Bhattacharya S, Frauenpreis A, Cherry C
… +5 more, Deutsch G, Glass IA, Mariani TJ, Al Alam D, Danopoulos S
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 40997082
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RATIONALE: Trisomy 21 (T21), resulting in Down syndrome (DS), is the most prevalent chromosomal abnormality worldwide. While pulmonary disease is a major cause of morbidity and mortality in DS, the ontogeny of pulmonary...RATIONALE: Trisomy 21 (T21), resulting in Down syndrome (DS), is the most prevalent chromosomal abnormality worldwide. While pulmonary disease is a major cause of morbidity and mortality in DS, the ontogeny of pulmonary complications remains poorly understood. We recently demonstrated that T21 lung anomalies, including airway branching and vascular lymphatic -abnormalities, are initiated in utero. Here, we aimed to describe molecular changes at the single-cell level in prenatal T21 lungs. METHODS: Single-cell RNA sequencing was used to generate transcriptomic profiles of individual human lung cells in tissue obtained from T21 (n = 5) and non-T21 (n = 4) prenatal lungs. Clustering of cells, marker identification, uniform manifold approximation and projection representation, and differential expression analysis were performed in Seurat. Cell type annotation and pathway analysis were annotated using ToppFun and a human fetal lung cell atlas. Spatial differences in cellular phenotypes were validated using immunofluorescence staining and fluorescent in situ hybridization. RESULTS: Our results detail changes in gene expression at the time of initiation of histopathological abnormalities in T21 prenatal lungs. Notably, we identify precocious differentiation of epithelial cells, widespread induction of key extracellular matrix molecules in mesenchymal cells, and hyperactivation of IFN signaling in endothelial cells. CONCLUSIONS: This single-cell dataset of T21 lungs greatly expands our understanding of antecedents to pulmonary complications and should facilitate efforts to mitigate respiratory disease in DS.
Huang R, Zhao Z, Liu S
… +11 more, Li M, Wang Y, Hu Y, Sun T, Duan Z, Ren C, Yang X, Zhang S, Jiang T, Yin J, Tan L
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 40996835
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RATIONALE: Ex vivo lung perfusion (EVLP) is a promising technique that allows organ preservation and repair, while the molecular mechanisms remain unknown. OBJECTIVES: This study aimed to establish a translational murine...RATIONALE: Ex vivo lung perfusion (EVLP) is a promising technique that allows organ preservation and repair, while the molecular mechanisms remain unknown. OBJECTIVES: This study aimed to establish a translational murine EVLP model and to unveil the molecular mechanisms responsible for EVLP beneficial effects. METHODS: We developed a murine EVLP system with four experimental groups: (1) without ischemia or EVLP (control), (2) 45 minutes of EVLP followed by 135 minutes cold ischemia (EVLP-CI), (3) 135 minutes of cold ischemia (CI) followed by 45 minutes of EVLP (CI-EVLP), and (4) 180 minutes of CI. Following 3-hour preservation, changes in lung weight (Δweight) and lung vascular filtration coefficient (Kf) were measured. Complementary in vitro studies utilized human pulmonary microvascular endothelial cells (hPMVECs) under simulated perfusion conditions. MEASUREMENTS AND MAIN RESULTS: Compared with the CI group, both EVLP intervention groups exhibited superior preservation outcomes, with an attenuated Δweight and Kf, and histological and microscopic evidence of lung damage. Proteomic profiling on mouse lungs revealed that EVLP regulated the Hippo signaling in response to CI. Pharmacological inhibition or genetic deletion of Yap1 or Lats1 specifically in endothelial cells (Yap1EN-KO or Lats1EN-KO) abrogated EVLP-mediated endothelial barrier protection. EVLP efficacy in lung preservation was enhanced by Yap1 phosphorylation activation using AICAR or metformin. In vitro perfusion models recapitulated these findings, where barrier function was disrupted with Yap1 phosphorylation inhibitor, with a decreased cytoplasmic localization of Yap1. CONCLUSIONS: Our findings establish the functional murine EVLP model and first demonstrate that mechanical perfusion preserves donor lung viability through Hippo signaling-mediated endothelial barrier stabilization.
