ABSTRACT
BACKGROUND: The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). METHODS: Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. RESULTS: The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK (mitogen-activated protein kinase) signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. CONCLUSIONS: Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.
Subject(s)
Hypertension, Pulmonary , Vascular Remodeling , Zinc Finger Protein GLI1 , Animals , Zinc Finger Protein GLI1/metabolism , Zinc Finger Protein GLI1/genetics , Mice , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/physiopathology , Hypertension, Pulmonary/pathology , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Mice, Inbred C57BL , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Pulmonary Artery/physiopathology , Mice, Transgenic , Male , Humans , Hypoxia/metabolism , Hypoxia/physiopathologyABSTRACT
Influenza A virus (IAV) infection mobilizes bone marrow-derived macrophages (BMDM) that gradually undergo transition to tissue-resident alveolar macrophages (TR-AM) in the inflamed lung. Combining high-dimensional single-cell transcriptomics with complex lung organoid modeling, in vivo adoptive cell transfer, and BMDM-specific gene targeting, we found that transitioning ("regenerative") BMDM and TR-AM highly express Placenta-expressed transcript 1 (Plet1). We reveal that Plet1 is released from alveolar macrophages, and acts as important mediator of macrophage-epithelial cross-talk during lung repair by inducing proliferation of alveolar epithelial cells and re-sealing of the epithelial barrier. Intratracheal administration of recombinant Plet1 early in the disease course attenuated viral lung injury and rescued mice from otherwise fatal disease, highlighting its therapeutic potential.
Subject(s)
Influenza A virus , Influenza, Human , Pneumonia, Viral , Animals , Female , Humans , Mice , Pregnancy , Lung , Macrophages, Alveolar , PlacentaABSTRACT
The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and AT2) progenitors during embryonic lung development are poorly defined. Current models of distal epithelial lineage formation fail to capture the heterogeneity and dynamic contribution of progenitor pools present during early development. Furthermore, few studies explore the pathways involved in alveolar progenitor specification and fate. In this paper, we build upon our previously published work on the regulation of airway epithelial progenitors by fibroblast growth factor receptor 2b (FGFR2b) signalling during early (E12.5) and mid (E14.5) pseudoglandular stage lung development. Our results suggest that a significant proportion of AT2 and AT1 progenitors are lineage-flexible during late pseudoglandular stage development, and that lineage commitment is regulated in part by FGFR2b signalling. We have characterized a set of direct FGFR2b targets at E16.5 which are likely involved in alveolar lineage formation. These signature genes converge on a subpopulation of AT2 cells later in development and are downregulated in AT2 cells transitioning to the AT1 lineage during repair after injury in adults. Our findings highlight the extensive heterogeneity of pneumocytes by elucidating the role of FGFR2b signalling in these cells during early airway epithelial lineage formation, as well as during repair after injury.
Subject(s)
Alveolar Epithelial Cells , Lung , Receptor, Fibroblast Growth Factor, Type 2 , Stem Cells , Animals , Mice , Embryonic Development , Receptor, Fibroblast Growth Factor, Type 2/genetics , Signal Transduction , Lung/embryology , Cell LineageABSTRACT
Repair-supportive mesenchymal cells (RSMCs) have been recently reported in the context of naphthalene (NA)-induced airway injury and regeneration. These cells transiently express smooth muscle actin (Acta2) and are enriched with platelet-derived growth factor receptor alpha (Pdgfra) and fibroblast growth factor 10 (Fgf10) expression. Genetic deletion of Ctnnb1 (gene coding for beta catenin) or Fgf10 in these cells using the Acta2-Cre-ERT2 driver line after injury (defined as NA-Tam condition; Tam refers to tamoxifen) led to impaired repair of the airway epithelium. In this study, we demonstrate that RSMCs are mostly captured using the Acta2-Cre-ERT2 driver when labeling occurs after (NA-Tam condition) rather than before injury (Tam-NA condition), and that their expansion occurs mostly between days 3 and 7 following NA treatment. Previous studies have shown that lineage-traced peribronchial GLI1+ cells are transiently amplified after NA injury. Here, we report that Gli1 expression is enriched in RSMCs. Using lineage tracing with Gli1Cre-ERT2 mice combined with genetic inactivation of Fgf10, we show that GLI1+ cells with Fgf10 deletion fail to amplify around the injured airways, thus resulting in impaired airway epithelial repair. Interestingly, Fgf10 expression is not upregulated in GLI1+ cells following NA treatment, suggesting that epithelial repair is mostly due to the increased number of Fgf10-expressing GLI1+ cells. Co-culture of SCGB1A1+ cells with GLI1+ cells isolated from non-injured or injured lungs showed that GLI1+ cells from these two conditions are similarly capable of supporting bronchiolar organoid (or bronchiolosphere) formation. Single-cell RNA sequencing on sorted lineage-labeled cells showed that the RSMC signature resembles that of alveolar fibroblasts. Altogether, our study provides strong evidence for the involvement of mesenchymal progenitors in airway epithelial regeneration and highlights the critical role played by Fgf10-expressing GLI1+ cells in this context.
