Orsini EM, Boulton AM, Abraham S
… +12 more, Brown A, Ricci H, Ahuja A, Pant B, Snyder CM, Reinhardt A, Attaway AH, Musich R, Grove LM, Olman MA, Vachharajani V, Scheraga RG
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42345538
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RATIONALE: Respiratory pathogens, such as Pseudomonas aeruginosa damage the alveolar-capillary barrier leading to lung injury and stiffness. Lung stiffness is a key macrophage signal for bacterial clearance, but it remai...RATIONALE: Respiratory pathogens, such as Pseudomonas aeruginosa damage the alveolar-capillary barrier leading to lung injury and stiffness. Lung stiffness is a key macrophage signal for bacterial clearance, but it remains unknown how stiffness-sensing mechanosensitive ion channels in macrophages are regulated during pneumonia. Macrophage Piezo1 is critical to bacterial clearance in experimental pneumonia in vivo; however, identification of putative matrix-derived signals and the mechanism of their effects remain to be determined. OBJECTIVES: We investigated the role of P. aeruginosa virulence factors on Piezo1 activity in macrophages on infected lung matrix stiffness. METHODS: Using bone-marrow derived macrophages, we measured Piezo1 abundance and function and bacterial clearance in response to P. aeruginosa virulence factors on pathophysiologic range lung stiffnesses and standard tissue culture conditions. MEASUREMENTS AND MAIN RESULTS: To our knowledge, our work is the first to show that during pneumonia, transcription of the mechanosensitive ion channel Piezo1 is increased in macrophages by the NF-κB transcription factor, p65, through its signaling adaptor protein, MyD88, leading to increased Piezo1 Ca2 + channel activity. Piezo1 mRNA abundance is increased in association with open chromatin at the Piezo1 promoter in macrophages. The enhanced level of Piezo1 increases the abundance of transcription factor EB (Tfeb) resulting in lysosome biogenesis and stiffness-dependent phagolysosome maturation, a critical step for macrophage bacterial clearance. CONCLUSIONS: Our data support the mechanism whereby transcription of macrophage Piezo1 is enhanced by p65 to augment bacterial clearance on an injured, stiffened lung matrix during pneumonia. Therefore, Piezo1 is a future therapeutic target against pneumonia-induced lung injury.
Zou X, Huang Q, Cao C
… +6 more, Shen S, Shao P, Zhang X, Bian Y, Xu W, Wu J
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42334931
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While human genetics implicate the interleukin-6 (IL6) signaling pathway as a potential therapeutic target in chronic obstructive pulmonary disease (COPD), its functional role in pulmonary macrophages remains paradoxical...While human genetics implicate the interleukin-6 (IL6) signaling pathway as a potential therapeutic target in chronic obstructive pulmonary disease (COPD), its functional role in pulmonary macrophages remains paradoxical given its established role in promoting cholesterol efflux. Here, integrating single-cell transcriptomics of human COPD lungs with mechanistic studies, we resolve this paradox by identifying a pathogenic rewiring of the IL6/STAT3 pathway. We discovered a disease-enriched macrophage subpopulation exhibiting co-activation of IL6/STAT3 signaling, cholesterol biosynthesis, and inflammatory pathways. In a murine COPD model, chronic cigarette smoke (CS) exposure recapitulated this immunometabolic phenotype. We defined a linear pathway wherein CS-induced IL6 activates STAT3, which directly transactivates the sterol regulatory element-binding protein 2 (SREBP2) to drive de novo cholesterol synthesis. This SREBP2-dependent cholesterol accumulation was essential for NLRP3 inflammasome activation and pro-inflammatory cytokine release. In vitro, pharmacological inhibition of STAT3 or SREBP2, as well as IL6 silencing, disrupted this cascade, suppressing cholesterol-driven inflammation. Critically, in vivo macrophage-specific Il6 knockdown attenuated pulmonary inflammation, cholesterol accumulation, and emphysema development by disrupting the entire IL6/STAT3/SREBP2 axis. Thus, we define the IL6/STAT3/SREBP2 axis as a core immunometabolic driver of COPD pathogenesis, which directly couples CS exposure to sustained macrophage inflammation via pathological cholesterol synthesis, thereby providing a mechanistic basis for targeting this druggable pathway.
