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

Emerging evidence identifies platelets as active participants in inflammation beyond their traditional hemostatic function. Mast cells, tissue-resident granulocytes, are key players in innate immunity. Recent studies reveal dynamic bidirectional interaction between these two cell types. An IL33-driven feed-forward circuit has been described, in which mast cell-derived leukotriene C₄ activates platelets through cysteinyl leukotriene receptor 2 (CysLT₂R), whereas platelet-derived nucleotides subsequently enhance mast cell activation through P2Y1-dependent signaling. This reciprocal exchange redefines platelets and mast cells as cooperative amplifiers of type 2 inflammation rather than isolated effectors. The model challenges classical hierarchical views of immune signaling, proposing reciprocity where feedback strength, not stimulus persistence, determines inflammatory stability. Several mechanistic questions emerge, including the physiological magnitude of platelet-derived ATP/ADP flux in vivo, the spatial context of platelet-mast cell interactions within airway microenvironments, and whether other epithelial alarmins such as IL-25 and thymic stromal lymphopoietin (TSLP) engage similar pathways. Conceptually, this bilateral circuitry positions platelets as integral components of cytokine-driven networks that sustain allergic and asthmatic inflammation. Therapeutically, it suggests opportunities to target CysLT₂R and P2Y1 signaling to locally dampen inflammatory amplification without impairing systemic hemostasis. In addition, platelets contribute to vascular leakage, shock, and tissue inflammation following cardiac surgery through perivascular mast cell activation mediated by platelet-activating factors. Moreover, mast cell and platelet-derived 5-hydroxyindoleacetic acid signal through the GPR35 receptor to promote eosinophil recruitment and fungal persistence during Cryptococcus neoformans infection. Collectively, these findings broaden our understanding of platelet function and underscore the importance of intercellular communication in maintaining or disrupting the balance between transient and chronic inflammation.

Proinflammatory 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 the 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 nonspliceable XBP1 mutant (δXBP1). The binding site sequences of XBP1s to the CCNB1 (cyclin B1 gene) promoter were 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.NEW & NOTEWORTHY Airway hyperreactivity and hASM cell proliferation (hyperplasia) are hallmark characteristics of asthma. The results of the present study showed that short-term exposure of nonasthmatic hASM to TNFα (a proinflammatory cytokine) can also induce hASM cell proliferation via activation of an ER stress pathway. Identification of the ER stress signaling pathway as important in mediating hASM hyperplasia may provide an important therapeutic target for treating asthma or acute inflammation.

MicroRNA-21 and microRNA-34a are implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, but their cell-specific expression patterns and interactions within individual airway cells remain unexplored. The objective of the study was to develop a single-cell microfluidic platform for dual, amplification-free detection of miR-21-5p and miR-34a-5p in primary small airway cells from patients with COPD. Small airway epithelial cells (SAEC) and fibroblasts (SAF) were isolated from patients with COPD and nonsmokers (n = 6-8 per group). A microfluidic chip with dual miRNA sandwich hybridization assays was used to quantify miR-21-5p and miR-34a-5p in single cells. Expression of miRNAs and their target genes was evaluated under oxidative stress using qPCR and Western blotting. Single-cell analysis revealed significantly higher miR-21-5p and miR-34a-5p expression in COPD-derived cells compared with controls. miR-21 exhibited greater variability than miR-34a, and their positive correlation in control cells was disrupted in COPD. Oxidative stress elevated miR-21 and miR-34a while reducing expression of miR-21 targets and increasing senescence markers (p21^Cip1/Waf1, p16^INK4a). miR-21 antagomir restored expression of suppressed targets in both cell types. Our novel single-cell microfluidic platform enables precise, simultaneous detection of miR-21 and miR-34a in single small airway cells. This allows the interrelationship between the miRNAs to be assessed within the same cell. miR-21 and miR-34a represent promising therapeutic targets for restoring gene regulatory balance in COPD.NEW & NOTEWORTHY This study demonstrates a novel microfluidic system that can precisely quantify two specific microRNAs in COPD samples at a single-cell resolution using small quantities of cells and reveal the cellular heterogeneity in cell populations. This dual detection system enables the interrelationship between microRNAs in the same cell to be studied as well as having the ability to monitor microRNA levels during disease progression and therapeutics.