Subject(s)
Mesenchymal Stem Cells , Mice , Animals , Zinc Finger Protein GLI1/genetics , Zinc Finger Protein GLI1/metabolism , Lung/metabolism , Stem Cells , Epithelium/physiology , Epithelial Cells/metabolismABSTRACT
Insulin-like growth factor (IGF) signaling controls the development and growth of many organs, including the lung. Loss of function of Igf1 or its receptor Igf1r impairs lung development and leads to neonatal respiratory distress in mice. Although many components of the IGF signaling pathway have shown to be dysregulated in idiopathic pulmonary fibrosis (IPF), the expression pattern of such components in different cellular compartments of the developing and/or fibrotic lung has been elusive. In this study, we provide a comprehensive transcriptional profile for such signaling components during embryonic lung development in mice, bleomycin-induced pulmonary fibrosis in mice and in human IPF lung explants. During late gestation, we found that Igf1 is upregulated in parallel to Igf1r downregulation in the lung mesenchyme. Lung tissues derived from bleomycin-treated mice and explanted IPF lungs revealed upregulation of IGF1 in parallel to downregulation of IGF1R, in addition to upregulation of several IGF binding proteins (IGFBPs) in lung fibrosis. Finally, treatment of IPF lung fibroblasts with recombinant IGF1 led to myogenic differentiation. Our data serve as a resource for the transcriptional profile of IGF signaling components and warrant further research on the involvement of this pathway in both lung development and pulmonary disease.
Subject(s)
Idiopathic Pulmonary Fibrosis , Animals , Bleomycin/pharmacology , Female , Idiopathic Pulmonary Fibrosis/chemically induced , Idiopathic Pulmonary Fibrosis/genetics , Idiopathic Pulmonary Fibrosis/metabolism , Insulin-Like Growth Factor Binding Proteins , Lung/metabolism , Mice , Organogenesis , Pregnancy , Signal TransductionABSTRACT
Fibroblast growth factor receptor 2b (Fgfr2b) signaling is essential throughout lung development to form the alveolar epithelial lineage. However, its role in alveolar epithelial type 2 cells (AT2s) homeostasis was recently considered dispensable. SftpcCreERT2; Fgfr2bflox/flox; tdTomatoflox/flox mice were used to delete Fgfr2b expression in cells belonging to the AT2 lineage, which contains mature AT2s and a novel SftpcLow lineage-traced population called "injury activated alveolar progenitors" or IAAPs. Upon continuous tamoxifen exposure for either 1 or 2 weeks to delete Fgfr2b, a shrinking of the AT2 population is observed. Mature AT2s exit the cell cycle, undergo apoptosis and fail to form alveolospheres in vitro. However, the lung morphometry appears normal, suggesting the involvement of compensatory mechanisms. In mutant lungs, IAAPs which escaped Fgfr2b deletion expand, display enhanced alveolosphere formation in vitro and increase drastically their AT2 signature, suggesting differentiation towards mature AT2s. Interestingly, a significant increase in AT2s and decrease in IAPPs occurs after a 1-week tamoxifen exposure followed by an 8-week chase period. Although mature AT2s partially recover their alveolosphere formation capabilities, the IAAPs no longer display this property. Single-cell RNA seq analysis confirms that AT2s and IAAPs represent stable and distinct cell populations and recapitulate some of their characteristics observed in vivo. Our results underscore the essential role played by Fgfr2b signaling in the maintenance of the AT2 lineage in the adult lung during homeostasis and suggest that the IAAPs could represent a new population of AT2 progenitors.