Celada SI, Ryter SW, Baughman RP
… +2 more, Van Kaer L, Celada LJ
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42320023
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Interstitial lung disease (ILD) disproportionately affects older adults, yet the contribution of immunosenescence to disease pathogenesis remains poorly understood. In fibrotic ILDs (fILDs), CD8 + T cells accumulate in f...Interstitial lung disease (ILD) disproportionately affects older adults, yet the contribution of immunosenescence to disease pathogenesis remains poorly understood. In fibrotic ILDs (fILDs), CD8 + T cells accumulate in fibrotic regions, where they may drive disease by promoting cytotoxic inflammation, impairing epithelial repair, and sustaining senescence. CD8 + T cell exhaustion (CD8 + Tex) has also emerged as a hallmark of chronic lung disease, although its relationship to immunosenescence in ILD remains unclear. Here, we highlight the heterogeneity among CD8 + T cells in fILD, including effector- and senescent-like subsets, and identify programmed death (PD)-1 as a protective "brake" limiting tissue-damaging immunopathology. Functional profiling indicates that CD8 + T cells in fILD exhibit features consistent with ex-tissue-resident and effector memory CD8 + T cell subsets. Recent evidence from severe and post-acute viral injury demonstrates that PD-1hiCD8 + T cells balance protective immunity with restraint of fibrotic sequelae while also driving maladaptive epithelial remodeling through expansion of dysplastic basal-like cells and impaired alveolar regeneration. These observations suggest that CD8 + T cells in fILD may directly regulate the balance between tissue repair and fibrosis. Our recent studies have shown that the antifibrotic effects of pirfenidone and nintedanib may arise from selective modulation of profibrotic programs in CD8+/CD4 + T cells, lymphoid endothelial cells and dendritic cells. Collectively, these findings support a paradigm shift in which fILD reflects a dysregulation of local immune networks rather an inevitable consequence of aging. Most importantly, these networks are modifiable, offering opportunities for early detection, patient stratification, and stage-specific immunomodulatory interventions, with maladaptive memory CD8 + T cell functional states serving as potential biomarkers of disease susceptibility.
Calyeca J, Liu L, Li K
… +7 more, Drake K, Najibi H, Dharmadhikari S, Carraro G, Reynolds SD, Stripp BR, Chiang T
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42301704
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A pseudostratified epithelium lines the upper airways and is maintained by stem cells that express a basal cell phenotype. These airway stem cells share molecular and functional similarities with other tissue stem cells,...A pseudostratified epithelium lines the upper airways and is maintained by stem cells that express a basal cell phenotype. These airway stem cells share molecular and functional similarities with other tissue stem cells, particularly those that are involved in development or postnatal maintenance of the esophagus and epidermis. Thus, analysis of gene function in the airway is complicated by the potential for lethal non-respiratory phenotypes. Conditional genetic approaches employing cell type-specific and/or temporally controlled expression systems provide some options, but limitations remain. To overcome these concerns, we tested a novel microsurgical approach in which a chimeric airway is created by orthotopic transplantation of transgenic mouse tissue into the trachea. We hypothesized that precise temporal and spatial control of gene expression in the pseudostratified tracheal epithelium would prevent life-threatening collateral tissue injury and allow analysis of essential genes. First, we demonstrated that the graft was revascularized allowing parenteral administration of tamoxifen. Second, we transplanted tracheal tissue from Krt5CreERT2; Rosa26mTmG or Col1a2CreERT2; Rosa26mTmG donors into wild type mice and established that tamoxifen-dependent recombination in basal cells or fibroblasts was highly efficient and restricted to the graft. Finally, we assessed the feasibility of knocking out an essential gene, Itgb1, by transplanting tracheal segments from Krt5CreERT2; Rosa26mTmG; Itgb1flox/flox transgenic mice into wild type mice. We demonstrated highly selective recombination in the graft and long-term survival. We conclude that the chimeric airway model allows analysis of essential gene function in the airway and has the potential to be a versatile tool for preclinical testing of targeted gene therapies.
