Wang Y, Kulkarni VV, Pantaleón García J
… +7 more, Ntita M, Chavez MA, Reese TC, Liu Y, Vila Ellis L, Tuvim MJ, Evans SE
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 41738189
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Pneumonias remain a leading cause of death worldwide. Seeking novel strategies to protect susceptible patients, we have reported that inhaled delivery of a diacylated lipopeptide and a synthetic CpG oligodeoxynucleotide...Pneumonias remain a leading cause of death worldwide. Seeking novel strategies to protect susceptible patients, we have reported that inhaled delivery of a diacylated lipopeptide and a synthetic CpG oligodeoxynucleotide (ODN) protects animals against a broad range of infectious pneumonias by stimulating antimicrobial responses from the lung epithelium. Toll-like receptor 9 (TLR9) is well-established as the primary cellular receptor for CpG ODNs. However, we recently reported that ODNs also stimulate TLR9-independent generation of antimicrobial mitochondrial reactive oxygen species. By testing a variety of synthetic ODN molecules, we found that ODNs containing a phosphorothioate backbone, but not those with a phosphodiester backbone, induce TLR9-independent pathogen killing in lungs and improve mouse survival. Phosphorothioate-backboned ODN binds mitochondrial protein voltage-dependent anion channel 1 (VDAC1) at its N-terminus, initiating pneumonia-protective metabolic reprogramming in lung epithelial cells that yield the protective antimicrobial effect. Thus, the phosphorothioate backbone of ODN is a critical structural pattern that activates TLR9-independent, metabolically modulated innate immune protection that may be harnessed to protect vulnerable patients against pneumonia.
Endo A, Enomoto Y, Horiguchi R
… +7 more, Meguro S, Kawasaki H, Kosugi I, Iijima K, Takabayashi S, Baba S, Iwashita T
Am J Respir Cell Mol Biol
· 2026 Jan · PMID 41738167
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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 experim...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.
Wang J, Shen X, Li Y
… +9 more, Xia Z, Yin T, Qiao J, Deng Y, He R, Guo Y, Zhang Y, Zhang G, Qu J
Am J Respir Cell Mol Biol
· 2026 May · PMID 41738162
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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...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-N6-methyladenosine (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 lactylation, diminishing m6A methylation by directly downregulating YTHDF1; m6A sequencing 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.
Jiang T, Wu L, Wang Y
… +10 more, Yang X, Huang R, Ren C, Zhang Q, Hu Y, Zhang S, Yang X, Yin J, Wang L, Tan L
Am J Respir Cell Mol Biol
· 2026 May · PMID 41738153
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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 contributio...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, a lung epithelial-specific CLEC2 knockout mouse (Clec1bAT2-KO) was generated and performed for ALI models. In both LPS- and acid-induced lung injury models, the ALI signs of Clec1bAT2-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 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 beta (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/IRF1-mediated cytokine/chemokine production and TGF-β-mediated ECM remodeling.
