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

Ozone (O₃) is a ubiquitous pollutant known to produce acute, transient inflammation through oxidative injury and inflammation. These effects are exacerbated in susceptible populations, such as the elderly and those exhibiting genetic mutations in central nodes of pulmonary function. To comprehend the impact of these predisposing factors, the present study examines structural, mechanical, and immunological responses to single acute O₃ exposure (0.8 ppm, 3 h) in young (8-14-wk old), middle-aged (44-52-wk old), and old (>80-wk old) mice. Furthermore, this work compares the impact of a clinically relevant mutation in the gene encoding for the alveolar epithelial type 2 specific surfactant protein C. Aging was associated with reduced lung resistance and increases in respiratory elastic properties, the latter of which was exacerbated in SP-C mutant mice. Ozone exposure produced focal injury localized at the terminal bronchiole-to-alveolar junctions and enlarged alveoli in aged SP-C mutant lungs. Flow cytometric analysis revealed increases in mononuclear myeloid abundance in aged SP-C mutant lungs, paired with a contraction in CD8⁺ expressing cells. Expansion of tertiary lymphoid tissues was also noted in aged groups, more evident in the mutant mice. Spatial transcriptomics of CD68⁺ macrophages and CD45^- nonimmune parenchymal cells highlighted age-dependent shifts in inflammatory and extracellular matrix organization signaling, and enrichment in senescence and chromatin remodeling pathways. These results illustrate the structural and immunological impact of O₃ in the aging wild-type and mutant lung and emphasize the significance of modeling environmental exposure in at-risk populations.NEW & NOTEWORTHY Environmental stress and genetic mutations in key functional nodes are linked to the pathogenesis and exacerbation of respiratory pathologies. These responses are exacerbated by aging, though the impact of these factors in combination is not clearly defined. Using a surfactant protein-C mutant line, our studies describe structural changes and phenotypic responses triggered by acute ozone exposure in the young/middle-aged/old lung. Spatial transcriptomics also found regionally distinct and enhanced activation in the aged lung.

Lung progenitor (LP) cells identified by the expression of transcription factor NK2 homeobox 1 (NKX2.1) are essential for the development of all lung epithelial cell types and hold tremendous potential for pulmonary research and translational regenerative medicine applications. Here, we present engineered hydrogels as a promising alternative to the naturally derived materials that are often used to differentiate human-induced pluripotent stem cells (iPSCs) into LP cells. Poly(ethylene glycol) norbornene (PEGNB) hydrogels with defined composition were used to systematically investigate the role of microenvironmental stiffness, cell origin, and splitting during the differentiation process. Results demonstrated that each factor impacted LP differentiation efficiency and that the soft hydrogels replicating healthy lung stiffness [elastic modulus (E) = 4.00 ± 0.25 kPa] produced the highest proportion of LP cells based on flow cytometric analysis results (54%) relative to the stiff hydrogels (48%) and Matrigel controls (32%) at the end of the nonsplit differentiation protocol. Collectively, these results showed that engineered hydrogels provide a well-defined microenvironment for iPSC-to-LP differentiation and perform as effectively as the current gold standard Matrigel-coated tissue culture plastic. Adopting engineered biomaterials in cell culture protocols may enable greater control over differentiation parameters and has the potential to enhance the clinical translation of iPSC-derived LP cells.NEW & NOTEWORTHY Standard iPSC differentiation protocols rely on Matrigel, a basement membrane extract from mouse sarcoma cells that is poorly defined and exhibits significant batch-to-batch variation. Due to these limitations, Matrigel-derived products have never been approved by the Food and Drug Administration. This study introduces a novel method for differentiating iPSCs into lung progenitor cells using well-defined hydrogel substrates. These biomaterials not only enhance differentiation efficiency but also streamline the regulatory pathway, facilitating their potential therapeutic application.

