Pulmonology
Abstract
YAP/TAZ signaling is required for initiation of lung alveolar repair, yet previous studies in idiopathic pulmonary fibrosis (IPF) predicted increased YAP/TAZ signaling in alveolar epithelial cells (AECs). We investigated whether persistent YAP/TAZ AEC signaling contributes to failed epithelial repair and persistent fibrotic remodeling. In IPF lungs, we identified increased YAP+/TAZ+ AECs and increased transcriptional target expression. Pharmacological YAP/TAZ activation in human AEC organoids and in murine AT2 cell organoids generated with genetic YAP/TAZ activation (YTactive) (via deletion of Hippo-kinases Stk3/4), resulted in phenotype shifts into aberrant transitional and airway-like states. Bleomycin injury of YTactive mice resulted in persistent fibrotic remodeling at 28- and 56-days post-bleomycin injury. Gene promoter activity associated with transitional cell markers (Krt19, Hopx, and Runx2) was increased in YTactive AT2 cells. Immunofluorescent staining showed a loss of AT2 associated Cebpa and increased Krt19 in YTactive lineage traced AT2 cells 28 days post-injury. Inhibition of YAP/TAZ using Verteporfin resulted in improved lung repair in YTactive mouse lungs, including restored Cebpa and decreased Krt19+ transitional cells. These findings demonstrate sustained YAP/TAZ activation drives abnormal alveolar repair and persistent fibrotic remodeling. Blocking aberrant persistent YAP/TAZ activity promotes adaptive repair and has potential as a therapeutic strategy for pulmonary fibrosis.
Authors
Isabella P. Gaona, A. Scott McCall, Natalie M. Geis, Arlo C. Colvard, Gianluca T. DiGiovanni, Taylor P. Sherrill, Ujjal K. Singha, David S. Nichols, Ana P. Serezani, Holly E. David, Jean-Philippe Cartailler, Shristi Shrestha, Sergey S. Gutor, Timothy S. Blackwell, Jonathan A. Kropski, Jason J. Gokey
Abstract
Fibroblasts in the lung mesenchyme produce growth factors and extracellular matrix components that guide formation of distal airspaces during the saccular stage of lung development. Inflammation in preterm infants disrupts this process, leading to bronchopulmonary dysplasia (BPD). To examine how mesenchymal inflammation contributes to BPD pathogenesis, we developed a transgenic mouse model (“IKKβTbx4”) in which expression of activated human IκB kinase beta (IKKβ), an upstream activator of NF-κB, was induced in Tbx4 lung enhancer-positive mesenchymal cells during the saccular stage of lung development (postnatal day 0 [PN0] - PN5). Saccular stage IKKβTbx4 mice exhibited a BPD-like phenotype with interstitial thickening and reduced distal airspaces at PN5, progressing to emphysematous enlargement of the distal lung at 2 mo of age. Mesenchymal NF-κB activity upregulated the chemokines CCL2 and CCL7, recruiting CCR2pos monocyte-derived macrophages to the lung. Recruited macrophages disrupted the elastin scaffold and impaired microvascular organization with reductions in CAP2 endothelial cells (aCaps) and pericytes. Blocking CCR2-dependent monocyte recruitment with a small molecule CCR2 antagonist rescued the abnormal lung phenotype. These findings identify mesenchyme-macrophage crosstalk as a mechanism by which inflammation disrupts saccular stage lung development, suggesting a role for this signaling axis in BPD pathogenesis.