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 40986758
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RATIONALE: PELI1, an E3 ubiquitin ligase, has been identified as a controller of the innate immune response. OBJECTIVES: This study was designed to expound the functional role of PELI1 in asthma (AS). METHODS: Newborn mi...RATIONALE: PELI1, an E3 ubiquitin ligase, has been identified as a controller of the innate immune response. OBJECTIVES: This study was designed to expound the functional role of PELI1 in asthma (AS). METHODS: Newborn mice were induced with house dust mite (HDM) to establish a mouse model of AS, and overexpression of Peli1 was performed specifically in airway epithelial cells by AAV6.2 vector. The human bronchial epithelial cell line 16HBE was induced with HDM, PELI1 was overexpressed in 16HBE cells by liposome transfection, and knockout was elicited through CRISPR/Cas9. MEASUREMENTS AND MAIN RESULTS: Peli1 expression was reduced in the airway epithelium of newborn mice induced with HDM, and overexpression of Peli1 alleviated airway inflammation, mitigated airway injury, and inhibited airway remodeling in AS mice. PELI1 induced protein degradation of IRAK2 through K63 ubiquitination modification. Ectopic expression of Irak2 abated the mitigating effect of Peli1 overexpression on airway inflammation by activating p38-mitogen-activated protein kinase 1 (MAPK)/NF-κB signaling. Blockade of MAPK/NF-κB signaling mitigated the exacerbation of inflammatory responses and cellular damage in 16HBE cells induced by IRAK2 overexpression. CONCLUSIONS: Taken together, this research reveals a functional role of PELI1 in IRAK2 degradation and airway inflammation, which provides novel insights into the treatment of pediatric AS.
Pinkaew D, Pal U, Fujise K
… +2 more, Dellis C, Folz RJ
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 40986750
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RATIONALE: Airway mucus is a complex process influenced by various factors and signaling pathways. A key player is mammalian inositol-requiring enzyme 1 beta (IRE1β), a paralog of IRE1 alpha (IRE1α), found only in epithe...RATIONALE: Airway mucus is a complex process influenced by various factors and signaling pathways. A key player is mammalian inositol-requiring enzyme 1 beta (IRE1β), a paralog of IRE1 alpha (IRE1α), found only in epithelial cells lining the mucosal surfaces of the gastrointestinal and respiratory tracts. IRE1β processes X-box binding protein 1 (XBP1) mRNA via its endoribonuclease (RNase) domain, generating the active XBP1 spliced form (XBP1s). XBP1s is crucial for mucin production, the main component of mucus. IRE1β is upregulated in human bronchial epithelial (HBE) cells from individuals with cystic fibrosis and asthma. Fortilin binds to IRE1α, blocking its kinase/RNase functions and preventing cell death. However, the interaction between fortilin and IRE1β, and its effects on airway mucus under basal conditions, remain unknown. OBJECTIVES: We investigate whether fortilin binds IRE1β, regulates its RNase activity, and is associated with IRE1β-mediated mucin expression. METHODS: The interaction between fortilin and IRE1β was examined by immunoprecipitation and microscale thermophoresis. IRE1β RNase and kinase activities were assessed using in vitro RNase and kinase assays in the presence of fortilin and the IRE1 inhibitor KIRA8. Functional effects of fortilin were evaluated in CRISPR/Cas9-mediated fortilin-knockout HBE cells cultured under air-liquid interface conditions. IRE1β-mediated mucin expression was assessed by RT-qPCR and western blot analyses. MEASUREMENTS AND MAIN RESULTS: We find that fortilin binds to the cytosolic domain of IRE1β, significantly increasing its RNase and kinase activities. Furthermore, fortilin depletion significantly attenuates mucin 5AC (MUC5AC) expression by reducing XBP1 splicing and AKT phosphorylation in differentiated HBE cells under air-liquid interface culture (ALI-HBE). IRE1 inhibitor KIRA8 blunts IRE1β kinase/RNase activities in ALI-HBE cells, inhibiting both XBP1 splicing and AKT phosphorylation regardless of fortilin presence. CONCLUSIONS: These data suggest that fortilin promotes IRE1β-mediated MUC5AC expression primarily via the IRE1β/XBP1 signaling pathway. The IRE1β-fortilin complex holds promise for developing innovative therapies to regulate mucin production in conditions characterized by airway mucus hypersecretion, including chronic obstructive pulmonary disease, asthma, bronchiectasis, and cystic fibrosis.