Dharmadhikari S, Calyeca J, Liu L
… +8 more, Wu Z, Schneller A, Hussein Z, Tan ZH, Breuer CK, Reynolds SD, Zhao K, Chiang T
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 41738144
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RATIONALE: Mechanical ventilation is integral to the medical management of the critically ill. However, prolonged exposure to pathologic airflow during mechanical ventilation places patients at risk for airway injury. He...RATIONALE: Mechanical ventilation is integral to the medical management of the critically ill. However, prolonged exposure to pathologic airflow during mechanical ventilation places patients at risk for airway injury. Here, we developed an innovative microsurgical mouse model to examine how changes in airflow affect cellular regeneration and repair in the tracheobronchial airway. We examined wall shear stress (WSS), a known consequence of airflow constriction, and evaluated clinical significance using Heliox, a helium-rich environment, which reduces pathologic airflow in human patients. METHODS: We modulated airflow by altering airway geometry. Specifically, orthotopic airway transplants replaced a tracheal segment with either a bronchial scaffold or a tracheal scaffold (control) (n = 10/group). After surgery, mice were subjected to room air (1 or 2 weeks) or Heliox (1 week). Computational fluid dynamics models were used to quantify WSS. Epithelial and fibroblast cell number and phenotype, as well as collagen density, were analyzed using histology. Statistical analysis was performed using 1-way analysis of variance with a multiple comparison test. RESULTS: Using our novel microsurgical model, we reduced airway cross-sectional area by 0.54-fold and increased WSS by 71.40-fold. This increase in WSS was associated with squamous epithelial differentiation, increased fibroblast activation, and significant fibrosis. These phenotypic changes were rescued when mice were recovered in Heliox. CONCLUSIONS: Our findings suggest a mechanical mechanism in which airflow regulates epithelial differentiation, fibroblast activation, and fibrosis in the tracheobronchial airway. These studies support the development of therapeutic interventions that modulate intraluminal WSS and have the potential to accelerate airway regeneration.
Whitehead GS, Nakano K, Wilkinson CL
… +8 more, Patterson AM, Upadhyay S, Massri AJ, Papas BN, Gruzdev A, Ray M, Nakano H, Cook DN
Am J Respir Cell Mol Biol
· 2026 May · PMID 41134983
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RATIONALE: The urokinase plasminogen activator receptor (uPAR) is a membrane-bound protein that can contribute to the activation and mobilization of leukocytes and is present at increased levels in asthmatics. However, i...RATIONALE: The urokinase plasminogen activator receptor (uPAR) is a membrane-bound protein that can contribute to the activation and mobilization of leukocytes and is present at increased levels in asthmatics. However, its role in allergic asthma remains poorly understood. METHODS: We used multiple mouse strains and different models of allergic airway disease to study the function of uPAR in the pathogenesis of this disease. MEASUREMENTS AND MAIN RESULTS: Plaur, the gene encoding uPAR, was rapidly induced following allergic sensitization through the airway and again following subsequent allergen challenge. Plaur-deficient mice displayed both increased numbers of eosinophils and heightened airway hyperresponsiveness (AHR) in multiple models of allergic asthma. Mice selectively lacking Plaur in eosinophils also had more robust eosinophilia than did wild-type (WT) mice, and eosinophils lacking Plaur displayed increased activity in an ex vivo assay of chemokine-dependent migration. However, those mice did not have increased AHR compared with WT mice. Conversely, although mice selectively lacking Plaur in lung epithelial cells did not have increased inflammation compared with WT mice, they displayed heightened AHR. CONCLUSIONS: These findings suggest that uPAR controls both airway inflammation and AHR, but through distinct mechanisms. Targeting uPAR might have therapeutic potential for treating inflammation and AHR in asthma.
Many GM, Sagendorf TJ, Mitchell H
… +19 more, Sanford JA, Cohen SE, Misra RS, Estevao I, Ludovico ID, Gaul DA, Lindholm ME, Ushakumary MG, Pino JC, Musi N, Nie J, Fernández FM, Ortlund EA, Esser KA, Bodine SC, Schenk S, Clair G, Adkins JN, MoTrPAC Study Group
Am J Respir Cell Mol Biol
· 2026 May · PMID 41134976
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Endurance exercise is broadly beneficial to cardiopulmonary function, with these benefits thought to be driven by extrapulmonary factors rather than direct structural changes in the lungs. Thus, to address how endurance...Endurance exercise is broadly beneficial to cardiopulmonary function, with these benefits thought to be driven by extrapulmonary factors rather than direct structural changes in the lungs. Thus, to address how endurance exercise training and sex impact molecular responses in the lungs, we used a multi-omics approach to study 6-month-old Fischer 344 rats that undertook a progressive endurance treadmill training protocol for 1 to 8 weeks. Specifically, we reannotated publicly accessible transcriptomics, metabolomics, proteomics and phosphoproteomics data from the Molecular Transducers of Physical Activity Consortium and integrated newly analyzed acetylproteomics data to assess multi-omic sex differences in sedentary and treadmill trained rats. Female rats displayed enrichment in immune-related features and pathways at the transcriptome and proteome level that were largely maintained with training. However, both sexes exhibited decreases in immune pathway activity following 8 weeks of training, although the effect was more pronounced in males. Shared responses to training included increased enrichment in transcriptomic pathways related to type I alveoli, proteomic pathways related to cilia, and decreased acetylation of pathways linked to mitochondrial function. Furthermore, features known to be enriched in lung diseases were attenuated with training in both sexes. Together, our findings provide novel insight into responses to endurance exercise training in the healthy rat lung and may offer translational insight into sex-specific differences in lung disease pathogenesis and treatment.
