Pulmonology
Abstract
Mucociliary clearance (MCC) is an innate defense mechanism that normally keeps airways clean but is dysfunctional in cystic fibrosis (CF) and other muco-obstructive pulmonary diseases. Previously we discovered that activating adenyl cyclase in combination with a cholinergic agonist increased MCC velocity (MCCV) synergistically in ex vivo WT and CF ferret and WT piglets. The present study extends and underpins our earlier findings by showing for the first time, in vivo synergistic MCC in WT rats and in CF sheep models and CF rats using inhalable β-adrenergic and cholinergic drugs approved for human use when delivered to the apical surface and a single dose is tolerated by humans. As for mechanisms via ex vivo experiments, we show the combined agonists increased net fluid secretion mainly by stimulating gland secretion and by inhibiting surface absorption, consequently increased ASL depth. They also increased net base secretion and increased ciliary beat frequency. Additional ex vivo and in vitro experiments show that the combined agonists have additive effects when combined with highly effective CF transmembrane conductance regulator (CFTR) modulator therapy (HEMT). The synergistic increase in MCCV induced by this combination of agonists offers therapeutic potential for treating muco-obstructive pulmonary diseases including CF.
Authors
Nam Soo Joo, Susan E. Birket, Johnathan D. Keith, Juan P. Ianowski, Xiaojie Luan, Jacquelyn Spano, Jennifer B. Bollyky, Marissa N. Dobry, Juan R. Sabater, Ryan W. Williams, John F. Engelhardt, Jeffrey J. Wine, Carlos E. Milla
Abstract
Cilia are cellular organelles extruding from the surface of various cell types, serving either sensory or motile functions. Retinitis pigmentosa GTPase regulator (RPGR) variants affect both photoreceptor sensory cilia and airway motile cilia, leading to retinitis pigmentosa (RP) and primary ciliary dyskinesia (PCD), respectively. Not all patients develop PCD, and it remains unclear which RPGR variants predispose patients to PCD. Here, we leverage 2D organoids, super-resolution microscopy, and live-cell imaging to characterize the multiciliated cells (MCCs) from patients with different RPGR variants and CRISPR-modified RPGR KO MCCs. We demonstrate that MCCs with RPGR variants have reduced ciliation, shorter cilia, impaired cilia beat, or cilia beat incoordination, potentially resulting in compromised mucociliary clearance and lung diseases. Moreover, we show that RPGR regulates motile cilia through interfering with F-actin dynamics, evidenced by the undissolved F-actin meshwork in RPGR-deficient MCCs, and the defects can be ameliorated with either Latrunculin A or Y27632 treatment. Though PCD was observed only in patients with variants that affect both isoforms, patients with RPGRORF15 variants also showed cilia and airway anomalies. All RPGR variants affect motile cilia one way or another, and the mechanisms involve the accumulation of apical F-actin.
Authors
Yang Wu, Erika Tavares, Binrun Liang, Wallace B Wee, Vito Mennella, Han-Chao Feng, Jiaying Cao, Pui Yee Wong, Jiayi Zheng, Mu He, Kirk AJ Stephenson, Liran Hanan Hochma, Janice Min Li, Nan-Peng Chen, Sharon D Dell, Elise Heon, ZHEN LIU
Abstract
Airway mucus clearance from the lungs occurs by two widely recognized mechanisms: cilia-mediated clearance and high-velocity airflow-mediated cough clearance. However, a potentially important third mechanism of mucus clearance, referred to as cilia-independent gas-liquid transport (GLT), was proposed based on in vitro model systems to occur during normal tidal breathing, but has largely been overlooked. To investigate the role of tidal breathing airflow rates in mucus clearance, we conducted a series of in vitro and in vivo studies. An in vitro airway culture bead-tracking model demonstrated airflow-dependent mucus transport at tidal breathing flow rates. As with other modes of mucus clearance, GLT was critically dependent on mucus concentration. In vivo studies in cilial beat-deficient mice demonstrated that GLT-mediated mucus clearance occurs during tidal-breathing in the absence of cough, and the rate of GLT mucus clearance was dependent on breathing frequency and body orientation. These studies demonstrated that GLT represents a third mechanism of mucus clearance and likely represents a significant mode of clearance in persons with cilial dysfunction. These findings indicate that increasing breathing rates through exercise, using mucus rehydrating agents or mucolytics, or combining these approaches may restore clinically and physiologically meaningful airway clearance in these patients.
