Pulmonology
Abstract
Mechanosensitive feedback between tissue stiffness and cellular contractile forces instructs cell identity. To characterize phosphorylation-mediated mechanosensing, we charted the global phosphoproteome dynamics of primary human lung fibroblasts on fibronectin-coated polydimethylsiloxane substrates of defined stiffness. We identified a key signaling threshold at 2–8 kPa, above which cells activated cytoskeletal remodeling, ECM secretion, and transition to a CTHRC1+/ACTA2+ myofibroblast state, accompanied by stiffness-dependent phosphorylation of the transcription factor NFATC4 at S213/S217. In micro-CT staged pulmonary fibrosis tissues, NFATC4 expression increased progressively, colocalizing with CTHRC1 and ACTA2 in myofibroblasts. Transcription factor regulon inference from a multicohort pulmonary fibrosis atlas confirmed elevated NFATC4 activity in disease fibroblasts, revealing a core 119-gene NFATC4-dependent fibrotic program with CTHRC1 as a top target. Phosphomimetic S213D/S217D mutants drove myofibroblast differentiation on soft substrates independently of TGFB, while phospho-dead S213A/S217A mutants blocked differentiation even on stiff matrix with TGFB, establishing the phospho-switch as both necessary and sufficient. Stiff matrix and TGFB converged on this JNK- and calcineurin-dependent switch to amplify the fibrotic response. This positions NFATC4 S213/S217 as a mechanosensitive checkpoint for CTHRC1+ myofibroblast fate and a candidate therapeutic target in multiorgan fibrosis.
Authors
Safwen Kadri, Laura F. Mattner, Zhen Zeng, Sai Rama Sridatta Prakki, Arun Kumar Verma, Umut Cetin, Christoph H. Mayr, Meshal Ansari, Xin Wei, Sara Asgharpour, Anita A. Wasik, Nikolaus Kneidinger, Mircea-Gabriel Stoleriu, Jürgen Behr, Julien Polleux, Ali Önder Yildirim, Laurens J. De Sadeleer, Wim A. Wuyts, Gerald Burgstaller, Matthias Mann, Martin Mück-Häusl, Herbert B. Schiller
Abstract
Authors
S. Marina Casalino-Matsuda, Vijeeth Guggilla, Catherine A. Gao, Kaitlyn E. DeMeulenaere, Luisa Cusick, Samuel W. Fenske, Zhan Yu, Ziyan Lu, Suchitra Swaminathan, Rogan A. Grant, Maxwell J. Schleck, Murali Prakriya, Sudarshan Hebbar, Kenneth Stauderman, Helen K. Donnelly, Chiagozie I. Pickens, Luisa Morales-Nebreda, The NU SCRIPT Study Investigators, Richard G. Wunderink, Alexander V. Misharin, Benjamin D. Singer, G.R. Scott Budinger
Abstract
Alveolar type 2 cells (AT2s) are critical to lung regeneration, and the absence of large-scale methods to expand AT2s has hindered regenerative medicine efforts. We report a microcarrier-based, large-scale expansion method that was used to generate hundreds of billions of human AT2s. Through our process, expanded AT2s largely retained their phenotype. Furthermore, we showed that culture medium, substrate composition, and stiffness are all critical to the maintenance of AT2s. Finally, we showed that expanded AT2s can differentiate into alveolar type 1–like cells, both in vitro and in a decellularized porcine lung, demonstrating the utility of these cells for lung tissue engineering.
