Search

Although alveolar epithelial cells type II (AEC2) are chronically injured in Idiopathic Pulmonary Fibrosis (IPF), they contribute to epithelial regeneration in IPF.We hypothezised that Notch signaling may contribute to AEC2 proliferation, de-differentiation characterized by loss of surfactant processing machinery and lung fibrosis in IPF Methods: We applied microarray analysis, kinome profiling, flow cytometry, immunofluorescence analysis, western blotting, qPCR, proliferation- and surface activity- analysis to study epithelial differentiation, proliferation and matrix deposition in vitro (AEC2 cell lines, primary murine/human AEC2), ex vivo (human IPF-derived precision cut lung slices; IPF-PCLS) and in vivo (bleomycin-, pepstatin-application, Notch1 ICD overexpression) Measurements and Main Results: We document here extensive surfactant protein (SP)-B/C processing defects in IPF AEC2, due to loss of Napsin A, resulting in increased intra-alveolar surface tension and alveolar collapse and induction of ER-stress in AEC2. In vivo pharmacological inhibition of Napsin A results in the development of AEC2 injury and overt lung fibrosis. We also demonstrate that Notch1 signaling is already activated early in IPF and determines AEC2 fate by inhibiting differentiation (reduced lamellar body compartment, reduced capacity to process hydrophobic SP) and by causing increased epithelial proliferation and development of lung fibrosis, putatively via altered JAK/Stat signaling in AEC2. Conversely, inhibition of Notch signaling in IPF-PCLS improved surfactant processing capacity of AEC2s and reversed fibrosis.Notch1 is a central regulator of AEC2 fate in IPF. It induces alveolar epithelial proliferation, loss of Napsin A and of surfactant proprotein processing and it contributes to fibroproliferation.

Idiopathic lung fibrosis (IPF) is a fatal lung disease characterized by chronic epithelial injury and exhausted repair capacity of the alveolar compartment, associated with the expansion of cells with intermediate alveolar epithelial cell (AT2) characteristics. Using SftpcCreERT2/+: tdTomatoflox/flox mice, we previously identified a lung population of quiescent injury activated alveolar epithelial progenitors (IAAPs) marked by low expression of the AT2 lineage trace marker tdTomato (Tomlow), and characterized by high levels of Pd-l1 (Cd274) expression. This led us to hypothesize that a population with similar properties exists in the human lung. To that end, we used flow cytometry to characterize the CD274 cell surface expression in lung epithelial cells isolated from donor and end-stage IPF lungs. The identity and functional behavior of these cells were further characterized by qPCR analysis, in vitro organoid formation, and ex vivo precision-cut lung slices (PCLS). Our analysis led to the identification of a population of CD274pos cells expressing intermediate levels of SFTPC, which was expanded in IPF lungs. While donor CD274pos cells initiated clone formation, they did not expand significantly in 3D organoids in AT2-supportive conditions. However, an increased number of CD274pos cells was found in cultured PCLS. In conclusion, we demonstrate that, similar to the IAAPs in the mouse lung, a population of CD274 expressing cells exists in the normal human lung and this population is expanded in the IPF lung and in an ex vivo PCLS assay, suggestive of progenitor cell behavior.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of the lung is progressively replaced by fibrotic scars. Myofibroblasts are the effector cells that excessively deposit extracellular matrix proteins thus compromising lung structure and function. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug metformin exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGFβ1 action, suppressing collagen formation, activating PPARγ signaling and inducing lipogenic differentiation in lung fibroblasts derived from IPF patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-to-lipofibroblast transdifferentiation. Detailed pathway analysis revealed a two-arm mechanism by which metformin accelerates fibrosis resolution. Our data report an antifibrotic role for metformin in the lung, thus warranting further therapeutic evaluation., Idiopathic pulmonary fibrosis is associated with myofibroblast activation in the lungs and metabolic alterations. Here, the authors show that the antidiabetic drug metformin has antifibrotic effects in human-derived samples and mouse models, by modulating a number of metabolic pathways to induce lipogenic transdifferentiation of myofibroblasts.