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42299646
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Benzodiazepines are widely used for sedation and anxiolysis but may also exert unrecognized beneficial effects on airway smooth muscle (ASM) tone in clinical settings where they are routinely administered. While their pr...Benzodiazepines are widely used for sedation and anxiolysis but may also exert unrecognized beneficial effects on airway smooth muscle (ASM) tone in clinical settings where they are routinely administered. While their primary mechanism involves GABAA receptor (GABAAR) modulation in the central nervous system, their direct effects on peripheral airways remain poorly understood. Using mouse precision-cut lung slices (PCLS) and video phase-contrast microscopy, we show that diazepam, lorazepam, and midazolam induce robust, reversible, and dose-dependent relaxation of methacholine (MCh)-constricted peripheral airways with IC50 of 12, 22, and 23 µM, respectively. We selected diazepam to further investigate the cellular and molecular mechanisms underlying airway relaxation. GABAAR antagonists picrotoxin and flumazenil failed to block relaxation, and diazepam inhibited MCh-induced airway constriction even in the absence of extracellular Ca2+, consistent with a GABAAR-independent mechanism. Diazepam-induced relaxation correlated with strong inhibition of concurrent intracellular Ca2+ oscillations in ASM cells. Diazepam inhibited airway constriction and Ca2+ transients elicited by intracellular IP3 uncaging but not by caffeine, indicating specific modulation of IP3-receptor mediated Ca2+ signaling. Furthermore, low concentrations of diazepam (1 µM) significantly prolonged terbutaline-induced airway relaxation, mirroring the effects of selective phosphodiesterase 4 (PDE4) inhibitor rolipram. Diazepam inhibited purified PDE4D2 activity and potentiated forskolin-induced cAMP accumulation in human ASM cells. These findings indicate that diazepam produces bronchodilation at least in part through direct PDE4 inhibition and modulation of IP3 receptor-mediated Ca2+ oscillations in ASM. The synergistic interaction between benzodiazepines and β2-adrenoreceptor agonists at therapeutic concentrations has important clinical implications for bronchodilation in high-risk patients.
Vaghasiya J, Sikarwar A, Dalvand A
… +10 more, Basu S, Bagan A, Varghese N, Duaqui AA, Pandey D, Mishra S, Ravandi A, Pascoe CD, Halayko AJ, Canadian Respiratory Research Network
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42296335
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Understanding mechanisms for β2 adrenergic receptor (β2AR) insensitivity in asthmatics is incomplete. We discovered that the accumulation of oxidized phosphatidylcholines (OxPC) in the lung correlates with airway hyperre...Understanding mechanisms for β2 adrenergic receptor (β2AR) insensitivity in asthmatics is incomplete. We discovered that the accumulation of oxidized phosphatidylcholines (OxPC) in the lung correlates with airway hyperresponsiveness, and OxPC induce contraction and cytokine synthesis in human airway smooth muscle (ASM) cells. Here, we test whether OxPC impair β2AR agonist bronchodilator responses and related mechanism(s) for β2AR insensitivity. Using tracheal rings from BALB/c mice, we assayed the effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on β2AR agonist (isoproterenol (Iso))-induced relaxation of methacholine (MCh)-contracted airways. OxPAPC pre-exposure attenuated Iso-induced airway relaxation, increasing Iso EC50 4.3-fold and reducing maximum relaxation by 12.2%. OxPAPC did not affect adenylyl cyclase (AC) mediated relaxation induced by forskolin. OxPAPC significantly inhibited Iso-induced bronchodilation in murine precision cut lung slices (PCLS). Intranasal OxPAPC pre-challenge significantly inhibited albuterol-mediated suppression of MCh-induced respiratory resistance in BALB/c mice. In cultured human ASM, OxPAPC dose-dependently inhibited Iso- (but not forskolin) induced phosphorylation of the Protein Kinase A substrate VASP by up to 53%. Live cell cADDis assay for intracellular cAMP confirmed that OxPAPC dose-dependently reduces Iso-elicited cAMP generation up to 50%. OxPAPC did not reduce the abundance of cell surface HA-tagged human β2AR in HEK-293 cells. However, inhibition of Protein Kinase C prevented the suppressive effects of OxPAPC on both Iso-induced relaxation of murine tracheal rings, and cAMP signaling in cultured HASM cells. In summary, OxPAPC impairs β2AR agonist induced bronchodilation by PKC-dependent suppression of β2AR-mediated cAMP signaling upstream of AC in ASM cells. These observations reveal a new mechanism for bronchodilator insensitivity.