Authors
Benjamin C. Crawford, Jessica Chauviere Lee, Bertha C. Elias, Shivangi Dave, Riet van der Meer, Wei Han, Alexandria L. Sharkey, David S. Nichols, Charles Shissias, Lauren Pate, Hayden Tan, Dawn C. Newcomb, Wei Shi, Lawrence S. Prince, Erin J. Plosa, Bradley W. Richmond, Timothy S. Blackwell, Susan H. Guttentag, John T. Benjamin
Abstract
Idiopathic Pulmonary Fibrosis (IPF) is a fatal, aging-related disease characterized by persistent lung fibroblast activation, progressive lung scarring and several vascular abnormalities. We have previously demonstrated that aging-associated vascular dysfunction drives maladaptive endothelial responses to injury and exacerbates lung fibrosis via secretion of pro-fibrotic endothelial-derived factors. However, regulatory mechanisms governing endothelial dysfunction during progressive lung fibrosis remain poorly understood. Here, using preclinical mouse models of progressive lung fibrosis as well as human IPF lungs, we demonstrate that miR-205-5p is overexpressed in lung ECs from fibrotic lungs, and coordinates gene expression programs implicated in endothelial dysfunction and progressive fibrosis. Mechanistically, miR-205-5p induces senescence in lung ECs, mirroring the senescent phenotype of IPF lung ECs. Consistently, conditioned medium derived from lung ECs overexpressing miR-205-5p promotes lung fibroblast activation. Importantly, miR-205-5p inhibition in IPF lung ECs attenuates endothelial senescence and limits paracrine fibroblast activation. Finally, inhibition of miR-205-5p in vivo preserves the pulmonary vascular network and attenuates lung fibrosis progression in aged mice challenged with bleomycin. Collectively, our findings support a novel connection between lung endothelial miR-205-5p, endothelial senescence and pro-fibrotic alteration of the endothelial secretome, and highlight miR-205-5p inhibition as a potential therapeutic intervention for pulmonary fibrosis.
Authors
Giuseppe Muscato, Benjamin B. Roos, Sharonda Harris, Xiaoyu Tracy Cai, Gina Civettini, Enrico Sciacca, Ahmed Raslan, Alessandra Castaldi, Sharon Elliot, Marilyn K. Glassberg, Carlo Vancheri, Daniel J. Tschumperlin, Giovanni Ligresti, Nunzia Caporarello
Abstract
Preclinical studies suggest beneficial effects of GLP-1 agonists in pulmonary arterial hypertension (PAH). This first-in-disease study evaluated acute hemodynamic effects of GLP-1 agonist, exenatide administered i.v. in patients with idiopathic PAH and CTEPH as well as in a PAH rodent model. Seventeen patients (9 idiopathic PAH) received an exenatide infusion during right heart catheterization, which included multisite sampling for circulating metabolites. Acute effects of exenatide were also assessed by cardiac magnetic resonance imaging in monocrotaline (MCT) PAH and control rats. In the clinical study, exenatide was well tolerated, reduced mean pulmonary artery pressure (45 ± 15 mmHg versus 40 ± 18 mmHg), and improved cardiac index (2.1 ± 0.6 L/min versus 2.4 ± 0.9 L/min/m2) and pulmonary vascular resistance (7.8 ± 8.0 WU versus 5.9 ± 5.0 WU) across all patients. Right ventricular (RV) contractility and afterload improved in a subset of patients undergoing pressure-volume measurements. In an exploratory metabolomics analysis, 47 metabolite levels changed after exenatide infusion, predominantly in free fatty acid pathways. Six metabolites with prognostic relevance in PAH within myocardial glycolytic and lipid oxidation pathways were also altered after exenatide. In MCT rats, exenatide improved RV stroke-volume, RV ejection fraction, and RV-arterial coupling. These findings support the further evaluation of exenatide within chronic studies as a potentially novel pulmonary vasodilator therapy.
Authors
Chinthaka B. Samaranayake, Marili Niglas, Nicoleta Baxan, Alexander Kempny, Ali Ashek, Michael Gatzoulis, Laura C. Price, Konstantinos Dimopoulos, Martin R. Wilkins, Stephen Wort, Christopher J. Rhodes, Lan Zhao, Colm McCabe
Abstract
Acute kidney injury (AKI) is a common and fatal complication of severe pneumonia, yet the mechanisms linking pulmonary inflammation to remote kidney injury remain poorly understood. Multicenter cohort data (n = 300) revealed that the incidence of severe pneumonia–associated AKI (SP-AKI) was 53.6%, with a mortality rate of 24.2%. SP-AKI was associated with elevated circulating levels of HMGB1, NETs, and IL-33. Murine experiments demonstrated that alveolar HMGB1 triggers the formation of IL-33–enriched NETs, which migrate to the kidney and activate tubular ST2/NF-κB signaling, driving inflammation and apoptosis. Genetic knockout of IL-33, ST2, or the NET-forming key enzyme PAD4, as well as pharmacological inhibition of HMGB1, IL-33, or NETs, all attenuated lung and kidney injury. Exogenous HMGB1 amplified NET-mediated IL-33 release, establishing a self-sustaining HMGB1/NET/IL-33 feed-forward loop. PAD4 deficiency completely blocked NET generation and disrupted HMGB1/IL-33 signaling. This study identified and validated a damage-associated molecular pattern–driven (DAMP-driven) HMGB1/NET/IL-33 signaling axis that mediates remote kidney injury in SP-AKI, redefining NETs from local effectors to cross-organ pathogenic carriers, thereby providing potential DAMP-targeted therapeutic avenues for SP-AKI.
