American Journal of Respiratory Cell and Molecular Biology最新文献

C-type lectin-like receptor 2 (CLEC2) is a transmembrane receptor highly expressed on platelets which regulates platelet aggregation and immune response. Yet, the function of CLEC2 in lung epithelium and its contribution to acute lung injury (ALI) is unclear. In this study, lung epithelial-specific CLEC2 knockout mouse (Clec1b  AT2-KO) was generated and performed for ALI models. In both lipopolysaccharide (LPS)- and acid-induced lung injury models, the ALI signs of Clec1b  AT2-KO mice were further exacerbated. The therapeutic application of epithelial-restricted CLEC2 overexpression using adeno-associated virus (AAV) or CLEC2 activation using its endogenous ligand podoplanin (PDPN) serves as a lung epithelial protective agent in the setting of ALI. Transcriptomic analyses reveal that CLEC2-regulated genes are highly enriched in chemotaxis, cytokine, and extracellular matrix (ECM) components. Lung injury was partially attenuated in Ccl5-/-, Csf3-/- and Cxcl1-/- mice pretreated with AAV-si-CLEC2, followed by LPS challenge. Loss of CLEC2 leads to ECM degradation, which could be reversed by exogenous transforming growth factor (TGF)-β. Furthermore, interferon regulatory factor 1 (IRF1) was identified as the key molecule that regulates CLEC2-related cytokine/chemokine production and ECM degradation. These findings suggest that epithelial CLEC2 protects against ALI by modulating spleen tyrosine kinase (Syk)/IRF1-mediated cytokine/chemokine production and TGF-β-mediated ECM remodeling.

Pleural mesothelial cells (PMCs) are a major component of the pleura. The involvement of visceral PMCs has been suggested in several pleural or subpleural lung diseases, thus highlighting the importance of animal experiments using these cells. However, in wild-type rodent lungs, no direct and pure isolation method for visceral PMCs has been established so far. Using reanalyzed single-cell RNA sequencing data, we identified that mesothelin (Msln), a specifically expressed gene in visceral PMC, can be useful for live cell sorting. After collecting cells by scraping the visceral pleura, MSLN+EpCAM-CD45-CD31-PDGFRα-CD146- cells with large size (≥1.4 times the median value of forward scatter in total cells) mostly exhibited WT1 protein (WT1-positivity in immunocytochemistry: 93.3% ± 0.8%). The sorted PMCs were culturable, and they responded generally predictably to several growth factors and cytokines, including genetic changes suggestive of a TGFβ-induced mesothelial-to-mesenchymal transition. In vivo experiments performed using PMC-specific reporter mice (Wt1-creERT2; tdTomato) demonstrated that intrapleural administration of bleomycin with carbon induced proliferation of PMCs in the visceral pleura. Importantly, some PMC-derived cells differentiated into αSMA-positive myofibroblasts with decreased MSLN expression, indicating that our method using MSLN is suitable only for uninjured lungs. In summary, we propose a novel and qualified method for visceral PMC isolation, which will aid in the elucidation of the mechanisms underlying pleura-related human lung diseases.

Pseudomonas aeruginosa infection poses a significant clinical challenge in respiratory diseases by subverting host defense mechanisms. While inflammatory responses in airway epithelial cells (AECs) during infection have been extensively studied, the interplay between epitranscriptomic regulation and metabolic reprogramming remains poorly understood. Here, we identify a lactylation-m6A axis that orchestrates ciliary function and antibacterial defense through dual-layer metabolic-epigenetic coordination. Using integrated in vivo and in vitro models, we demonstrate that P. aeruginosa infection depletes host lactic acid through direct consumption via lactate dehydrogenase and virulence factor-mediated glycolytic suppression. This metabolic perturbation reduces histone H3K18 lactyaltion, dimishing m6A methylation by directly downregulating YTHDF1; m6A-seq analysis reveals preferential hypomethylation of dynein axonemal heavy chain 5 (DNAH5) mRNA, a critical regulator of ciliary motility. Mechanistically, YTHDF1 recognizes m6A-modified DNAH5 transcripts to stabilize translation. The lactylation-YTHDF1-DNAH5 axis proves essential for maintaining ciliary beat frequency and mucociliary clearance capacity. This metabolic-epitranscriptomic circuitry significantly impacts host defense, as evidenced by increased bacterial burden in conditional YTHDF1 knockout mice. Our findings extend the paradigm of lactylation-mediated gene regulation to airway pathophysiology, revealing a novel mechanism where microbial-induced metabolic perturbations reprogram RNA modification landscapes to disable ciliary defenses. This study establishes a conceptual framework for understanding how opportunistic pathogens exploit host metabolic-epigenetic networks to establish persistent infections, suggesting therapeutic potential for targeting the lactate-YTHDF1 axis in P. aeruginosa-associated pulmonary disorders.