Secondhand smoke exposure (SHSe) is a public health threat for people with cystic fibrosis (CF) and other lung diseases. Primary smoking reduces CF transmembrane conductance regulator (CFTR) channel function, the causative defect in CF. We reported that SHSe worsens respiratory and nutritional outcomes in CF by disrupting immune responses and metabolic signaling. Recently, electronic cigarette (e-cigs) usage by caregivers and peers has increased rapidly, causing new secondhand e-cig vape exposures. Primary vaping is associated with immunologic deficits in healthy people, but it is unknown whether e-cigs similarly impacts CF immune function or how it differs from SHSe. Human CF and non-CF blood monocyte-derived macrophages (MDMs) and bronchial epithelial cells (HBECs) were exposed to flavored and unflavored e-cigs. The effect of e-cigs on CFTR expression and function, bacterial killing, cytokine signaling, lipid mediators, and metabolism was measured during treatment with CFTR modulators. E-cigs decreased CFTR expression and function in CF and non-CF MDMs and negated CFTR functional restoration by elexacaftor/tezacaftor/ivacaftor (ETI). E-cigs also negated the restoration of anti-inflammatory PGD₂ expression in CF MDMs treated with ETI compared with controls. Flavored but not unflavored e-cigs increased proinflammatory cytokine expression in CF MDMs and e-cigs promoted glycolytic metabolism. E-cigs did not impact bacterial killing. Overall, HBECs were less impacted by e-cigs compared with MDMs. E-cigs reduced macrophage CFTR expression and hindered functional CFTR restoration by CFTR modulators, promoting a glycolytic, proinflammatory state. E-cigs are an emerging public health threat that may limit the efficacy of CFTR modulators in people with CF.NEW & NOTEWORTHY New research reveals that e-cigarettes pose a serious health risk for individuals with cystic fibrosis (CF). Exposure to electronic cigarette (e-cig) vapors decreases CF transmembrane conductance regulator (CFTR) function and undermines the effectiveness of CFTR modulators, potentially worsening inflammation and metabolic responses. This highlights an urgent need for awareness around e-cig use, especially among caregivers and peers of those with CF. E-cigarettes may further complicate the management of this chronic lung disease.

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea, heightened chemosensory discharges of the carotid body (CB), which contributes to potentiate the ventilatory hypoxic response and elicits hypertension. We aimed to determine 1) whether the persistence of cardiorespiratory alterations found in long-term CIH depends on the inputs from the CB and 2) in what extension the activation of glial cells and neuroinflammation in the caudal region of the nucleus of the solitary tract (NTS) require functional CB chemosensory activity. To evaluate these hypotheses, we exposed male mice to CIH for 60 days. At 50 days of CIH, CBs were denervated and animals were kept in CIH for 10 additional days. At the end of the experiments, we measured arterial blood pressure, breathing regularity, and hypoxic ventilatory response (HVR) and assessed astrocyte and microglia cell activation. Compared to sham treatment, CIH induced hypertension [mean arterial blood pressure (MABP): 83.47 ± 1.39 vs. 95.00 ± 2.18 mmHg] and disordered breathing [irregularity score (IS): 7.77 ± 0.49 vs. 12.56 ± 1.66], increased the HVR [1.69 ± 0.17 vs. 4.31 ± 0.87 change in minute ventilation (ΔV̇e)/min], and produced an early transient activation of astrocytes followed by a late and persistent activation of microglia in the NTS. In addition, CIH increased IL-1β, IL-6, and TNF-α levels in the NTS. Bilateral CB denervation after 50 days of CIH results in the restoration of normal glial cell activation in the NTS, lower levels of IL-6 and TNF-α, and reductions in arterial blood pressure (83.47 ± 1.38 mmHg) and HVR (1.63 ± 0.43 ΔV̇e/min). The present results suggest that CB inputs to the NTS during long-term CIH contribute to maintain the cardiorespiratory alterations and the formation of a neuroinflammatory niche at the NTS by modifying glial cell activity.NEW & NOTEWORTHY Chronic intermittent hypoxia (CIH), a feature of obstructive sleep apnea, causes cardiorespiratory alterations (i.e. hypertension) linked to oxidative stress, inflammation, and sympathoexcitation. In the present study, we highlight the role of enhanced carotid body (CB) chemosensory afferent discharges to the nucleus of the solitary tract (NTS) in long-term CIH-induced cardiorespiratory disorders. Indeed, we provide evidence that supports the notion that increased CB afferent activity contributes to persistent CIH-induced hypertension, likely triggering neuroinflammation in the NTS.