Subject(s)
Lung , Receptor, Fibroblast Growth Factor, Type 2 , Alveolar Epithelial Cells , Animals , Cell Differentiation , Homeostasis , Lung/metabolism , Mice , Receptor, Fibroblast Growth Factor, Type 2/genetics , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Tamoxifen/pharmacologyABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a progressive and often lethal interstitial lung disease of unknown aetiology. IPF is characterised by myofibroblast activation, tissue stiffening, and alveolar epithelium injury. As current IPF treatments fail to halt disease progression or induce regeneration, there is a pressing need for the development of novel therapeutic targets. In this regard, tri-dimensional (3D) models have rapidly emerged as powerful platforms for disease modelling, drug screening and discovery. In this review, we will touch on how 3D in vitro models such as hydrogels, precision-cut lung slices, and, more recently, lung organoids and lung-on-chip devices have been generated and/or modified to reveal distinct cellular and molecular signalling pathways activated during fibrotic processes. Markedly, we will address how these platforms could provide a better understanding of fibrosis pathophysiology and uncover effective treatment strategies for IPF patients.
Subject(s)
Idiopathic Pulmonary Fibrosis , Lung Diseases, Interstitial , Drug Evaluation, Preclinical , Fibrosis , Humans , Idiopathic Pulmonary Fibrosis/drug therapy , Idiopathic Pulmonary Fibrosis/pathology , Lung/pathology , Lung Diseases, Interstitial/pathology , MyofibroblastsABSTRACT
Bronchopulmonary dysplasia (BPD) is a neonatal lung disease developing in premature babies characterized by arrested alveologenesis and associated with decreased Fibroblast growth factor 10 (FGF10) expression. One-week hyperoxia (HYX) exposure of newborn mice leads to a permanent arrest in alveologenesis. To test the role of Fgf10 signaling to promote de novo alveologenesis following hyperoxia, we used transgenic mice allowing inducible expression of Fgf10 and recombinant FGF10 (rFGF10) protein delivered intraperitoneally. We carried out morphometry analysis, and IF on day 45. Alveolospheres assays were performed co-culturing AT2s from normoxia (NOX) with FACS-isolated Sca1Pos resident mesenchymal cells (rMC) from animals exposed to NOX, HYX-PBS, or HYX-FGF10. scRNAseq between rMC-Sca1Pos isolated from NOX and HYX-PBS was also carried out. Transgenic overexpression of Fgf10 and rFGF10 administration rescued the alveologenesis defects following HYX. Alveolosphere assays indicate that the activity of rMC-Sca1Pos is negatively impacted by HYX and partially rescued by rFGF10 treatment. Analysis by IF demonstrates a significant impact of rFGF10 on the activity of resident mesenchymal cells. scRNAseq results identified clusters expressing Fgf10, Fgf7, Pdgfra, and Axin2, which could represent the rMC niche cells for the AT2 stem cells. In conclusion, we demonstrate that rFGF10 administration is able to induce de novo alveologenesis in a BPD mouse model and identified subpopulations of rMC-Sca1Pos niche cells potentially representing its cellular target.
Subject(s)
Bronchopulmonary Dysplasia , Hyperoxia , Animals , Animals, Newborn , Bronchopulmonary Dysplasia/genetics , Bronchopulmonary Dysplasia/metabolism , Fibroblast Growth Factor 10/genetics , Fibroblast Growth Factor 10/metabolism , Humans , Hyperoxia/metabolism , Infant, Newborn , Lung/metabolism , Mice , Mice, TransgenicABSTRACT
Organoid models have been shown to be valuable tools for studying epithelial-mesenchymal crosstalk during biological and pathological settings. Our data identified ACTA2+ PDGFRα+ repair-supportive mesenchymal cells as an important component of the conducting airway niche. Here, we provide a detailed protocol for culturing airway organoids, or bronchiolospheres, which provide an assessment of the ability of mesenchymal cells to support club-cell growth. For complete details on the use and execution of this protocol, please refer to Moiseenko et al. (2020).