Authors
Siddharth K. Shenoy, Mark Gutay, Ian Brown, Troy D. Rogers, Kane Banner, Nico Olegario, Nicholas Griffin, Henry P. Goodell, Bryan Yoder, David S. Lalush, David A. Edwards, Richard C. Boucher, Barbara R. Grubb, Brian Button
Abstract
Germline loss-of-function folliculin (FLCN) gene mutations cause Birt-Hogg-Dubé (BHD) syndrome, in which pulmonary cysts are present in up to 90% of the patients. The pathogenic mechanisms underlying lung cyst development in BHD are almost entirely unknown because of the limited availability of BHD patient lung samples and the lack of authentic BHD lung disease models. We generated lung mesenchyme–specific and lung epithelium–specific Flcn-knockout mice using a Cre/loxP approach. We found that deletion of Flcn in lung mesenchymal cells, but not in lung epithelial cells, resulted in alveolar enlargement starting from early postnatal life, with evidence of cyst formation in adult mice, resembling the pulmonary disease in human BHD. These changes were associated with increased mechanistic target of rapamycin complex 1 (mTORC1) activity in the lungs of both patients with BHD and Flcn-knockout mice. Attenuation of mTORC1 activity by knocking out Raptor gene (Rptor) or pharmacologic inhibition using rapamycin substantially rescued the pulmonary pathology caused by Flcn deletion in mice. Taken together, these human and mouse data support a model in which mTORC1 hyperactivation drives pulmonary cystic pathology in BHD.
Authors
Ke Cao, Hui Chen, Ling Chu, Hong-Jun Wang, Jianhua Zhang, Yongfeng Luo, Joanne Chiu, Damir Khabibullin, Nicola Alesi, Matthew E. Thornton, Brendan H. Grubbs, Ali Ataya, Nishant Gupta, Francis X. McCormack, Kathryn A. Wikenheiser-Brokamp, Elizabeth P. Henske, Wei Shi
Abstract
Cells exhibit diverse sizes and shapes, tailored for functional needs of tissues. Lung alveoli are lined by large, extremely thin epithelial alveolar type-1 cells (AT1s). Their characteristic morphology is essential for lung function and must be restored after injury. The mechanisms underlying small, cuboidal alveolar type-2 cells (AT2s) differentiation into thin AT1s remain elusive. Here, we demonstrated that AT2s undergo a stepwise morphological transformation characterized by the development of a unique thick microtubule (MT) bundle organization, critical for AT1 morphology. Using AT2 cultures and in vivo genetic loss of function models, we found that MT bundling process occurs in a transitional cell state during AT2 differentiation and was regulated by the TP53/TAU signaling axis. Notably, TAU underwent a linear clustering process, forming beads-on-a-string-like pattern that preceded thick MT-bundle formation. Genetic gain or loss of function of TAU in mouse or human models, prevented the formation of thick MT-bundles, highlighting the critical role of precise TAU levels in generating ultra-thin AT1s. This defect was associated with increased tissue fibrosis following bleomycin-induced injury in vivo. GWAS analysis revealed risk variants in MAPT locus in lung diseases. Moreover, TP53 controlled TAU expression and its loss phenocopied TAU deficiency. This work revealed an unexpected role for TAU in organizing MT-bundles during AT2 differentiation.