Identifying molecular biomarkers of pulmonary fibrosis (PF) would improve monitoring the disease progression and response to treatment. Hermansky-Pudlak syndrome (HPS)PF is an inherited type of progressive PF with accelerated onset of PF in patients with HPS type 1 (HPS-1). HPSPF could serve as a model to study biomarkers of progressive PF, given that all individuals with HPS-1 eventually develop HPSPF. We used a multiomics strategy to discover progressive blood biomarkers that can recognize factors contributing to the fibrotic cascade in the lungs of individuals with HPS. Metabolomic and cytokine/chemokine profiling were performed on serum samples from patients with HPS-1, HPS-1 with PF (HPSPF), HPS-3, HPS-5, or idiopathic PF and healthy volunteers. Metabolomics, cytokine/chemokine, pulmonary function, and age data from subjects with HPS-1 and HPSPF were integrated into a multiomics network. The analysis highlighted alterations in the transsulfuration pathway, arginine metabolism, and redox balance with the progression of PF in HPS-1. Among those, CCL22 and choline were significantly elevated in HPSPF compared with HPS-1 in two independent cohorts together with age and were associated with decline of pulmonary function. In receiver operating characteristic curve analysis, both CCL22 and choline demonstrated high accuracy in predicting PF in subjects with HPS-1 and therefore could serve as prognostic blood biomarkers of HPSPF. We noted similarity in molecular signatures of CCL22 in progressive idiopathic PF and HPSPF. We found that inducible nitric oxide synthase is an upstream regulator of releasing profibrotic mediators (CCL22, CCL24, IL-18, IL-1α, IL-1β), suggesting the therapeutic potential of inducible nitric oxide synthase inhibition in progressive HPSPF.

Prenatal exposure to cadmium (Cd) and arsenic (As) can severely impair fetal lung development, leading to lifelong adverse effects. As two of the most common and toxic heavy metals, Cd and As pose risks to many communities through food and water consumption. We have shown that prenatal coexposure to Cd and As at levels relevant to human intake inhibits branching morphogenesis, yet cell type-specific mechanisms remain elusive. Here, we examined early embryonic (Embryonic Day [E]12) lungs from mice exposed prenatally to either 0 (control) or 250 (treated) ppb of both Cd and As. Through single-cell multiome sequencing (single-cell transposase-accessible chromatin with high-throughput sequencing + single-cell RNA sequencing) and high-resolution metabolomics, we present a multifaceted landscape of Cd- and As-induced molecular and cellular disruption. We identified 19 cell states that exhibited state-specific changes in gene expression related to cell proliferation and differentiation. Velocity analysis integrating RNA splicing and chromatin kinetics showed profound disruptions in cell fate, particularly affecting differentiation of Sox2+ proximal progenitors and Wnt2+ mesenchymal progenitors. Gene regulatory network analysis pinpointed the diminished function of Gata6 and Gli2 as central to these disruptions, which was further confirmed by their reduced protein expression in exposed E12, E14.5, and E17 lungs. Additionally, metabolomic alterations in polyamine, tyrosine, and fatty acid biosynthesis correlated with changes in gene expression of catalytic enzymes. These findings demonstrate that Cd and As at levels relevant to human exposure impair early airway formation across multiple regulatory levels, including chromatin accessibility, transcription, and cell metabolism, and they provide insights into the factors central to cell resilience during this vulnerable stage of lung development.

