The cell type specific sequences of transcriptional programs during lung regeneration have remained elusive. Using time-series single cell RNA-seq of the bleomycin lung injury model, we resolved transcriptional dynamics for 28 cell types. Trajectory modeling together with lineage tracing revealed that airway and alveolar stem cells converge on a unique Krt8 + transitional stem cell state during alveolar regeneration. These cells have squamous morphology, feature p53 and NFkB activation and display transcriptional features of cellular senescence. The Krt8+ state appears in several independent models of lung injury and persists in human lung fibrosis, creating a distinct cell–cell communication network with mesenchyme and macrophages during repair. We generated a model of gene regulatory programs leading to Krt8+ transitional cells and their terminal differentiation to alveolar type-1 cells. We propose that in lung fibrosis, perturbed molecular checkpoints on the way to terminal differentiation can cause aberrant persistence of regenerative intermediate stem cell states.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor prognosis and limited therapeutic options. The incidence of IPF increases with age, and ageing-related mechanisms such as cellular senescence have been proposed as pathogenic drivers. The lung alveolar epithelium represents a major site of tissue injury in IPF and senescence of this cell population is probably detrimental to lung repair. However, the potential pathomechanisms of alveolar epithelial cell senescence and the impact of senolytic drugs on senescent lung cells and fibrosis remain unknown. Here we demonstrate that lung epithelial cells exhibit increasedP16andP21expression as well as senescence-associated β-galactosidase activity in experimental and human lung fibrosis tissue and primary cells.Primary fibrotic mouse alveolar epithelial type (AT)II cells secreted increased amounts of senescence-associated secretory phenotype (SASP) factorsin vitro, as analysed using quantitative PCR, mass spectrometry and ELISA. Importantly, pharmacological clearance of senescent cells by induction of apoptosis in fibrotic ATII cells orex vivothree-dimensional lung tissue cultures reduced SASP factors and extracellular matrix markers, while increasing alveolar epithelial markers.These data indicate that alveolar epithelial cell senescence contributes to lung fibrosis development and that senolytic drugs may be a viable therapeutic option for IPF.

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic interstitial lung disease (ILD) characterized by lung tissue scarring and high morbidity. Lung epithelial injury, myofibroblast activation, and deranged repair are believed to be key processes involved in disease onset and progression, but the exact molecular mechanisms behind IPF remain unclear. Several drugs have been shown to slow disease progression, but treatments that halt or reverse IPF progression have not been identified. Ex vivo models of human lung have been proposed for drug discovery, one of which is precision-cut lung slices (PCLS). Although PCLS production from IPF explants is possible, IPF explants are rare and typically represent end-stage disease. Here we present a novel model of early fibrosis-like changes in human PCLS derived from patients without ILD/IPF using a combination of profibrotic growth factors and signaling molecules (transforming growth factor-β, tumor necrosis factor-α, platelet-derived growth factor-AB, and lysophosphatidic acid). Fibrotic-like changes of PCLS were qualitatively analyzed by histology and immunofluorescence and quantitatively by water-soluble tetrazolium-1, RT-qPCR, Western blot analysis, and ELISA. PCLS remained viable after 5 days of treatment, and fibrotic gene expression (FN1, SERPINE1, COL1A1, CTGF, MMP7, and ACTA2) increased as early as 24 h of treatment, with increases in protein levels at 48 h and increased deposition of extracellular matrix. Alveolar epithelium reprogramming was evident by decreases in surfactant protein C and loss of HOPX. In summary, using human-derived PCLS, we established a novel ex vivo model that displays characteristics of early fibrosis and could be used to evaluate novel therapies and study early-stage IPF pathomechanisms.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo)fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored. Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF. Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies. Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-β in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A. Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Repetitive injury and reprogramming of the lung epithelium are thought to be critical drivers of disease progression, contributing to fibroblast activation, extracellular matrix remodeling, and subsequently loss of lung architecture and function. To date, Pirfenidone and Nintedanib are the only approved drugs known to decelerate disease progression, however, if and how these drugs affect lung epithelial cell function, remains largely unexplored. We treated murine and human 3D ex vivo lung tissue cultures (3D-LTCs; generated from precision cut lung slices (PCLS)) as well as primary murine alveolar epithelial type II (pmATII) cells with Pirfenidone or Nintedanib. Murine 3D-LTCs or pmATII cells were derived from the bleomycin model of fibrosis. Early fibrotic changes were induced in human 3D-LTCs by a mixture of profibrotic factors. Epithelial and mesenchymal cell function was determined by qPCR, Western blotting, Immunofluorescent staining, and ELISA. Low μM concentrations of Nintedanib (1 μM) and mM concentrations of Pirfenidone (2.5 mM) reduced fibrotic gene expression including Collagen 1a1 and Fibronectin in murine and human 3D-LTCs as well as pmATII cells. Notably, Nintedanib stabilized expression of distal lung epithelial cell markers, especially Surfactant Protein C in pmATII cells as well as in murine and human 3D-LTCs. Pirfenidone and Nintedanib exhibit distinct effects on murine and human epithelial cells, which might contribute to their anti-fibrotic action. Human 3D-LTCs represent a valuable tool to assess anti-fibrotic mechanisms of potential drugs for the treatment of IPF patients.