Authors
Satoshi Konishi, Khaliun Enkhbayar, Shuyu Liu, Naoya Miyashita, Yoshihiko Kobayashi, Vera Hutchison, Ashna Sai, Pankaj Agarwal, Jonathan Witonsky, Nathan D. Jackson, Max A. Seibold, Jichao Chen, Aleksandra Tata, Purushothama Rao Tata
Abstract
BACKGROUND. Critically ill patients with acute respiratory distress syndrome (ARDS) and sepsis exhibit distinct inflammatory phenotypes with divergent clinical outcomes, but the underlying molecular mechanisms remain poorly understood. These phenotypes, derived from clinical data and protein biomarkers, were associated with metabolic differences in a pilot study. METHODS. We performed integrative multi-omics analysis of blood samples from 160 ARDS patients in the ROSE trial, randomly selecting 80 patients from each latent class analysis-defined inflammatory phenotype (Hyperinflammatory and Hypoinflammatory) with phenotype probability >0.9. Untargeted plasma metabolomics and whole blood transcriptomics at Day 0 and Day 2 were analyzed using multi-modal factor analysis (MEFISTO). The primary outcome was 90-day mortality, with validation in an independent critically ill sepsis cohort (EARLI). RESULTS. Multi-omics integration revealed four molecular signatures associated with mortality: (1) enhanced innate immune activation coupled with increased glycolysis (associated with Hyperinflammatory phenotype), (2) hepatic dysfunction and immune dysfunction paired with impaired fatty acid beta-oxidation (associated with Hyperinflammatory phenotype), (3) interferon program suppression coupled with altered mitochondrial respiration (associated with Hyperinflammatory phenotype), and (4) redox impairment and cell proliferation pathways (not associated with inflammatory phenotype). These signatures persisted through Day 2 of trial enrollment. Within-phenotype analysis revealed distinct mortality-associated pathways in each group. All molecular signatures were validated in the independent EARLI cohort. CONCLUSIONS. Inflammatory phenotypes of ARDS reflect distinct underlying biological processes with both phenotype-specific and phenotype-independent pathways influencing patient outcomes, all characterized by mitochondrial dysfunction. These findings suggest potential therapeutic targets for precise treatment strategies in critical illness. FUNDING. This work is the result of NIH funding.
Authors
Narges Alipanah-Lechner, Lucile Neyton, Pratik Sinha, Carolyn Leroux, Kim Bardillon, Sidney A. Carrillo, Suzanna Chak, Olivia Chao, Taarini Hariharan, Carolyn Hendrickson, Kirsten Kangelaris, Charles R. Langelier, Deanna Lee, Chelsea Lin, Kathleen Liu, Liam Magee, Angelika Ringor, Aartik Sarma, Emma Schmiege, Natasha Spottiswoode, Kathryn Sullivan, Melanie F. Weingart, Andrew Willmore, Hanjing Zhuo, Angela J. Rogers, Kathleen A. Stringer, Michael A. Matthay, Carolyn S. Calfee
Abstract
Authors
Karen Hoffmann, Ulrike Behrendt, Peter Pennitz, Holger Kirsten, Jessica Pohl, Elena Lopez-Rodriguez, Chantal Weissfuss, Jens Kollmeier, Mario Tönnies, Sebastian Brill, Konrad Steinestel, Martin Witzenrath, Werner Wenzel, Christian Zobel, Geraldine Nouailles
Abstract
Influenza and other respiratory viral pathogens remain leading causes of mortality and morbidity. Circadian rhythms play a critical role in regulating immune responses and can confer temporal protection from influenza infection. Here, we investigated whether this protection requires rhythmic function after the initial infection by manipulating environmental cycles. We found that disrupting environmental lighting cues within a critical window of vulnerability abrogated the time-of-day-specific protection. This poor outcome was mediated by a dysregulated immune response, as evidenced by the accumulation of inflammatory monocytes and CD8+ T cells in the lungs and a transcriptomic profile indicative of an exaggerated inflammation. Disruption of the light cycle did not affect outcomes in a clock mutant, indicating that it acts through the host’s circadian clock. Importantly, rhythmic meal timing mitigated the adverse effects of disrupted light cycles, supporting the idea that external cues acting through different body clocks can compensate for one another. Together, these findings underscore the critical interplay between environmental timing cues and endogenous circadian rhythms in determining influenza outcomes and offer translational insight into optimizing care for critically ill patients with respiratory viral infections.