Nishiura K, Yokokawa T, Ichimura S
… +14 more, Miura S, Sato A, Shimizu T, Misaka T, Oikawa M, Yoshihisa A, Sugimoto K, Muto S, Suzuki H, Ueda K, Ikeda K, Nakazato K, Ishida T, Takeishi Y
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 40986747
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BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary arterial pressure and right ventricular failure. The perivascular macrophages in the lungs play a crucial rol...BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary arterial pressure and right ventricular failure. The perivascular macrophages in the lungs play a crucial role in the development of PAH. Here, we tested the hypothesis that colony-stimulating factor 1 receptor (CSF1R), essential for macrophage proliferation and polarization, contributed to the progression of PAH, and targeting CSF1R could offer a potential therapeutic strategy. METHODS AND RESULTS: In the lungs of patients with PAH, we found that the number of perivascular CSF1R-positive macrophages and M2 macrophages significantly increased. In the experimental sugen/hypoxia-induced PAH model, knockdown of CSF1R in the lungs decreased right ventricular systolic pressure and the number of perivascular macrophages. Pharmacological inhibition with a CSF1R inhibitor, pexidartinib, and anti-CSF1R neutralizing antibody blocked perivascular macrophage accumulation and improved the severity of pulmonary hypertension in the murine PAH models. Mechanistically, C-C motif chemokine ligand 2 (CCL2) produced by M2 macrophages was identified as a key driver for pulmonary artery smooth muscle cell proliferation, leading to pulmonary arterial remodeling. Activation of CSF1R and c-Jun N terminal kinase (JNK) transcriptionally regulated Ccl2 expressions in macrophages. CONCLUSION: Our study suggests that CSF1R and M2 macrophages have critical roles in the progression of PAH through CCL2.
Habibovic A, Lin MC, Morris CR
… +10 more, Schiffers C, da Cruz LC, van der Ploeg IS, Steinvoort F, Utermohlen O, Krönke M, Kumar A, Anathy V, Janssen-Heininger YMW, van der Vliet A
Am J Respir Cell Mol Biol
· 2026 Feb · PMID 40986746
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Fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF) are caused by various forms of environmental injury and involve reciprocal interactions between activated (myo)fibroblasts and recruited monocyte-derived...Fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF) are caused by various forms of environmental injury and involve reciprocal interactions between activated (myo)fibroblasts and recruited monocyte-derived macrophages (MoMacs), collectively leading to progressive tissue remodeling. Extending previous findings implicating redox-based mechanisms in IPF pathogenesis, we here highlight the involvement of the NADPH oxidase (NOX) homolog DUOX1 in pulmonary fibrosis, based on observed increases in DUOX1 expression within fibrotic regions of IPF lung tissues or lung tissues from mice with experimentally induced pulmonary fibrosis, localized primarily to (myo)fibroblasts and recruited MoMacs. Building on a previous report implicating DUOX1 in myofibroblast activation, conditional DUOX1 ablation from myeloid cells (including macrophages) using LysM-Cre was found to dramatically attenuate fibrosis, highlighted by impaired MoMac recruitment, reduced collagen production, and improved oxygen saturation. A macrophage-intrinsic role of DUOX1 was further supported by its observed contribution to in vitro migration of bone marrow-derived macrophages (BMDM) and to profibrotic BMDM activation, the latter including production of several epidermal growth factor receptor ligands involved in macrophage-fibroblast cross-talk. Finally, these DUOX1-mediated actions were associated with oxidative activation of Src kinase via cysteine oxidation, and were inhibitable by saracatinib, a clinically used Src inhibitor. Collectively, our findings highlight the involvement of DUOX1 in macrophage-(myo)fibroblast cross-talk in the pathogenesis and/or progression of pulmonary fibrosis, implicating it as a putatively novel therapeutically targetable feature of this devastating disease.