Ghani H, Thillai M, Jenkins D
… +12 more, Bussell E, Ruggiero A, Walsh S, Screaton N, Bunclark K, Cannon J, Sheares K, Taboada D, Graves M, Toshner M, Ng C, Pepke-Zaba J
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 41124339
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Pulmonary blood volumes (PBVs), currently not assessed by computed tomography pulmonary angiography (CTPA), could provide additional information to routine investigations performed for chronic thromboembolic pulmonary hy...Pulmonary blood volumes (PBVs), currently not assessed by computed tomography pulmonary angiography (CTPA), could provide additional information to routine investigations performed for chronic thromboembolic pulmonary hypertension (CTEPH). We investigated CTPA-based PBVs in evaluating hemodynamic outcome from pulmonary endarterectomy (PEA). A deep learning-based CTPA vascular segmentation model, differentiating arteries and veins, was applied for automated PBV measurements in patients with CTEPH who underwent PEA at the United Kingdom's national CTEPH service. Pulmonary arteries were compartmentalized into "central" (main pulmonary and proximal lobar) and "intrapulmonary." Mean pulmonary arterial pressure >30 mm Hg post-PEA defined "clinically relevant" residual pulmonary hypertension (PH). Logistic regression models applying CTPA-based PBV to identify residual PH were trained and tested on the discovery and validation cohorts, respectively. Paired pre- and postoperative CTPA in the discovery (n = 71) and validation (n = 102) cohorts showed that central pulmonary artery volume and total artery to vein volume ratio (A-VR) decreased and pulmonary vein volume increased with hemodynamic improvement post-PEA. Preoperative central pulmonary artery volume and A-VR helped identify patients at risk for clinically relevant residual PH post-PEA (area under the receiver operating characteristic curve [AUROC], 0.88 and 0.82 in the discovery and validation cohorts, respectively). Postoperative central pulmonary artery volume, A-VR, and pulmonary vein volume helped to noninvasively identify patients without clinically relevant residual PH (AUROC, 0.91 and 0.88 in the discovery and validation cohorts). Automated quantification of CTPA-based PBV at diagnosis can help stratify risk for residual PH in patients managed with PEA. Utilizing CTPA-derived PBV post-PEA to identify patients without residual PH can potentially reduce the need for routine postoperative right heart catheterization.