Surface complexation of cell iron following particle exposure can be relevant to coal mine dust lung disease. We tested the postulate that 1) coal dust and humic substances (HS), a specific component of coal, complex intracellular iron from cultured cells to initiate a functional metal deficiency, 2) the functional cell iron deficiency which results after exposure to coal dust and HS impacts an increased release of both superoxide-related products and pro-inflammatory mediators, and 3) the disruption in iron homeostasis after coal dust exposure is associated with pneumoconiosis in miners. Cell exposures to coal dust and HS initiated a functional iron deficiency, reflected by elevated expression of an importer (divalent metal transporter-1), which increased metal uptake measured as cell non-heme concentrations. Cell exposure to coal dust and HS increased 1) generation of superoxide, measured using Nitroblue Tetrazolium reduction and an Amplex Red assay, and 2) release of interleukin (IL)-6 and IL-8. These measures of oxidative stress and inflammatory mediator release were diminished with co-exposure to iron supporting a relationship of both with a functional cell deficiency of the metal. Using a cohort of retired miners, blood ferritin levels were elevated in those diagnosed with a positive B read for coal workers' pneumoconiosis. Elevated blood ferritin concentrations correlated with progression of disease on B reads obtained one year later in the miners. It is concluded that coal dust and HS can initiate a disruption of iron homeostasis associated with superoxide generation, release of pro-inflammatory cytokines, and pneumoconiosis.

Interest in respiratory stimulants has increased over the years. Research have intensified after the introduction of opioids that cause respiratory depression. In the most recent years, the indiscriminate consumption of opioids has generated concern and, consequently, there has been a growing number of studies focusing on respiratory stimulants that can mitigate opioid-induced respiratory depression (OIRD) without inducing withdrawal and identifying the molecular mechanisms. Carotid bodies (CBs) are polymodal sensors capable of detecting and responding to a wide variety of chemical stimuli, such as hypoxia, hypercapnia, hypoglycemia, hyperinsulinemia, and hyperleptinemia, among others. CBs have emerged as a potential therapeutic target to alleviate or eliminate OIRD. In this review, we present the most recent data on the mechanisms by which CBs may counteract OIRD. We also discuss whether CBs' stimulation may be a therapeutic target to relieve OIRD without affecting analgesia.

Lung diseases are major global causes of morbidity and mortality, yet the molecular basis of their observed sex differences remains unclear. Beyond their roles in reproductive biology, estrogens are central regulators of pulmonary homeostasis through three principal receptors: 1) estrogen receptor α (ERα), 2) estrogen receptor β (ERβ), and 3) the G-protein-coupled estrogen receptor 1 (GPER1). These receptors are widely expressed across the airway epithelium, smooth muscle, fibroblasts, lung endothelium, and immune cells, where they integrate slow, genomic transcriptional programs and rapid, membrane-initiated signaling cascades to regulate inflammation, oxidative balance, and tissue remodeling. ERβ, often the dominant pulmonary isoform, tends to preserve extracellular matrix integrity and attenuate maladaptive inflammation, whereas ERα frequently amplifies proinflammatory transcriptional programs. GPER1 mediates rapid nongenomic responses that modulate vascular tone, airway smooth-muscle reactivity, and innate immune function, and is both an important regulator of allergic inflammation and a modulator of oncogenic signaling. Together, estrogen receptor subtype balance, subcellular localization, and ligand context determine whether estrogenic signaling is protective or pathogenic. Clinically, this framework helps explain life course and sex differences, such as postpubertal female predominance of asthma, menstrual and pregnancy-related exacerbations, and enhanced chronic obstructive pulmonary disease (COPD) susceptibility in women at lower tobacco exposure. In this review, we synthesize mechanistic and clinical evidence across lung diseases; delineate areas where data remain incomplete or contradictory; and outline opportunities for experimental and translational innovation. These include development of receptor-selective or biased ligands, inhaled or localized delivery, and implementation of sex-aware clinical trial designs to leverage estrogen-receptor biology for precision respiratory therapeutics.