Ganzleben I, Zhang J, Lane CP
… +6 more, Chow Ming Chia D, Zhou H, Segal A, Shih AR, Yun SH, Medoff BD
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42286788
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Pathologically altered physical properties of the extracellular matrix are increasingly recognized as an active player in fibrosis inception and progression. Fibroblasts produce an increasingly stiff matrix, which in tur...Pathologically altered physical properties of the extracellular matrix are increasingly recognized as an active player in fibrosis inception and progression. Fibroblasts produce an increasingly stiff matrix, which in turn perpetuates fibroblast activation and transdifferentiation into myofibroblasts. Yet, there is still an unmet need for accessible technologies allowing detailed characterization of tissue stiffness to study this relationship. In our current study, we demonstrate the feasibility of a Brillouin microscopy-based quantitative spatial stiffness measurement for the characterization of lung tissue samples with variable degrees of fibrosis. First, we validated our Brillouin microscopy setup using hydrogels with defined levels of stiffness. We then devised a workflow to measure native murine lung tissue cryosections and successfully characterized stiffness levels of lung tissue sections with fibrosis and in non-fibrotic controls. Finally, we successfully applied our setup to fibrotic human lung tissue sections. In conclusion, our proof-of-concept study shows the feasibility of quantitative spatial lung tissue stiffness assessment by Brillouin microscopy in a setup that can easily be integrated into common research workflows. Stiffness measurements through label- and contact-free Brillouin microscopy in combination with techniques such as immunofluorescence staining, RNA-scope, spatial transcriptomics, spatial proteomics, and others have great potential to generate new insights into the mechanobiology of pulmonary fibrosis and a multitude of other diseases in the future.
Greaney AM, Orr JC, Moore SKL
… +3 more, Ikonomou L, Weiss DJ, Ryan AL
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42286350
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Over the past two decades there have been remarkable advances in stem cell biology, bioengineering, and lung regenerative research, transforming our understanding of pulmonary biology from development to repair, and dise...Over the past two decades there have been remarkable advances in stem cell biology, bioengineering, and lung regenerative research, transforming our understanding of pulmonary biology from development to repair, and disease. Strategies using endogenous lung progenitor cells, pluripotent stem cell technologies, and engineered tissue platforms have become central tools for interrogating lung biology. Major breakthroughs have included the identification of diverse cell populations that coordinate lung homeostasis and repair, facilitated by the extensive adoption of single cell, multiomic and spatialomics approaches. Simultaneous progress in biomaterials, organoid systems, decellularized lung scaffolds, and lung-on-chip platforms has uncovered how extracellular matrix composition, mechanical forces, and tissue architecture contribute to the regulation of cell fate and function. These advances have enabled increasingly physiologically relevant in vitro, and ex vivo models while informing tissue engineering strategies aimed ultimately at functional lung replacement. Translation toward the clinic has advanced through both cell-based and cell-free therapeutic strategies. Early efforts focused largely on mesenchymal stromal cell-based approaches and extracellular vesicles, which have demonstrated safety and context-dependent efficacy in inflammatory lung diseases, alongside emerging preclinical evidence of functional engraftment of induced pluripotent stem cell-derived lung lineages. The past twenty years of progress, captured at the 20th Anniversary Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, highlights the power of interdisciplinary collaboration in advancing lung regeneration from foundational discovery toward therapeutic reality.