Alveolar type 2 (AT2) cells are heterogeneous cells; where specialized AT2 subpopulations within this lineage exhibit stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage, which get activated during lung regeneration, is unknown.SftpcCreERT2/+; tdTomatoflox/flox mice were used for the labelling of AT2 cells and labeled subpopulations were analyzed by flow cytometry, qPCR, ATAC-seq, gene arrays, pneumonectomy, and culture of precision-cut lung slides. Human lungs from donor and IPF were also analyzed.In mice, we detected two distinct AT2 subpopulations with low tdTomato level (TomLow) and high tdTomato level (TomHigh). TomLow express lower level of AT2 differentiation markers, Fgfr2b and Etv5, while TomHigh, as bona fide mature AT2 cells, show higher level of Sftpc, Sftpb, Sftpa1, Fgfr2b, and Etv5. ATAC-seq analysis indicates that TomLow and TomHigh constitute two distinct cell populations with specific silencing of Sftpc, Rosa26 and cell cycle gene loci in TomLow. Upon pneumonectomy, TomLow but not TomHigh cells proliferate and upregulate the expression of Fgfr2b, Etv5, Sftpc, Ccnd1 and Ccnd2 compared to sham. TomLow cells overexpress PD-L1, an immune inhibitory membrane receptor ligand, which is used by flow cytometry to differentially isolate these two sub-populations. In the human lung, PD-L1 and HTII-280 antibodies are used by flow cytometry to differentially sort mature AT2 (HTII-280-high, PD-L1-low) as well as an additional subpopulation of epithelial cells characterized by HTII-280-Low and PD-L1-high.We have identified a novel population of AT2 quiescent immature progenitor cells in mouse that proliferate upon pneumonectomy and provided evidence for the existence of such cells in human.Significance of the workThe characterization and mechanism of the proliferation of a novel and relevant pool of AT2 progenitor cells for the repair/regeneration process after injury are critical to improving respiratory function in patients with lung disease.

Rationale: Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological functions. Their role in pulmonary arterial hypertension (PAH) remains to be explored. Objectives: To elucidate the role of TYKRIL (tyrosine kinase receptor–inducing lncRNA) as a regulator of p53/ PDGFRβ (platelet-derived growth factor receptor β) signaling pathway and to investigate its role in PAH. Methods: Pericytes and pulmonary arterial smooth muscle cells exposed to hypoxia and derived from patients with idiopathic PAH were analyzed with RNA sequencing. TYKRIL knockdown was performed in above-mentioned human primary cells and in precision-cut lung slices derived from patients with PAH. Measurements and Main Results: Using RNA sequencing data, TYKRIL was identified to be consistently upregulated in pericytes and pulmonary arterial smooth muscles cells exposed to hypoxia and derived from patients with idiopathic PAH. TYKRIL knockdown reversed the proproliferative (n = 3) and antiapoptotic (n = 3) phenotype induced under hypoxic and idiopathic PAH conditions. Owing to the poor species conservation of TYKRIL, ex vivo studies were performed in precision-cut lung slices from patients with PAH. Knockdown of TYKRIL in precision-cut lung slices decreased the vascular remodeling (n = 5). The number of proliferating cell nuclear antigen–positive cells in the vessels was decreased and the number of terminal deoxynucleotide transferase–mediated dUTP nick end label–positive cells in the vessels was increased in the LNA (locked nucleic acid)-treated group compared with control. Expression of PDGFRβ, a key player in PAH, was found to strongly correlate with TYKRIL expression in the patient samples (n = 12), and TYKRIL knockdown decreased PDGFRβ expression (n = 3). From the transcription factor–screening array, it was observed that TYKRIL knockdown increased the p53 activity, a known repressor of PDGFRβ. RNA immunoprecipitation using various p53 mutants demonstrated that TYKRIL binds to the N-terminal of p53 (an important region for p300 interaction with p53). The proximity ligation assay revealed that TYKRIL interferes with the p53–p300 interaction (n = 3) and regulates p53 nuclear translocation. Conclusions: TYKRIL plays an important role in PAH by regulating the p53/PDGFRβ axis.

Idiopathic pulmonary fibrosis (IPF) is associated with progressive destruction of the lung parenchyma with a median survival after diagnosis of three years and a five-year survival of

Alveolar epithelial cell type II (AEC2) injury underlies idiopathic pulmonary fibrosis (IPF). Here we show increased Notch1 signaling in AEC2s in human IPF and IPF models, causing enhanced proliferation and de-differentiation of AEC2s. As a result, we observed defective surfactant protein (SP)-B/C processing, elevated alveolar surface tension, repetitive alveolar collapse and development of lung fibrosis. Similar changes were encountered upon pharmacological inhibition of SP-B/C processing in vivo by pepstatin A. Inhibition of Notch signaling in cultured human IPF precision cut lung slices improved surfactant processing capacity of AEC2s and reversed fibrosis. Notch1 therefore offers as novel therapeutic target.One sentence summaryNotch1 inhibition restores alveolar epithelial differentiation and surface tension and reverses matrix deposition in lung fibrosis