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disorder characterized by neutrophil-dominant, corticosteroid-refractory airway inflammation involving the IL-23/IL-17A axis. IL-23 primarily activates the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway through TYK2 and JAK2, whereas IL-17A and other pro-inflammatory cytokines can activate JAK1. However, the contribution of these JAK-dependent pathways to neutrophil-driven inflammation in COPD remains incompletely understood. In this study, we investigated how the IL-23/IL-17A axis modulates neutrophil function and evaluated the therapeutic potential of the dual TYK2/JAK1 inhibitor brepocitinib in COPD. Gene expression and flow cytometric analyses revealed increased TYK2 and JAK1 expression and phosphorylation in sputum cells and neutrophils from patients with COPD and smokers. IL-23 and IL-17A enhanced neutrophil activation and stimulated IL-8 release from bronchial epithelial cells, effects that were abrogated by brepocitinib. Neutrophils from patients with COPD and smokers also exhibited elevated GRβ expression, a mechanism associated with corticosteroid resistance, which was recapitulated by IL-23/IL-17A stimulation and reversed by brepocitinib. In vivo, brepocitinib suppressed neutrophil recruitment induced by IL-23 or LPS in acute inflammation models. Overall, these findings demonstrate that TYK2/JAK1 inhibition mitigates IL-23/IL-17A-induced neutrophil-driven inflammation and GRβ upregulation in COPD. This highlights the JAK/STAT pathway as a promising therapeutic target to overcome severe airway inflammation and restore GRα/GRβ balance in neutrophils.NEW & NOTEWORTHY This study reveals that the IL-23/IL-17A axis drives neutrophil activation and GRβ upregulation in COPD through TYK2/JAK1-mediated signaling. Inhibition of TYK2/JAK1 with brepocitinib reduced neutrophilic inflammation and restored the GRα/GRβ balance in neutrophils, identifying TYK2/JAK1 as promising therapeutic targets for severe COPD.

Lung surfactant (LS) plays an essential role in preventing lung collapse due to physical forces by forming surface-active lipid-protein membranous films at the respiratory air-liquid interface. Throughout its biological cycle, LS exists in a variety of metabolically related, conspicuous morphological forms. Epithelial alveolar type II cells store LS as intracellular, tightly packed, multilayered organelles known as lamellar bodies. These are secreted as still-condensed material in the form of lamellar body-like particles, which, upon adsorption, give rise to the interfacial film and surface-associated structures. Surfactant material purified from bronchoalveolar lavage fluids has been extensively examined by conventional transmission electron microscopy (TEM), providing important information about LS ultrastructure. However, potential artifacts associated with classical TEM preparation methods-such as staining, dehydration, resin embedding, and sectioning-hinder the observation of surfactant biological samples in their truly native state. In this work, we have taken advantage of cutting-edge cryo-microscopy techniques to visualize the structural complexity present in LS preparations without fixation, in a frozen-hydrated state, and thus closer to physiological conditions. The implementation of cryopreservation approaches has allowed us to unveil unprecedented ultrastructural details of the diverse morphological states in which LS is present in the alveolar spaces, such as the presence of a protein-based pore connecting the lumen of the lamellar body-like particles (LBPs) with the external milieu, and an onion-like structure that suggests a mechanism that uses the energy accumulated upon LB assembly in the pneumocytes for a rapid release of the membranous complexes to the exterior. These morphological features shed light on the dynamic processes by which LS is unpacked from secreted condensed states to the more disorganized, interconnected membranous networks that sustain breathing mechanics.NEW & NOTEWORTHY We have applied some of the most advanced methodologies in cryo-electron microscopy and X-ray tomography to the characterization of native pulmonary surfactant. We still do not understand the way lung surfactant membranes unravel, once secreted, at the respiratory air-liquid interface, and current models are still based on structural observations made when the methodologies available 30 years ago required extensive manipulation/perturbation of membrane materials. Our study reveals new features on the architecture of this system.

