the role of BMP2 in mediating fibroblast-to-myofibroblast differentiation and matrix generation. Treatment of fibroblasts to PM_2.5 resulted in a dose-dependent rise in BMP2 mRNA and protein secretion, which was specific to BMP2 and not observed with other BMP family members. In normal quiescent fibroblasts, BMP2 induced an increase in collagen and α-smooth muscle actin expression. Interestingly, BMP2 exerted an opposite effect in TGF-β1-differentiated myofibroblasts, whereby BMP2 downregulated collagen levels. These differential responses aligned with variations in p38 and ERK1/2 phosphorylation. Fibroblasts treated with high concentrations of PM_2.5 demonstrated reduced collagen and α-smooth muscle actin expression, an effect reversed by BMP2 silencing or gremlin, a BMP2 antagonist. Overall, PM_2.5 was observed to induce BMP2 production in fibroblasts, and this was associated with suppression of fibroblast activation and matrix production by PM_2.5. These findings highlight a potential mechanism whereby PM_2.5 contributes to lung disease through impairment of fibroblast regenerative and repair capabilities.NEW & NOTEWORTHY Particulate matter <2.5 µm (PM_2.5) from air pollution contributes to many different lung diseases, but the mechanisms are not fully understood. Here, we demonstrated that PM_2.5 caused an upregulation of bone morphogenetic protein 2 (BMP2) in lung fibroblasts. BMP2 can promote myofibroblast differentiation or inhibit collagen expression, depending on the context, and can be a means by which PM_2.5 contributes to fibrotic and nonfibrotic lung diseases.

Defining preclinical models is of utmost importance for pleural mesothelioma (PM) to improve prognosis and predict therapeutic response. Using cells isolated from pleural fluid (PF) and diagnostic pleural biopsy (PB), we generated PM patient-derived organoids (PM-PDOs) and reactive-mesothelial (RM) patient-derived organoids (RM-PDOs) aiming at assessing the proportion of successful cultures both from PF and PB. We also compared the architectural and immune-histochemical features of PM-PDOs with those of parental tissues and evaluated the PM-PDOs response to chemoimmunotherapy. We obtained 11 successful PM-PDOs from 15 PF/PB (73.3%). The rate of success was higher in epithelioid PM (88.8%) compared with biphasic PM (40.0%) (P = 0.175), and when using PF (60.0%) compared with PB (20.0%) (P = 0.001). We also obtained 3 RM effective cultures from 6 asbestos-exposed patients (50%) with nonspecific pleuritis. Transcriptome analysis identified gene expression profile in PM-PDOs, which differentiate from RM-PDOs. PM-PDOs successfully maintained the histological architecture and molecular markers of their parental tumor tissues. The macrophagic component (CD68⁺ and CD163⁺) was an important component in RM-PDOs and was present in all three PM histotypes. Epithelioid PM-PDOs showed resistance to both Cis/PeMtx and pembrolizumab plus peripheral blood mononuclear cells (PBMCs), whereas both biphasic and sarcomatoid subtypes were sensitive to immunotherapy. Notably, immunotherapy induced an upregulation of PD-L1 expression and activated the STAT3/NF-κB signaling pathway, suggesting a mechanism of immune evasion. PF offers a valuable source of cancer and stromal cells to generate PDO, reinforcing its clinical utility for patients who cannot undergo invasive procedures.NEW & NOTEWORTHY Using cells isolated from pleural effusion and pleural biopsy, we established an efficient 3-D culture system for generating PM and reactive mesothelial (RM) patient-derived organoids. PM-PDOs expressed a specific gene profile, preserved the histological architecture, showing markers of the parental tumor tissues and recapitulated the tumor microenvironment (e.g., macrophages and tumor lymphocytes), which is an important factor influencing responses to therapy. This approach will be useful for drug screening, contributing to a more accurate selection of therapeutic options.