Authors
Mengqing Ma, Hao Zhang, Weijuan Deng, Xia Du, Mengxing Chen, Dawei Chen, Binbin Pan, Zhaowei Wang, Ting Chen, Caimei Chen, Xin Wan, Changchun Cao
Abstract
Idiopathic pulmonary fibrosis (IPF) is characterized by parenchymal scarring reflecting an imbalance between collagen deposition by myofibroblasts (MFs) and its turnover. Although collagen clearance is essential for fibrosis resolution, this process and its potential for therapeutic modulation in IPF are poorly understood. Here we evaluated internalization of degraded collagen and the role of its requisite endocytic receptor mannose receptor C-type 2 (MRC2), in lung tissue and MFs from IPF patients and bleomycin-injured mice. Fibrotic human and murine lung tissue exhibited an accumulation of degraded collagen, highlighting a failure of its clearance. MFs from fibrotic lung demonstrated a reduced capacity to internalize extracellular degraded collagen, with a concomitant reduction in MRC2 expression and endolysosomal activity. Both diminished collagen uptake and MRC2 expression recovered to baseline levels during spontaneous resolution of bleomycin fibrosis. In vitro treatment of IPF or TGF-β-elicited MFs with a variety of mechanistically distinct agents known to effect phenotypic dedifferentiation restored defective collagen internalization. Although enhanced uptake was MRC2-dependent, it involved increased endolysosomal activity rather than increased MRC2 expression. These results implicate defective MRC2-dependent collagen internalization and endolysosomal function in MFs as important factors contributing to fibrosis that may be therapeutically targeted to promote resolution.
Authors
Natalie M. Walker, Sean M. Fortier, Jennifer Speth, Steven K. Huang, Sergey Gutor, Timothy S. Blackwell, Marc Peters-Golden
Abstract
The lungs have a remarkable capacity to undergo homoeostatic repair and regeneration after injury, which often occurs in patients with acute respiratory distress syndrome (ARDS) and in the single-dose bleomycin mouse model. Fibroblasts are critical mediators of fibrotic disease and RNA sequencing has identified significant heterogeneity within pulmonary fibroblast populations. However, the contribution of distinct fibroblast subsets to the repair process has been understudied compared to their role in fibrosis initiation and progression. Therefore, we sought to define the transcriptional landscape of three phenotypically-defined fibroblast subsets that occupy discrete spatial locations in naïve lungs. Using TdTomato-lineage tracing approaches, we identified and interrogated collagen1a1+ (Col1a1) fibroblasts, perilipin 2+ (Plin2) alveolar fibroblasts, and a-smooth muscle actin+ (Acta2) myofibroblasts during fibrosis development and resolution after single-dose bleomycin. Quantification of fibroblast numbers showed that all three subsets expand during fibrosis and contract towards naïve levels with resolution. Principal component and gene-set enrichment analyses indicated that each subset undergoes major transcriptomic shifts during fibrosis development, converging on a similar pro-fibrotic transcriptional profile. However, during resolution, Plin2+ and Acta2+ fibroblasts revert towards a pre-fibrotic transcriptional state, whereas Col1a1+ fibroblasts acquire a distinct program that suggests suggesting an active role in mediating the repair processes.
Authors
Daniel G. Foster, Nomin Javkhlan, Bart P. Black, Brian E. Vestal, David W.H. Riches, Elizabeth F. Redente
Abstract
CHI3L1, a chitinase-like protein, is implicated in pulmonary fibrosis, yet its mechanisms incompletely understood. In this study, we demonstrated that CHI3L1 coordinates profibrotic macrophage activation and invasive myofibroblast differentiation, and their crosstalk. In vitro, CHI3L1 drove M2-like macrophage polarization as evidenced by increased CD163, CD206, and PD-L1, and amplified TGF-β1-induced fibroblast responses, including myofibroblast transformation, migration, and invasion. Mechanistically, CHI3L1 enhanced TGF-β1 signaling through SMAD, AKT, and ERK pathways, and PD-L1 was required for CHI3L1/TGF-β1-driven myofibroblast transformation. Co-culture studies further demonstrated the ability of CHI3L1 to induce profibrotic macrophage activation that enhanced myofibroblast transformation mediated via a CD44–PD-L1 axis. In vivo, following bleomycin challenge, CHI3L1 transgenic mice exhibited increased PD-L1+ M2 macrophages, PD-L1+/PDGFRα+ fibroblasts, and PD-1+ immune cells compared with wild-type controls. Therapeutically, combined anti-CHI3L1 and anti-PD-1 antibodies, as well as a bispecific anti-CHI3L1-anti-PD-1 antibody, produced greater anti-fibrotic efficacy than monotherapy. These findings demonstrate crosstalk between CHI3L1 and the PD-1/PD-L1 pathway that promotes profibrotic macrophage activation and invasive fibroblast differentiation and support dual targeting of CHI3L1 and PD-1/PD-L1 as a promising therapeutic strategy for pulmonary fibrosis.