Authors
Oindrila Paul, Thomas G. Brooks, Alisha Shetty, Y. Jane Choi, Martina Towers, Lora J. Assi, James P. Garifallou, Kaitlyn Forrest, Alecia Cameron, Amita Sehgal, Gregory Grant, Shaon Sengupta
Abstract
A greater understanding of chronic lung allograft dysfunction (CLAD) pathobiology, the primary cause of mortality after lung transplantation (LTx), is needed to improve outcomes. The complement system links innate to adaptive immune responses and is activated early post-lung transplantation to form the C3 convertase, a critical enzyme that cleaves the central complement component C3. We hypothesized that LTx recipients with a genetic predisposition to enhanced complement activation have worse CLAD-free survival mediated through increased adaptive alloimmunity. We interrogated a known functional C3 polymorphism (C3R102G) that increases complement activation through impaired C3 convertase inactivation in two independent LTx recipient cohorts. C3R102G, identified in at least one out of three LTx recipients, was associated with worse CLAD-free survival, particularly in the subset of recipients who developed donor-specific antibodies (DSAs). In a mouse orthotopic lung transplant model, impaired recipient complement regulation led to B cell-dependent CLAD pathology despite moderate differences in graft-infiltrating effector T cells. Dysregulated complement regulation promoted intragraft accumulation of memory B cells and antibody-secreting cells, leading to increased local and circulating DSA levels in mice. In summary, genetic predisposition to complement activation is associated with an increased humoral response and worse CLAD-free survival.
Authors
Hrishikesh S. Kulkarni, Laneshia K. Tague, Daniel R. Calabrese, Fuyi Liao, Zhiyi Liu, Lorena Garnica, Nishanth R. Shankar, Xiaobo Wu, Devesha H. Kulkarni, Aayusha Thapa, Dequan Zhou, Yan Tao, Victoria E. Davis, Cory T. Bernadt, Derek E. Byers, Catherine Chen, Howard J. Huang, Chad A. Witt, Ramsey R. Hachem, Daniel Kreisel, John P. Atkinson, John R. Greenland, Andrew E. Gelman
Abstract
The interaction between cells and extracellular matrix (ECM) has been recognized in mechanism of fibrotic diseases. Collagen type VII (collagen VII) is an ECM component which plays an important role in cell-ECM interaction, particularly in cell anchoring and maintaining ECM integrity. Pleural mesothelial cells (PMCs) drive inflammatory reactions and ECM production in pleura. However, the role of collagen VII and PMCs in pleural fibrosis was poorly understood. In this study, collagen VII protein was found increase in pleura of patients with tuberculous pleural fibrosis. Investigation of cellular and animal models revealed that collagen VII began to increase at early stage in pleural fibrotic process. Increase of collagen VII occurred ahead of collagen I and α-SMA in PMCs and pleura of animal models. Inhibition of collagen VII by mesothelial cell-specific deletion of collagen VII gene (WT1-Cre+-COL7A1flox/flox) attenuated mouse experimental pleural fibrosis. At last, it was found that excessive collagen VII changed collagen conformation which resulted in elevation of ECM stiffness. Elevation of ECM stiffness activated integrin/PI3K-AKT/JUN signaling and promoted more ECM deposition, as well as mediated pleural fibrosis. In conclusion, excessive collagen VII mediated pleural fibrosis via increasing extracellular matrix stiffness.
Authors
Qian Li, Xin-Liang He, Shuai-Jun Chen, Qian Niu, Tan-Ze Cao, Xiao-Ling Cui, Zi-Heng Jia, He-De Zhang, Xiao Feng, Ye-Han Jiang, Li-Mei Liang, Pei-Pei Cheng, Shi-He Hu, Liang Xiong, Meng Wang, Hong Ye, Wan-Li Ma
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)