Alveolar type 2 (AT2) cells are heterogeneous cells, with specialised AT2 subpopulations within this lineage exhibiting stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage that are activated during lung regeneration is unknown.SftpcCreERT2/+;tdTomatoflox/flox mice were used for the labelling of AT2 cells and labelled subpopulations were analysed by flow cytometry, quantitative PCR, assay for transposase-accessible chromatin using sequencing (ATAC-seq), gene arrays, pneumonectomy and culture of precision-cut lung slices. Single-cell RNA-sequencing (scRNA-seq) data from human lungs were analysed.In mice, we detected two distinct AT2 subpopulations, with low tdTomato level (TomLow) and high tdTomato level (TomHigh). TomLow cells express lower levels of the AT2 differentiation markers Fgfr2b and Etv5, while TomHigh, as bona fide mature AT2 cells, show higher levels of Sftpc, Sftpb, Sftpa1, Fgfr2b and Etv5 expression. ATAC-seq analysis indicates that TomLow and TomHigh cells constitute two distinct cell populations, with specific silencing of Sftpc, Rosa26 and cell cycle gene loci in the TomLow population. Upon pneumonectomy, the number of TomLow but not TomHigh cells increases and TomLow cells show upregulated expression of Fgfr2b, Etv5, Sftpc, Ccnd1 and Ccnd2 compared to Sham. TomLow cells overexpress programmed cell death 1 ligand 1 (PD-L1), an immune inhibitory membrane receptor ligand, which is used by flow cytometry to differentially isolate these two subpopulations. In the human lung, data mining of a recent scRNA-seq AT2 data set demonstrates the existence of a PD-L1Pos population. Therefore, we have identified a novel population of AT2 quiescent, immature progenitor cells in mouse that expand upon pneumonectomy and we have provided evidence for the existence of such cells in human.

Idiopathic pulmonary fibrosis is a fatal, incurable lung disease in which the intricate alveolar network of the human lung is progressively replaced by fibrotic scars, eventually leading to respiratory failure. Myofibroblasts are the effector cells that lead to abnormal deposition of extracellular matrix proteins and therefore mediate fibrotic disease not only in the lung but also in other organs. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug, metformin, exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGFβ1 action, suppressing collagen formation, activating PPARγ signaling and inducing lipogenic differentiation in lung myofibroblasts derived from human patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-tolipofibroblast transdifferentiation. Detailed pathway analysis showed that the reduction of collagen synthesis was largely AMPK-dependent, whereas the transdifferentiation of myo- to lipofibroblasts occurred in a BMP2-PPARγ-dependent fashion and was largely AMPK-independent. Our data report an unprecedented role for metformin in lung fibrosis, thus warranting further therapeutic evaluation.

Summary: Idiopathic pulmonary fibrosis is a common form of interstitial lung disease resulting in alveolar remodeling and progressive loss of pulmonary function because of chronic alveolar injury and failure to regenerate the respiratory epithelium. Histologically, fibrotic lesions and honeycomb structures expressing atypical proximal airway epithelial markers replace alveolar structures, the latter normally lined by alveolar type 1 (AT1) and AT2 cells. Bronchial epithelial stem cells (BESCs) can give rise to AT2 and AT1 cells or honeycomb cysts following bleomycin-mediated lung injury. However, little is known about what controls this binary decision or whether this decision can be reversed. Here we report that inactivation of Fgfr2b in BESCs impairs their contribution to both alveolar epithelial regeneration and honeycomb cysts after bleomycin injury. By contrast overexpression of Fgf10 in BESCs enhances fibrosis resolution by favoring the more desirable outcome of alveolar epithelial regeneration over the development of pathologic honeycomb cysts. : FGF10-FGFR2B signaling in bronchial epithelial stem cells is critical to drive both alveolar epithelial regeneration and the development of honeycomb cysts after bleomycin injury. Overexpression of Fgf10 in bronchial epithelial stem cells enhances fibrosis resolution by favoring the more desirable outcome of alveolar epithelial regeneration over the development of pathologic honeycomb cysts. Keywords: lung stem cells, lung regeneration, lung fibrosis, Fgf signaling

Background Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts. Methods FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed. Results Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects. Conclusions Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling. Electronic supplementary material The online version of this article (doi:10.1186/s12931-015-0242-2) contains supplementary material, which is available to authorized users.