Subject(s)
Bronchioles/cytology , Animals , Coculture Techniques , Epithelial-Mesenchymal Transition , Mice , Naphthalenes/toxicity , Organoids/cytology , Tamoxifen/administration & dosageABSTRACT
Resident mesenchymal cells (rMCs defined as Cd31Neg Cd45Neg EpcamNeg ) control the proliferation and differentiation of alveolar epithelial type 2 (AT2) stem cells in vitro. The identity of these rMCs is still elusive. Among them, Axin2Pos mesenchymal alveolar niche cells (MANCs), which are expressing Fgf7, have been previously described. We propose that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1Pos Fgf10Pos ) are equally important to maintain AT2 stem cell proliferation. The alveolosphere model, based on the AT2-rMC co-culture in growth factor-reduced Matrigel, was used to test the efficiency of different rMC subpopulations isolated by FACS from adult murine lung to sustain the proliferation and differentiation of AT2 stem cells. We demonstrate that rMC-Sca1Pos Fgf10Pos cells are efficient to promote the proliferation and differentiation of AT2 stem cells. Co-staining of adult lung for Fgf10 mRNA and Sftpc protein respectively, indicate that 28% of Fgf10Pos cells are located close to AT2 cells. Co-ISH for Fgf7 and Fgf10 indicate that these two populations do not significantly overlap. Gene arrays comparing rMC-Sca1Pos Axin2Pos and rMC-Sca1Pos Fgf10Pos support that these two cell subsets express differential markers. In addition, rMC function is decreased in obese ob/ob mutant compared to WT mice with a much stronger loss of function in males compared to females. In conclusion, rMC-Sca1Pos Fgf10Pos cells play important role in supporting AT2 stem cells proliferation and differentiation. This result sheds a new light on the subpopulations of rMCs contributing to the AT2 stem cell niche in homeostasis and in the context of pre-existing metabolic diseases.
Subject(s)
Alveolar Epithelial Cells , Spinocerebellar Ataxias , Animals , Cell Differentiation , Cell Proliferation , Female , Homeostasis , Male , MiceABSTRACT
Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a knock-in Fgf10 Cre-ERT2 line (with the Cre-ERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10 Ki-v1, to target FGF10Pos cells. While this line allowed fairly efficient and specific labeling of FGF10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus of our research. We report here the generation and validation of a new knock-in Fgf10 Cre-ERT2 line (called thereafter Fgf10 Ki-v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. Fgf10 Ki-v2/+ heterozygous mice exhibited comparable Fgf10 expression levels to wild type animals. However, a mismatch between Fgf10 and Cre expression levels was observed in Fgf10 Ki-v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10 Ki-v2 mice. Bioinformatic analysis shows that the 3'UTR, where the Cre-ERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Importantly, Fgf10 Ki-v2 mouse is capable of significantly targeting FGF10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10 Ki-v2 line opens the way for future mechanistic experiments involving the postnatal lung.
ABSTRACT
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-L1 Pos 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.
Subject(s)
B7-H1 Antigen , Pneumonectomy , Alveolar Epithelial Cells , Animals , Chromatin , Lung , MiceABSTRACT
Three-dimensional (3D) organoid culture systems have rapidly emerged as powerful tools to study organ development and disease. The lung is a complex and highly specialized organ that comprises more than 40 cell types that offer several region-specific roles. During organogenesis, the lung goes through sequential and morphologically distinctive stages to assume its mature form, both structurally and functionally. As branching takes place, multipotent epithelial progenitors at the distal tips of the growing/bifurcating epithelial tubes progressively become lineage-restricted, giving rise to more differentiated and specialized cell types. Although many cellular and molecular mechanisms leading to branching morphogenesis have been explored, deeper understanding of biological processes governing cell-fate decisions and lung patterning is still needed. Given that these distinct processes cannot be easily analyzed in vivo, 3D culture systems have become a valuable platform to study organogenesis in vitro. This minireview focuses on the current lung organoid systems that recapitulate developmental events occurring before and during branching morphogenesis. In addition, we also discuss their limitations and future directions.
ABSTRACT
BACKGROUND: The technology of single cell RNA sequencing (scRNA-seq) has gained massively in popularity as it allows unprecedented insights into cellular heterogeneity as well as identification and characterization of (sub-)cellular populations. Furthermore, scRNA-seq is almost ubiquitously applicable in medical and biological research. However, these new opportunities are accompanied by additional challenges for researchers regarding data analysis, as advanced technical expertise is required in using bioinformatic software. RESULTS: Here we present WASP, a software for the processing of Drop-Seq-based scRNA-Seq data. Our software facilitates the initial processing of raw reads generated with the ddSEQ or 10x protocol and generates demultiplexed gene expression matrices including quality metrics. The processing pipeline is realized as a Snakemake workflow, while an R Shiny application is provided for interactive result visualization. WASP supports comprehensive analysis of gene expression matrices, including detection of differentially expressed genes, clustering of cellular populations and interactive graphical visualization of the results. The R Shiny application can be used with gene expression matrices generated by the WASP pipeline, as well as with externally provided data from other sources. CONCLUSIONS: With WASP we provide an intuitive and easy-to-use tool to process and explore scRNA-seq data. To the best of our knowledge, it is currently the only freely available software package that combines pre- and post-processing of ddSEQ- and 10x-based data. Due to its modular design, it is possible to use any gene expression matrix with WASP's post-processing R Shiny application. To simplify usage, WASP is provided as a Docker container. Alternatively, pre-processing can be accomplished via Conda, and a standalone version for Windows is available for post-processing, requiring only a web browser.