Improvements in radiation therapy (RT) for thoracic cancers have increased survival; thus, preventing radiotoxicity in normal lung tissue becomes even more important. Respiratory infection is a lung stressor that increases the risk of RT toxicity. However, this risk factor remains understudied with no effective treatment approaches. Although RT is toxic to tissue-resident alveolar macrophages, recruited monocyte-derived macrophages (MDMs) drive fibrogenesis. We therefore investigated how these macrophage populations are impacted by a respiratory infection subsequent to lung RT. Mice received whole-thorax RT (5-12.5 Gy), then were infected with influenza A virus (IAV) 1 or 20 weeks later. Chronic lung injury and acute and chronic macrophage responses were evaluated. RT plus IAV was lethal at doses that were well tolerated when either was administered singly. IAV potentiated chronic pathology from even a benign RT dose of 5 Gy, even when IAV was delayed for 20 weeks. Macrophage dynamics shifted toward more predominant proinflammatory, profibrotic MDM responses. Acutely, RT plus IAV amplified loss of tissue-resident alveolar macrophages but increased inflammatory MDMs. Expression of maturation receptors and antigen presentation factors by inflammatory MDMs decreased, whereas profibrotic factors increased. These novel findings warrant further investigation of the risks of respiratory infection for those receiving thoracic radiation.

Ozone (O3) is a criteria pollutant that is anticipated to increase over the next decade because of climate-related activity. Varying amounts of nitrogen oxides (NOx) are produced as byproducts during O3 generation from oxygen depending on the method of production, including the source and oxygen purity. A review of the current literature confirms a lack of consistent monitoring and reporting of potential nitrogen species produced with different methods of experimental O3 generation. The lack of consistent monitoring and reporting is potentially a factor that can explain divergence of reported experimental O3 exposure outcomes from different research groups. In the present report, we compare the effects of O3 generation from either a filtered air (FA-ozone) or a pure oxygen (Oxy-ozone) source on NOx generation and measures of O3-induced lung injury. We also consider if this impacts mixed exposures with O3 and ultrafine carbon black (CB) based on if the O3 was generated from a filtered air (FA-ozone-CB) versus a pure oxygen (Oxy-ozone-CB) source. Comparing FA-ozone versus Oxy-ozone, we observed increased lung inflammation and injury in the FA-ozone group. In the FA-ozone-CB group, compared with the Oxy-ozone-CB group, the FA-ozone-CB inhalation exposure resulted in the formation of a greater amount of NOx and induced protein nitrotyrosine in the lungs. Moreover, the FA-ozone-CB group had evidence of eosinophil recruitment not observed in the Oxy-ozone-CB group. Overall, this suggests that the source of oxygen for O3 generation impacts experimental outcomes. Furthermore, measurement and reporting of nitrogen species in O3 exposure should be considered.

Streptococcus pneumoniae (Spn) infection secondary to influenza A virus (IAV) frequently leads to an increase in morbidity and mortality of IAV. Our recent work establishes that IAV infection disrupts bacterial host defense in the lung epithelium through loss of cystic fibrosis transmembrane conductance regulator protein (CFTR) function, causing an acidification of the airway surface liquid (ASL) and subsequently increasing susceptibility to Spn. Infection with IAV and other respiratory pathogens causes a robust endoplasmic reticulum (ER) stress response. However, the role of this acute ER stress response in predisposing the airway epithelium to susceptibility to bacterial infections remains unknown. Using a primary differentiated human bronchial airway epithelial cell (HBEC) culture system, we found that both IAV-induced ER stress and ER stress alone increased susceptibility to Spn in the airway epithelium and led to a loss of CFTR activity, subsequently causing a disruption in the rheostatic properties of the ASL. Importantly, in HBECs without functional CFTR, modulation of ER stress in the presence and absence of IAV has no effect on susceptibility to Spn. Restoration of ASL pH after ER stress in HBECs with functional CFTR reduces Spn, suggesting that ER stress increases susceptibility to bacterial infection by disrupting CFTR and causing an acidification of the ASL. Here, we demonstrate a clear role for ER stress in disruption of both the airway epithelium and bacterial host defense mechanisms during respiratory viral infection. Clinical trial registered with www.clinicaltrials.gov (NCT04164212).