Ding H, Zhu X, Pan Y
… +5 more, Zhang Q, Feng M, Yu Y, Fan Y, Zhu L
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 40986744
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RATIONALE: Neurotensin receptor 1 (NTSR1) is a high-affinity receptor for neurotensin. Its abnormal expression correlates with cancer development. However, its mechanisms in promoting the malignant progression and irinot...RATIONALE: Neurotensin receptor 1 (NTSR1) is a high-affinity receptor for neurotensin. Its abnormal expression correlates with cancer development. However, its mechanisms in promoting the malignant progression and irinotecan resistance in lung adenocarcinoma (LUAD) remain unelucidated. METHODS: NTSR1 expression in LUAD and its relationship with patients' prognosis were analyzed by bioinformatics analysis. NTSR1 expression in a human normal pulmonary epithelial cell line and LUAD cell lines was detected by RT-qPCR and Western blot. Cell proliferation ability was examined using the CCK-8 assay and colony formation assay. Flow cytometry was employed to detect cell cycle and apoptosis. The Transwell assay was undertaken to assess cell migration and invasion ability. DNA damage was detected using the comet assay and γ-H2AX immunofluorescence. Dot-blot and methylated RNA immunoprecipitation-qPCR were employed to examine m6A methylation levels. The interaction between IGF2 mRNA binding protein (IGF2BP3) and NTSR1 was verified by RNA immunoprecipitation and dual luciferase experiments. Immunohistochemistry was applied to analyze protein expression in mouse tumor tissues. MEASUREMENTS AND MAIN RESULTS: NTSR1 was upregulated in LUAD cells, affecting patients' dismal overall survival. NTSR1 knockdown hindered cell proliferation, migration, and invasion, and reinforced apoptosis and irinotecan sensitivity. Mechanistically, IGF2BP3 interacted with NTSR1 and induced m6A methylation modification to enhance transcriptional stability, advancing the malignant progression of LUAD and irinotecan resistance. Additionally, NTSR1 knockdown enhanced the sensitivity of LUAD to irinotecan in mice and induced DNA damage. CONCLUSIONS: Overall, IGF2BP3-mediated NTSR1 m6A methylation expedites LUAD malignant progression and reinforces irinotecan resistance. Targeting this pathway may be an effective method for treating LUAD.
Zhang Y, Tang M, Xie R
… +16 more, Yang Y, Sun P, Luo Y, Wang R, Hu X, Ma T, Zhang Y, Zou C, Liu H, Wu M, Ren Y, Li H, Li J, Bian C, Tang Y, Zhou X
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 40986663
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Effective immune activation is essential for host defense against pathogenic microorganism infection. However, excessive or uncontrolled immune activation can cause tissue damage. Negative regulatory factors and immune h...Effective immune activation is essential for host defense against pathogenic microorganism infection. However, excessive or uncontrolled immune activation can cause tissue damage. Negative regulatory factors and immune homeostasis regulatory molecules play important roles in immune activation. CircRNAs are known to be involved in a variety of pathological and physiological processes, but their role in the regulation of host immune activation remains unclear. In this study, we identified a novel circRNA, circSlc7a11, in the lung using a Pseudomonas aeruginosa pulmonary infection model. circSlc7a11 functions as a negative regulator that prevents excessive immune activation in the host response to bacterial infection by regulating the IL-1β signaling axis through PUF60 in macrophages.
Chatterjee P, Ahmad A, Dhillon E
… +7 more, Matthaiou EI, Abd El-Hafeez AA, Chiu W, Illek B, Stevens DA, Milla C, Hsu JL
Am J Respir Cell Mol Biol
· 2026 Feb · PMID 40965296
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RATIONALE: Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity disease triggered by inhaled Aspergillus -fumigatus (Af) spores (i.e., conidia), in persons with cystic fibrosis (CF). High iron levels have...RATIONALE: Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity disease triggered by inhaled Aspergillus -fumigatus (Af) spores (i.e., conidia), in persons with cystic fibrosis (CF). High iron levels have been commonly described in CF respiratory secretions. Af proteases cause a severe allergic immune response, leading to pulmonary exacerbations and progressive lung function decline. The stimuli that promote Af proteases in ABPA are poorly understood. OBJECTIVES: Determine if airway iron is a critical factor in CF-ABPA by regulating fungal protease production. METHODS: We used transcriptomics and proteomics to investigate the role of iron in stimulating the production of Af proteases and evaluated the impact of these iron-induced Af proteases on the Th2 immune response in CF bronchial epithelial (CFBE) cells and in a CF-ABPA mouse model. MEASUREMENTS AND MAIN RESULTS: Transcriptional analysis showed a significant upregulation of Af proteases with exposure to iron and a downregulation with iron chelation. This finding was validated in secretome studies showing that Af-culture filtrates (Af-cf) from iron-exposed conidia contained significantly higher levels of 14 proteases compared to controls. Exposure to iron-primed Af-cf increased the production of Th2 promoting cytokines (IL-33, IL-25 and TSLP) in CFBE cells compared to the wild type cells. In vivo, inoculation of CF mice with iron-primed Af conidia led to higher levels of serum IgE and Th2 cytokines (IL-4, IL-5, IL-9, IL-13) in lung tissue. CONCLUSIONS: These findings suggest that airway iron is a potent stimulus for Af proteases and represents a modifiable risk factor and potential therapeutic target for CF-ABPA.