Authors
Lauren K. Rochelle, Rachael S. Van, Richard J. Ottman, Daren F. Robinson, Ashley R. Dockham, Amy K. Smith, Daniel P. Keeley, Jia C. Wang, Darell W. McCoy, Tyler R. Zimmerman, Bryan A. Fioret, Ryan W. Bonvillain, Thomas H. Petersen, Sarah S. Hogan, Laila C. Roudsari
Abstract
Cellular plasticity is a hallmark of cancer, enabling tumor cells to alter identity and evade therapeutic pressure. In invasive mucinous adenocarcinoma of the lung (IMA), NK2 homeobox 1 (NKX2-1) loss triggers a pulmonary to gastric switch marked by aberrant activation of hepatocyte nuclear factor 4 alpha (HNF4α), a master regulator of gastrointestinal/hepatic differentiation. We show that HNF4α promotes IMA growth and activates a gastric pit cell-like program. Loss of HNF4α enables forkhead box A1/A2 (FoxA1/2) transcription factors to bind de novo sites and activate alternative, non-gastric identities in IMA. HNF4α also establishes a mucinous program associated with tolerance to KRAS blockade, and loss of HNF4α enhances response to KRASG12D inhibition. Mechanistically, HNF4α blocks cell cycle exit in drug-tolerant persister cells and promotes activity of the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (NRF2). NRF2 activation partially rescues effects of Hnf4a deletion on KRASG12D inhibition, whereas NRF2 inhibition enhances sensitivity to KRASG12D blockade. Thus, HNF4α is a key regulator of growth, identity, and primary response to KRASG12D inhibition in IMA.
Authors
Headtlove Essel Dadzie, Yangsook Song Green, Soledad Camolotto, Henry U. Arnold, Matthew Gumbleton, Minzhe Guo, Mari Mino-Kenudson, Yutaka Maeda, Benjamin T. Spike, Eric L. Snyder
Abstract
Alveolar type II (AT2) progenitor cell exhaustion and impaired regenerative capacity are key pathogenic hallmarks in idiopathic pulmonary fibrosis (IPF). Nicotinamide adenine dinucleotide (NAD+) functions as a central regulator of cellular energy metabolism. We have reported that downregulation of NAD+-dependent sirtuin signaling contributes to the impaired progenitor function of IPF AT2 cells. In this study, we identified that a key NAD+ biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT), is significantly downregulated in IPF AT2 cells. NAMPT deficiency impaired AT2 renewal and enhanced lung fibrosis through downregulation of SIRT7 and SOD2, which results in increased oxidative stress, mitochondrial dysfunction, accumulated aberrant transitional cells, and impaired differentiation from AT2 to alveolar type I (AT1) cells. A mouse model with AT2-specific deletion of Nampt showed severely impaired AT2 renewal capacity and increased susceptibility to bleomycin lung injury. Activation of NAMPT by small molecule activators promoted IPF AT2 renewal and reversed lung fibrosis in wild-type mice. NAMPT activation is a potential promising therapeutic strategy for restoring AT2 progenitor function and halting or reversing progressive pulmonary fibrosis.
Authors
Xuexi Zhang, Xue Liu, Yujie Qiao, Anas Rabata, Ningshan Liu, Changfu Yao, Tanyalak Parimon, Danica Chen, Cory M. Hogaboam, Peter Chen, Barry R. Stripp, Stephen J. Gardell, Dianhua Jiang, Paul W. Noble, Jiurong Liang
Abstract
Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disorder with limited treatment options. Macropinocytosis is one of the key cellular processes involved in nutrient consumption from the extracellular environment under stress conditions. Here, we studied the role of macropinocytosis in experimental pulmonary fibrosis models. We found that macropinocytosis is increased in human lung fibroblasts (HLFs) derived from IPF patients. The inhibition of macropinocytosis with 5-(n-ethyl-n-isopropyl)-amiloride (EIPA) inhibited profibrotic responses in IPF-derived and TGF-1-stimulated HLFs and reduced pulmonary fibrosis in bleomycin (Bleo)-injured mice. EIPA exerted its antifibrotic effects by regulating amino acid (AA) uptake, mammalian target of rapamycin complex 1 (mTORC1) activation and mesenchyme homeobox1 (MEOX1) expression in activated HLFs. Fittngly, genetic inhibition of macropinocytosis also ameliorated lung fibroblast activation and pulmonary fibrosis in mice. Using IPF-derived precision cut lung slices (PCLS), we observed robust repression of profibrotic gene expression programs in EIPA-treated PCLS across different fibroblast subpopulations. Finally, we found that imipramine (Imi), a tricyclic antidepressant approved by the Food and Drug Administration (FDA), effectively inhibited macropinocytosis and ameliorated profibrotic responses in lung fibroblasts, Bleo-injured mice and IPF-derived PCLS. Taken together, our results suggest macropinocytosis inhibition can be considered as a potential therapeutic strategy to treat pulmonary fibrosis.