Obesity is a risk factor for asthma morbidity, associated with less responsiveness to inhaled corticosteroids. CD4+ T cells are central to the immunology of asthma and may contribute to the unique obese asthma phenotype. We sought to characterize the single-cell CD4+ transcriptional profile differences in obese children with asthma compared with normal-weight children with asthma. Eight normal-weight and obese participants with asthma were clinically phenotyped and matched based on asthma control. Peripheral blood (PB) CD4+ T cells were sorted, and single-cell RNA sequencing was conducted. Cell clusters were identified by canonical gene expression and differential gene expression and reactome pathway analysis was applied. The obese PB bulk transcriptomic signature from the U-BIOPRED pediatric cohort was assessed in our cohort as well. Obese children with asthma have a distinct CD4+ transcriptional profile with differential gene expression. There were more activated protein tyrosine phosphate receptor type C (PTPRC)^high cells and less PTPRC^low in children with obesity. Children with obesity had higher enrichment of the neutrophil degranulation, interleukin-7 (IL-7) receptor, and IL-7-related janus kinase-signal transducer and activator of transcription signaling pathways. Genes previously associated with more severe asthma, IL-32, FKBP5 gene expression, IL-6, and Rho transcriptional signaling, were also enriched in obese children with asthma. Our findings shed insight into the molecular mechanisms underpinning more severe and steroid-resistant asthma among children with obesity. Further investigation is needed to identify potential new therapeutic targets for this group.NEW & NOTEWORTHY This study identified unique contributors to asthma in children with obesity and found novel pathways. Increased expression of IL-7R, IL-32, PARP-1, FKBP5 gene, IL-6, and Rho transcriptional signaling were observed in obese individuals with asthma.

Normal shear stress produced by blood flow is sensed by the vascular endothelium and required for maintenance of the homeostatic functions of the endothelium in systemic conduit and resistance vessels. Many critical illnesses are characterized by periods of abnormally reduced or absent shear stress in the lung (e.g., hemorrhagic shock, embolism, ischemia reperfusion injury, and lung transplantation) and are complicated by pulmonary edema following reperfusion due to microvascular leak. The role of shear stress in regulating the pulmonary microvascular endothelial barrier in the intact vascular bed has not been previously examined. We tested the hypothesis that, in lungs injured by a period of ischemia and reperfusion (IRI), reduced shear stress contributes to increased pulmonary microvascular endothelial barrier permeability and edema formation. Furthermore, we examined the role of vascular endothelial-derived growth factor receptor 2 (VEGFR2) as a mechanosensor mediating the endothelial response to this altered shear stress. Following IRI, we perfused isolated ventilated mouse lungs with a low viscosity solution (LVS) or a higher, physiological viscosity solution (PVS) at constant flow to produce differing endothelial shear stresses in the intact microcirculation. Lungs perfused with LVS developed pulmonary edema due to increased endothelial permeability whereas those perfused with PVS were protected from edema formation by reduced endothelial permeability. This effect of PVS required normal VEGFR2 mechanoreceptor function. These data show for the first time that shear stress has an important role in restoring endothelial barrier function in the intact pulmonary microcirculation following injury and have important implications for the treatment of pulmonary edema in critically ill patients.NEW & NOTEWORTHY Critical illnesses are frequently complicated by noncardiogenic pulmonary edema. Many such illnesses include periods of reduced blood flow, often accompanied by hemodilution, which together reduce endothelial shear stress. We report that in ischemia-reperfusion injury reduced shear stress contributes to increased permeability of the in situ pulmonary microvascular endothelium and worsens alveolar edema. Restoring shear stress toward normal reduces endothelial permeability and edema formation, an effect that requires the normal mechanoreceptor function of VEGFR2.