Jendzjowsky N, Kuo CS, Reznikov LR
… +24 more, Chen Z, Li P, Prescott S, Liu Y, Seeholzer L, Tschumperlin DJ, Taylor-Clark TE, Rajagopal J, Haber AL, Noël A, Kotas M, Drake M, Pabelick C, Rosenblatt J, Mouradian G, Su Y, Gomez C, Lin Q, Lu Q, Mongodin EF, Vuga L, Zhou G, Prakash YS, Sun X
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42265991
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The lung, one the largest branching organs in our body and vital for survival at first breath, is commonly known as the gas-exchange organ. Recent novel findings on lung sensing cells, molecules, and lung-brain crosstalk...The lung, one the largest branching organs in our body and vital for survival at first breath, is commonly known as the gas-exchange organ. Recent novel findings on lung sensing cells, molecules, and lung-brain crosstalk in the emerging discipline of interoception have highlighted the consideration of the lung as a sensory organ. Acknowledging the central importance of lung sensing to human health and disease, NHLBI convened a workshop to synthesize the past, current, and future of lung sensing research, with in-person presentations by ∼20 interdisciplinary experts and a large virtual audience. Here, we highlight the key topics discussed and summarize a blueprint for the future in this important field.
Karunamurthy P, Morin J, Tangamornsuksan W
… +8 more, Macleod M, Kuhar E, Ali S, Levings M, Jahandideh F, Stewart DJ, Fergusson DA, Lalu MM
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42246928
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Acute respiratory distress syndrome (ARDS) is a life-threatening inflammatory lung injury. Regulatory T-cells (Tregs) and their extracellular vesicles (Treg-EVs) possess immunomodulatory properties that may be therapeuti...Acute respiratory distress syndrome (ARDS) is a life-threatening inflammatory lung injury. Regulatory T-cells (Tregs) and their extracellular vesicles (Treg-EVs) possess immunomodulatory properties that may be therapeutic in ARDS. However, evidence is scattered across individual studies, hindering the assessment of their safety and efficacy. To address this gap, we applied a systematic review (SR) methodology, a common approach in clinical research that has increasingly been recognized for its value in preclinical evidence synthesis. Specifically, we synthesized studies of Tregs/Treg-EVs in animal models of acute lung injury (ALI) or patients with ARDS. Importantly, we provide explanatory text throughout for readers less familiar with SRs. The primary preclinical and clinical outcomes were histological lung injury and mortality, respectively. Study selection and data extraction were performed in duplicate. Twenty-two preclinical and two clinical studies met the inclusion criteria, all using whole-cell Tregs. Meta-analysis of animal studies demonstrated that, compared to controls, Treg treatment significantly reduced histological lung injury (≤7 days: standardized mean difference (SMD) = -2.06 [95% confidence interval (CI): -2.96, -1.15]; >7 days: SMD = -2.18 [95% CI: -3.28, -1.08]). Tregs also reduced bronchoalveolar lavage fluid pro-inflammatory cytokines, total protein, total cells, and neutrophil counts. Clinically, early-phase study designs precluded meta-analysis; however, safety and tolerability of Tregs for ARDS were supported. Identified mechanisms underlying Treg effects included immunomodulation, cytokine regulation, and epigenetic pathways. Our review demonstrates the utility of formal preclinical evidence synthesis and supports the therapeutic potential of Tregs. Further investigations are justified to refine Tregs as a cellular therapy for ARDS.