Authors
Ivan O. Rosas, Aaron K. McDowell-Sanchez, Santiago Sanchez, Juan D. Cala-Garcia, Alan R. Waich Cohen, Elisa Ruiz-Echartea, Scott A. Ochsner, Daniel C. Kraushaar, Lindsay J. Celada, Dandan Sun, Francesca Polverino, Cristian Coarfa, Neil J. McKenna, Konstantin Tsoyi
Abstract
Integrative multiscale imaging bridges the gap between macroscopic organ structures and microscopic cellular processes, enabling holistic visualization of anatomy and function across scales. Photoacoustic imaging (PAI) leverages melanin’s potent contrast for label-free melanoma detection, yet its potential in lung imaging, challenged by air-tissue acoustic impedance mismatch, remains unexplored for melanoma lung metastases (MLMs). We used hierarchical multiscale PAI, transitioning from whole-body macroscale to localized mesoscale and single-cell-resolution microscale. PAI also guided photoablation interventions in the first and second near-infrared windows, requiring only 10.4 pg intracellular melanin/cell. Bioinformatic analysis of human MLM tissues revealed perturbed signaling pathways compared with normal skin and lung tissues, accounting for dysfunctional melanogenesis to enable label-free PAI with high sensitivity and specificity. Malignant MLM lesions in living mice, resected mouse lungs, and human lungs were delineated with margins closely conforming to histology. The high sensitivity allowed visualization of low-cellularity microsatellite foci down to a few tens of cell clusters, with sufficient penetration in the lungs of mice and Bama minipigs. The multiscale imaging methodology streamlines a theranostic workflow and specifically identifies MLM burden in a progressive, label-free manner, which may aid real-time tumor ablation in the future.
Authors
Wei Xing, Yujia Zhou, Katja Haedicke, Chenyixin Wang, Karla Ximena Vazquez-Prada, Hong Wu, Zhijun Lin, Chrysafis Andreou, Qize Zhang, Ke Shang, Ruoyang Hu, Moritz Kircher, Xingdong Ye, Jan Grimm, Jiang Yang
Abstract
Regulatory T (Treg) cells expressing Forkhead Box P3 (FOXP3) play crucial roles in maintaining immune tolerance and tissue integrity. EZH2, a Histone H3 lysine 27 (H3K27) methyltransferase, is known as a key regulator of Treg cell identity and suppressive function upon activation. Here, we demonstrate that the H3K27 lysine demethylase KDM6B, which catalyzes the opposing reaction to EZH2, was also required for Treg cell identity and function after activation. Treg-specific deletion of Kdm6b impaired tissue Treg cell fate and function. KDM6B was upregulated following T cell antigen receptor (TCR) signaling in Treg cells and contributed to the regulation of Treg-associated gene expression through both direct and indirect mechanisms. A subset of Treg functional genes were direct targets of KDM6B and were co-occupied by FOXP3 at cis-regulatory regions, where KDM6B recruitment limited H3K27me3 accumulation. More broadly, KDM6B-dependent H3K27 demethylation facilitated Treg gene expression programs that supported tissue Treg homeostasis.
Authors
Minghong He, Beisi Xu, Pria G. Bose, Morgan J. McCullough, Rani S. Sellers, Xinying Zong, Wenjie Qi, Brianna L. Banten, Miriya K. Tune, Matthew P. Zimmerman, Genevieve N. Mullins, Brian C. Miller, J. Justin Milner, Jason K. Whitmire, Ageliki Tsagaratou, Karl B. Shpargel, Claire M. Doerschuk, Yong-Dong Wang, Jacob A. Steele, Shondra M. Pruett-Miller, Yongqiang Feng, Jason R. Mock
Abstract
Authors
Lawrence W. Rasmussen, Deepali Luthra, Diego Moncada-Giraldo, Crystal Lewis, Yixel M. Soto-Vazquez, Zhuo Li, Buqu Hu, Brian S. Dobosh, Delores A. Stacks, Jonathan L. Koff, Amit Gaggar, Rabindra Tirouvanziam, Camilla Margaroli
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
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)