Microgravity is known to promote muscle loss and impair physical performance. The carotid body (CB) chemoreceptors are sensitive to several stimuli and have been associated with peripheral vascular control and deterioration in exercise performance. Accordingly, it is plausible that the CB chemoreflex drive may modify the microgravity-induced muscle changes and, consequently, exercise performance. Thus, it is reasonable to propose that a microgravity environment can alter the CB chemoreflex drive, affecting exercise performance. Hence, we aimed to determine the effects of simulated microgravity, through the hind-limb suspension model, on hypoxic ventilatory chemoreflex drive and to examine whether modulation of the CB chemoreflex function influences exercise performance. Adult male Wistar Kyoto rats underwent hind-limb suspension (HLS; n = 6) or the sham condition (n = 4) for 2 wk. A separate group of rats received bilateral injection of two adeno-associated viruses in the CB bifurcation (AVV-TH-Cre-SV40 and AVV-hSyn-DREADD(Gi)-mCherry) (HLS + CB-G_i, n = 4) to partially inhibit the CB chemosensory responses. Clozapine-N-oxide (1 mg/kg/day) was administered via osmotic minipump to activate the inhibitory DREADD-Gi receptor. Before and after exposure to HLS, we measured the hypobaric-hypoxic ventilatory response (HHVR), muscle performance, and V̇o_2peak. HLS promotes a significant increase in HHVR and a decrease in body weight, back leg muscle strength, soleus mass, and V̇o_2peak. Notably, CB inhibition reduced the HLS-induced deterioration in muscle mass and strength, as well as body weight loss. Our findings suggest a novel role for CB chemoreceptors in mediating the decline in muscle strength induced by HLS, reduced muscle mass, and body weight loss.NEW & NOTEWORTHY This study reveals that carotid body (CB) chemoreceptors contribute to muscle dysfunction induced by simulated microgravity. In rats, hind-limb suspension increased hypoxic ventilatory drive and impaired muscle strength, mass, and V̇o_2peak. Inhibiting CB chemosensory activity significantly attenuated these effects. These findings identify a novel physiological role for CBs in mediating microgravity-induced muscle decline, suggesting that the CB chemoreflex may be a potential target for preserving physical performance during spaceflight or similar conditions.

Cystic fibrosis (CF) is caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, leading to abnormal anion transport and consequent airway dehydration and hyper-viscous mucus. Potential difference (PD) testing measures voltage across the epithelium and can be a sensitive marker for changes in ion transport reflective of CFTR activity. By the conventional method, agar gel salt-bridge-based probes in combination with calomel electrodes have been used to measure transepithelial PD across the respiratory mucosa, allowing discrimination between healthy controls and CF. This method is known to be cumbersome and subject to errors due to discontinuity in salt bridges as a result of entrained air that are difficult to detect and a lack of real time visual guidance for probe placement, adversely affecting quality control and data analysis. These limitations are particularly relevant to endobronchial PD, where visualization is less precise, and the chance of electrical discontinuity with extended salt bridges is greater. We developed a novel portable probe system with onboard silver-silver chloride electrodes, integrated gas removal to extract gas bubbles, and optical coherence tomography-mediated visual guidance to provide a platform for improved accuracy and sensitivity of CFTR functional testing that can be adapted for endobronchial PD testing. We also developed a bedside electrocell simulator for the validation of probe performance, ensuring real-time external validation and use of probes that exhibit optimal performance characteristics before human measurements. In a pilot nasal PD study in CF subjects and non-CF controls (n = 10), measurements with the new probe were feasible with discrimination between disease groups. Bland-Altman suggested limited agreement (mean difference: -2.44, SD 4.79; 95% limits of agreement -11.84 to 6.95), but the Deming regression demonstrated a consistent linear relationship despite proportional bias (b = 1.21, P < 0.001) and the Somers' D indicated moderate concordance in rank ordering (0.56; 95% CI: -0.24 to 0.90). These results establish proof of principle of the new device and support the need for further validation in a larger sample.NEW & NOTEWORTHY Cystic fibrosis (CF) impairs CFTR protein function, disrupting ion transport and airway hydration. Traditional potential difference (PD) testing uses salt-bridge probes and calomel electrodes, but is error-prone due to air bubbles and poor visual guidance, especially in endobronchial applications. A novel probe with integrated silver-silver chloride electrodes, gas removal, and OCT guidance improves accuracy and usability. Validation through benchtop and preliminary human nasal testing shows 55% concordance with conventional methods, supporting its clinical potential.