Huang R, Feng Z, Zhang W
… +14 more, Zhong Y, Su G, Lai X, Li H, Fang Z, Zhao Z, Zhang M, Li S, Yang R, Gao X, Xiao H, Jiang Y, Duan J, Lai T
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42244110
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Macrophages play central role in driving airway inflammation during asthma pathogenesis, though the molecular mechanism governing their function remains incompletely understood. Here, we demonstrate that lysine acetyltra...Macrophages play central role in driving airway inflammation during asthma pathogenesis, though the molecular mechanism governing their function remains incompletely understood. Here, we demonstrate that lysine acetyltransferase 8 (KAT8) plays a critical role in papain-induced neutrophilic airway inflammation through post-translational modification of poly(C)-binding protein 1 (PCBP1). We show that papain exposure significantly upregulates KAT8 expression in lung macrophages both in vivo and in vitro. Myeloid-specific Kat8 deficiency conferred protection against papain-induced airway inflammation, reducing CXCL1 and CXCL2 production and consequent neutrophil recruitment. Mechanistically, KAT8 interacted with PCBP1 via its C2HC zinc finger domain and acetylated it at lysine 119 (K119), an essential modification for CXCL1 and CXCL2 secretion. Moreover, KAT8 enhanced M1 macrophage polarization in papain-exposed mice. Importantly, pharmacological inhibition of KAT8 significantly attenuated neutrophil infiltration and allergic airway inflammation in vivo. Our findings establish KAT8 as a pivotal regulator of neutrophilic airway inflammation through PCBP1 acetylation and highlight the KAT8-PCBP1 axis as a promising therapeutic target for severe asthma.
Bodmer NK, Choudhury M, Mirza H
… +6 more, Pradhan S, Yin Y, Mecham RP, Brody SL, Ornitz DM, Koenitzer JR
Am J Respir Cell Mol Biol
· 2026 Jun · PMID 42236262
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The identification of clinically predictive serum biomarkers for pulmonary fibrosis is a significant challenge and important goal. Multiple recent proteomic biomarker studies have identified latent transforming growth fa...The identification of clinically predictive serum biomarkers for pulmonary fibrosis is a significant challenge and important goal. Multiple recent proteomic biomarker studies have identified latent transforming growth factor binding protein-2 (LTBP2) as a circulating factor associated with disease progression in fibrotic lung diseases in humans (including IPF), but its role in the development of fibrosis is incompletely defined. LTBP2 competes with the large latent transforming growth factor-beta (TGFβ) complex (LLC) for binding to the N-terminus of fibrillin and is thought to promote the release of active TGFβ. We hypothesized that LTBP2 deficiency would promote LLC sequestration in matrix and reduce TGFβ signaling. We recently reported an LTBP2 knockout (Ltbp2-/-) mouse with no baseline lung abnormalities. Here we show that Ltbp2-/- mice exposed to either bleomycin or silica have a significant reduction in fibrosis compared to wild type controls. Consistent with reduced fibrosis, after bleomycin Ltbp2-/- mouse lungs have reduced TGFβ signaling and isolated fibroblasts from Ltbp2-/- mice exhibit impaired migration in an in vitro wound closure assay. Transcriptomic analysis of bleomycin-treated control and Ltbp2-/-mouse lung tissue identified multiple LTBP2-regulated genes, including the lncRNA antisense of IGFR2 non-coding RNA (Airn) which has reported antifibrotic effects. Interestingly, we also observed that Ltbp2-/-mice had impaired epithelial repair after bleomycin treatment, a phenotype that also occurred in a naphthalene model of club cell injury. These findings provide evidence that LTBP2 is profibrotic and facilitates TGFβ signaling but is also required for normal airway epithelial repair.