Cystic fibrosis (CF) is an autosomal recessive disease caused by variants in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Delivery of a functional CFTR transgene to airway epithelial cells...Cystic fibrosis (CF) is an autosomal recessive disease caused by variants in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Delivery of a functional CFTR transgene to airway epithelial cells (AEC) offers the potential to provide durable restoration of normal CFTR function. Adeno-associated virus (AAV) vectors are the leading platform for the delivery of in vivo gene therapy; however, wild-type AAV vectors exhibit a limited capacity to transduce airway cells and evade pre-existing human neutralizing antibodies (NAb). We therefore employed a directed evolution platform to invent a novel AAV capsid (A101) with the capacity to efficiently transduce AECs, including in the presence of NAbs, following aerosolized administration to nonhuman primates (NHP). We then engineered 4D-710, a gene therapy comprising the A101 vector and a CFTR transgene with a partial deletion in the regulatory domain (CFTRΔR) to facilitate vector packaging. 4D-710 exhibited efficient transduction of human bronchial epithelial (HBE) cell air-liquid interface (ALI) cultures in vitro and robust functional activity in CF HBE ALI cultures. Aerosolized administration of 4D-710 to NHPs was well tolerated and resulted in dose-dependent transgene expression and increased CFTR protein in diverse AEC types compared with vehicle controls. No significant differences in CFTRΔR mRNA levels were observed in lung samples from NHPs with pre-existing serum anticapsid NAbs compared with NAb-negative NHPs. These findings demonstrate the tolerability and feasibility of A101-mediated transgene delivery and expression in primate airways. A clinical trial evaluating aerosol delivery of 4D-710 in adults with CF (NCT05248230) is underway.
Karpurapu M, Yan J, Chung S
… +8 more, Posham L, Fritz JR, Englert JA, Pannu SR, Parinandi N, Berdyshev E, Zhang L, Christman JW
Am J Respir Cell Mol Biol
· 2026 May · PMID 41124277
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Extracellular vesicles (EVs) have emerged as versatile carriers of therapeutic cargo, including nucleic acids, proteins, and small molecules. However, their potential to deliver bioactive lipid mediators remains largely...Extracellular vesicles (EVs) have emerged as versatile carriers of therapeutic cargo, including nucleic acids, proteins, and small molecules. However, their potential to deliver bioactive lipid mediators remains largely unexplored. Here, we present a novel synthetic biology-based strategy to selectively load EVs with proresolving lipid mediators of the resolvin D- and E-series by coexpressing the resolvin biosynthetic enzymes cyclooxygenase 2, 5-lipoxygenase, and 15-lipoxygenase using a custom-designed multigene expression vector. Human embryonic kidney 293 T cells transfected with the multigene expression vector and cultured in the presence of fatty acid free bovine serum albumen-complexed docosahexaenoic acid, eicosapentaenoic acid, and aspirin produced multiple members of the resolvin D, aspirin-triggered resolvin D-series, and resolvin E1 and E2, along with their biosynthetic precursors, which were subsequently packaged into EVs (referred to as resolvin EVs). Resolvin EVs attenuated neutrophil adhesion to endothelial cells both under static and flow conditions and preserved endothelial barrier integrity by upregulating VE-cadherin. In macrophages, resolvin EVs suppressed nuclear factor κB reporter activity and the release of IL6 and TNFα. Effects of resolvin EVs on endothelial permeability and macrophage activation were abrogated by pharmacologic inhibition of EV uptake using nystatin and cytochalasin D. Furthermore, resolvin EVs enhanced efferocytosis in THP-1-derived macrophages compared to control EVs. Notably, postinjury administration of resolvin EVs attenuated pulmonary inflammation in lipopolysaccharide-treated mice without inducing systemic or pulmonary toxicity. Together, these findings establish a novel, scalable platform for generating resolvin-loaded EVs and highlight their therapeutic potential for acute lung injury and other chronic inflammatory disorders.