Authors
Han-Seok Jeong, Takayuki Sadanaga, Joyce H. Lee, Suchitra Kamle, Bing Ma, Yang Zhou, Sung Jae Shin, Jack A. Elias, Chun Geun Lee
Abstract
Heterozygous TBX4 variants are the second most common genetic cause of pediatric pulmonary hypertension (PH), yet mechanisms underlying TBX4-related lung disease remain poorly understood. This study developed a lung mesenchyme-specific Tbx4 loss-of-function (Tbx4cKO) mouse model that bypasses embryonic lethality to investigate this condition. Adult Tbx4cKO mice demonstrated significantly impaired pulmonary flow acceleration consistent with PH. Three-dimensional analysis of embryonic lungs revealed reduced lobe volumes and decreased distance between pleural edges and muscularized vessels. In adult Tbx4cKO lungs, we identified extensive vascular remodeling characterized by medial thickening and the extension of muscularized arteries into normally non-muscularized subpleural parenchymal zones. Contrary to previous reports suggesting vascular simplification, three-dimensional analysis demonstrated an elaborated pulmonary artery (PA) tree in addition to pathologic wall muscularization. Depletion of a single Tbx5 allele in addition to both Tbx4 alleles exacerbated histologic phenotypes with worsened right ventricular dilation. This model also demonstrated dysregulated airway smooth muscle patterning and prominent subpleural smooth muscle bands, similar to those in human TBX4 syndrome. We identify TBX4 as a critical regulator of smooth muscle differentiation and patterning across multiple lung compartments. Our model recapitulates key features of human TBX4 syndrome and identifies dysregulated smooth muscle differentiation as a potential future therapeutic target.
Authors
Lea C. Steffes, Kaylie A. Chiles, Sehar R. Masud, Aleen Rahman, Madeline Dawson, Csaba Galambos, Maya E. Kumar, Ripla Arora
Abstract
Survival after lung transplantation is limited by chronic, progressive graft failure, termed chronic lung allograft dysfunction (CLAD). Graft-resident mesenchymal cells (MCs) drive CLAD pathogenesis and exhibit stable dysregulated signaling, yet the transcriptomic and epigenomic drivers underlying this fibrogenic transformation remain elusive. We used single-cell multi-omic profiling to characterize gene expression and chromatin accessibility in MCs isolated from lavage fluid of lung transplant recipients with and without CLAD, collected early post-transplantation or after disease onset. MCs obtained after CLAD onset demonstrated a distinct transcriptomic signature compared with non-CLAD controls, enabling classification of disease status at the single-cell level with > 98% accuracy using signature genes. Chromatin accessibility analyses identified enrichment of CCAAT-enhancer-binding protein family transcription factors, specifically CEBPD, in CLAD MCs. Early post-transplant MCs showed minimal accessibility differences, suggesting that CEBPD-associated regulatory changes emerge over time. Integration analyses identified eight MC states and a CLAD-specific shift towards a fibrotic state. CEBPD, SOX4, and FOXP2 were identified as putative regulators of this state with substantial overlap in predicted targets. Targeting CEBPD reversed fibrotic phenotypes of CLAD MCs (decreased ECM expression, contractility, proliferation, and migration). Together, these data provide insights into transcriptomic and epigenomic changes in post-transplant MCs, nominating biomarkers and therapeutic targets.
Authors
Lu Lu, A. Patrick McLinden, Natalie M. Walker, Ragini Vittal, Yichen Wang, Fatemeh Fattahi, Stephen T. Russell, Michael P. Combs, Joshua D. Welch, Vibha N. Lama
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