Tartrate-resistant acid phosphatase (TRAP, gene Acp5) is highly expressed in alveolar macrophages with proposed roles in lung inflammation and lung fibrosis development. We previously showed that its expression and activity are higher in lung macrophages of smokers and COPD patients, suggesting involvement in smoke-induced lung damage. In this study we explored the function of TRAP and regulation of its different mRNA transcripts (Acp5 201-206) in lung tissue exposed to cigarette smoke to elucidate its function in alveolar macrophages. In mice exposed to cigarette smoke or air for 4-6 weeks, higher Acp5 mRNA expression in lung tissue after smoking was mainly driven by transcript Acp5-202, which originates from macrophages. Expression of Acp5-202 correlated with transcription factors previously found to drive proliferation of macrophages. Treating fetal liver progenitors-derived alveolar-like macrophages (MPI macrophages) with cigarette smoke extract resulted in more proliferation compared to nontreated cells. In contrast, Acp5-deficient MPI macrophages and MPI macrophages treated with a TRAP inhibitor proliferated significantly less than control macrophages. Mechanistically this lack of proliferation after TRAP inhibition was associated with higher presence of phosphorylated β-catenin compared to nontreated controls. Phosphorylation of β-catenin is known to mark it for ubiquitination and degradation by the proteasome, preventing its activity in promoting cell proliferation. In conclusion, our findings provide strong evidence for TRAP stimulating alveolar macrophage proliferation by dephosphorylating β-catenin. By driving proliferation, TRAP likely helps sustain alveolar macrophage populations during smoke exposure, either compensating for their loss due to smoking or increasing their numbers to better manage smoke-induced damage.

Bronchial epithelial cells derived from the tracheo-bronchial regions of human airways (HBECs) provide a valuable in vitro model for studying pathological mechanisms and evaluating therapeutics. This cell population comprises a mixed population of basal cells (BCs), the predominant stem cell in airways capable of both self-renewal and functional differentiation. Despite their potential for regenerative medicine, BCs exhibit significant phenotypic variability in culture. To investigate how culture conditions influence BC phenotype and function, we expanded three independent BC isolates in three media: airway epithelial cell growth medium (AECGM), dual-SMAD inhibitor (DSI)-enriched AECGM, and Pneumacult Ex plus (PEx+). Analysis through RNA sequencing, immune assays and impedance measurements revealed that PEx+ media significantly drove cell proliferation and a broad pro-inflammatory phenotype in BCs. In contrast, BCs expanded in AECGM, displayed increased expression of structural and extracellular matrix components at higher passage. AECGM increased expression of some cytokines at high passage, while DSI suppressed inflammation implicating the involvement TGF-β in BC inflammatory processes. Differentiation capacity of BCs declined with time in culture irrespective of expansion media. This was associated with an increase in PLUNC expressing secretory cells in AECGM and PEx+ media consistent with the known immune modulatory role of PLUNC in the airways. These findings highlight the profound impact of media conditions on inflammatory niche established by, and function of, in vitro expanded BCs. The broad pro-inflammatory phenotype driven by PEx+ media, in particular, should be considered in the development of cell-based models for airway diseases and therapeutic application.

In this research, we delve into the association between epigenetic aging and idiopathic pulmonary fibrosis (IPF), a debilitating lung disease that progresses over time. Utilizing the Illumina MethylationEPIC array, we assessed DNA methylation levels in donated human lung tissue from IPF patients, categorizing the disease into mild, moderate, and severe stages based on clinical assessments. We employed seven epigenetic clocks to determine age acceleration, which is the discrepancy between biological (epigenetic) and chronological age. Our findings revealed a notable acceleration of biological aging in IPF tissues compared to healthy controls, with four clocks-Horvath's, Hannum's, PhenoAge, and DunedinPACE-showing significant correlations. DunedinPACE, in particular, indicated a more rapid aging process in the more severe regions within the lungs of IPF cases. These results suggest that the biological aging process in IPF is expedited and closely tied to the severity of the disease. The study underscores the potential of DNA methylation as a biomarker for IPF, providing valuable insights into the underlying methylation patterns and the dynamics of epigenetic aging in affected lung tissue. This research supports the broader application of epigenetic clocks in clinical prognosis and highlights the critical role of biological age in the context of medical research and healthcare.