Shirakawa K, Sano M, Sakane I
… +11 more, Yamanoi K, Kusumoto D, Goto S, Moriyama H, Daigo K, Sugai K, Katsumata Y, Endo J, Ikuta K, Minato N, Ieda M
Am J Respir Cell Mol Biol
· 2026 May · PMID 41092115
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RATIONALE: Osteopontin, also known as secreted phosphoprotein 1 (SPP1), is a critical mediator of lung fibrosis. However, the cellular sources of SPP1 and the mechanisms by which SPP1-producing cells promote fibrotic pro...RATIONALE: Osteopontin, also known as secreted phosphoprotein 1 (SPP1), is a critical mediator of lung fibrosis. However, the cellular sources of SPP1 and the mechanisms by which SPP1-producing cells promote fibrotic progression remain poorly defined. Objectives To define the functional roles and regulatory mechanisms of SPP1-producing macrophages in lung fibrosis. METHODS: Publicly available single-cell RNA sequencing datasets from patients with idiopathic pulmonary fibrosis were analyzed to investigate macrophage-fibroblast interactions. A bleomycin-induced lung fibrosis model was used in Spp1-enhanced green fluorescent protein (EGFP) knock-in reporter mice to identify Spp1-producing cells in vivo. Multi-omics approaches were applied to characterize profibrotic macrophage populations. The contribution of interleukin-7 receptor (IL7R)signaling in macrophages was evaluated using Il7r fl/fl Csf1r-iCre mice. MEASUREMENTS AND MAIN RESULTS: Single-cell transcriptomic analyses revealed fibrogenic interactions between SPP1-expressing macrophages and fibroblasts in human pulmonary fibrosis. In mice, interstitial macrophages (IMs) were identified as the predominant source of Spp1 in fibrotic lungs and promoted fibrosis by enhancing fibroblast activation. Spp1-EGFP + IMs expanded rapidly, peaking 7 days after bleomycin administration, and subsequently engrafted as inflammatory resident macrophages. Multi-omics analyses demonstrated that these cells produced glycoprotein non-metastatic melanoma protein B (Gpnmb), a profibrotic and proinflammatory mediator. SPP1-producing macrophages expressed IL7R in fibrotic lungs of both humans and mice. Macrophage-specific deletion of Il7r reduced Spp1 and Gpnmb expression and significantly attenuated lung fibrosis. CONCLUSIONS: SPP1-producing interstitial macrophages are key drivers of lung fibrosis through IL-7R-dependent mechanisms. Targeting the IL-7/macrophage/SPP1 axis may represent a promising therapeutic strategy for lung fibrosis.
Blumhagen RZ, Kurche JS, Cool CD
… +8 more, Heinz D, Ma X, Davidson EJ, Fingerlin TE, Huber JP, Dobrinskikh E, Schwartz DA, Yang IV
Am J Respir Cell Mol Biol
· 2026 May · PMID 41091081
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RATIONALE: IPF is an irreversible and progressive type of lung fibrosis that is pathologically characterized as spatially heterogeneous. Despite the identified dominant risk factor for IPF as the gain-of-function MUC5B p...RATIONALE: IPF is an irreversible and progressive type of lung fibrosis that is pathologically characterized as spatially heterogeneous. Despite the identified dominant risk factor for IPF as the gain-of-function MUC5B promoter variant, little is understood for how MUC5B drives lung fibrosis. OBJECTIVES: We used spatial transcriptomics from idiopathic pulmonary fibrosis (IPF) and unaffected control lung tissue to further understand the pathogenesis of MUC5B-driven lung fibrosis. METHODS: We captured 43 fields of view in 15 IPF and 13 controls with and without the MUC5B promoter variant using the CosMx® platform and identified 19 cell types via semi-supervised clustering. MEASUREMENTS AND MAIN RESULTS: MUC5B was ectopically expressed in AT2 cells in controls with the risk variant. We observed a decreased proportion of AT2 cells in controls and an increased proportion of aberrant basaloid cells in IPF associated with the MUC5B risk variant. We identified co-localized expression of MUC5B in respiratory bronchioles with 13 genes including the endoplasmic reticulum (ER) stress marker XBP1 and distal secretory markers SCGB3A1 and SCGB1A1. Experimentally, we demonstrated a direct relationship between MUC5B expression and ER stress in bronchiolar epithelia in vitro and validated the co-expression of MUC5B and XBP1 in the IPF lung. CONCLUSIONS: Based on our results, we conclude that MUC5B injures alveolar and bronchiolar epithelia that results in loss of AT2 cells and an increase in aberrant basaloid cells which initiates ER stress and a secretory phenotype in the terminal respiratory bronchiole, establishing a persistently injured distal airspace.