Supplemental oxygen is an essential therapy during critical illness. However, patients with severe hypoxemic respiratory failure and/or acute respiratory distress syndrome often require high oxygen concentrations, exposi...Supplemental oxygen is an essential therapy during critical illness. However, patients with severe hypoxemic respiratory failure and/or acute respiratory distress syndrome often require high oxygen concentrations, exposing lungs to alveolar hyperoxia despite systemic hypoxemia, with consequent pulmonary oxygen toxicity. Pulmonary oxygen toxicity causes disruption of surfactant, which is essential for maintenance of alveolar functional anatomy, as well as efficient and effective gas exchange and immune regulation. This surfactant dysregulation can increase alveolar surface tension, causing alveolar collapse with atelectasis, resulting in poor lung compliance and impaired gas exchange. Hyperoxia-induced lung injury mechanisms may interact with mechanisms of harm associated with infections and mechanical ventilation. The intricate relationship between these different, interrelated stressors and altered surfactant metabolism and function is not yet delineated, particularly in humans. This review examines the current understanding of hyperoxia-induced surfactant dysregulation. We discuss potential mechanisms, including biochemical/compositional and functional changes to lipids and proteins including surfactant proteins A and D, epithelial atrophy, impaired surfactant synthesis/metabolism, redox imbalances, phospholipase A2, and altered macrophage clearance. Key areas for future research are outlined, emphasizing the need for clinically relevant human models that discriminate between the effects of oxygen therapy dose and duration, as well as other iatrogenic effects and underlying disease processes. We propose a roadmap to progress current knowledge and outline opportunities for well-designed human studies, novel surfactant preparations resistant to functional inhibition and breakdown, and technological developments, with the potential for leveraging these to identify innovative biomarkers, individualized therapeutic targets, and novel therapies in the future.
Su X, Yadav SK, Blagdon C
… +5 more, Singh AK, Milman-Krentsis I, Shoshan E, Alon R, Reisner Y
Am J Respir Cell Mol Biol
· 2026 Feb · PMID 40938399
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Lung cell transplantation has demonstrated remarkable regenerative potential in various mouse models of lung injury, including pulmonary fibrosis. However, early processes governing donor cell lung homing and fate after...Lung cell transplantation has demonstrated remarkable regenerative potential in various mouse models of lung injury, including pulmonary fibrosis. However, early processes governing donor cell lung homing and fate after transplantation remain poorly understood. This study interrogates mechanisms underlying donor cell homing, extravasation, and formation of regenerative patches inside recipient lungs after intravenous infusion of CD45- lung cells. Naphthalene (NA) and total body irradiation (TBI) were used to induce lung injury. Donor-derived lung cell suspensions were infused intravenously, and donor cell localization was analyzed at various time points using flow cytometry and immunofluorescence. The functional roles of VLA-4/VCAM-1 and CXCR4/CXCL12 interactions in early donor cell homing to injured lungs were assessed by incubating donor cells with anti-VLA-4 or anti-CXCR4 blocking antibodies or by pretreatment of recipient mice with anti-VCAM-1 antibody. At 24 hours after infusion, only 0.8% of infused cells accumulated inside the lungs, with approximately a third of the cells within the pulmonary vasculature. By Day 7, 97% of donor cells were found in the lung parenchyma. These donor cells were highly proliferative and formed regenerative patches by Day 21. Blocking VLA-4 or CXCR4 inhibited adhesion of infused cells to blood vessels early after infusion and interfered with subsequent formation of regenerative donor-derived lung patches at 6 weeks after infusion. This study highlights the sequential roles of VLA-4/VCAM-1 and CXCR4/CXCL12 interactions in facilitating donor lung cell homing and regenerative patch formation in injured lungs.