Subject(s)
Single-Cell Analysis , Software , Computational Biology , Gene Expression Profiling , RNA-Seq , Sequence Analysis, RNAABSTRACT
Tissue regeneration requires coordinated and dynamic remodeling of stem and progenitor cells and the surrounding niche. Although the plasticity of epithelial cells has been well explored in many tissues, the dynamic changes occurring in niche cells remain elusive. Here, we show that, during lung repair after naphthalene injury, a population of PDGFRα+ cells emerges in the non-cartilaginous conducting airway niche, which is normally populated by airway smooth muscle cells (ASMCs). This cell population, which we term "repair-supportive mesenchymal cells" (RSMCs), is distinct from conventional ASMCs, which have previously been shown to contribute to epithelial repair. Gene expression analysis on sorted lineage-labeled cells shows that RSMCs express low levels of ASMC markers, but high levels of the pro-regenerative marker Fgf10. Organoid co-cultures demonstrate an enhanced ability for RSMCs in supporting club-cell growth. Our study highlights the dynamics of mesenchymal cells in the airway niche and has implications for chronic airway-injury-associated diseases.
Subject(s)
Epithelial Cells/metabolism , Guided Tissue Regeneration/methods , Mesenchymal Stem Cells/metabolism , Animals , Epithelial Cells/pathology , Female , Humans , MiceABSTRACT
Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three-dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self-organization of FACS-sorted bronchioalveolar stem cells (BASCs) upon co-culture with lung-resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single-cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue-resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial-mesenchymal-myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro.
Subject(s)
Lung Diseases/pathology , Lung/growth & development , Organoids/growth & development , Stem Cells/physiology , Animals , Ataxin-1/genetics , Ataxin-1/metabolism , Cell Differentiation/genetics , Cells, Cultured , Endothelial Cells/cytology , Epithelial Cell Adhesion Molecule/genetics , Epithelial Cell Adhesion Molecule/metabolism , Epithelial Cells/cytology , Fibroblasts , Humans , Lung/cytology , Mesenchymal Stem Cells , Mice , Morphogenesis/genetics , Morphogenesis/physiology , Organogenesis/physiology , Organoids/cytology , Pulmonary Alveoli/cytology , Pulmonary Alveoli/growth & development , RNA, Messenger/metabolism , Regeneration/genetics , Regeneration/physiologyABSTRACT
Branching morphogenesis is the basic developmental mode common to organs such as the lungs that undergo a process of ramification from a rudimentary tree. However, the precise molecular and cellular bases underlying the formation of branching organs are still unclear. As inactivation of fibroblast growth factor receptor 2b (Fgfr2b) signaling during early development leads to lung agenesis, thereby preventing the analysis of this pathway at later developmental stages, we used transgenic mice to induce expression of a soluble form of Fgfr2b to inactivate Fgfr2b ligands at embryonic day (E) 14.5, corresponding to the mid-pseudoglandular stage of lung development. We identified an Fgfr2b signaling signature comprised of 46 genes enriched in the epithelium, some of which were common to, but most of them distinct from, the previously identified Fgfr2b signaling signature at E12.5. Our results indicate that Fgfr2b signaling at E14.5 controls mostly proliferation and alveolar type 2 cell (AT2) differentiation. In addition, inhibition of Fgfr2b signaling at E14.5 leads to morphological and cellular impairment at E18.5, with defective alveolar lineage formation. Further studies will have to be conducted to elucidate the role of Fgfr2b signaling at successive stages (canalicular/saccular/alveolar) of lung development as well as during homeostasis and regeneration and repair after injury.
Subject(s)
Lung/embryology , Lung/metabolism , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Signal Transduction , Transcription, Genetic , Animals , Cell Differentiation , Cell Proliferation , Fibroblast Growth Factors/metabolism , Mice , Transcriptome/geneticsSubject(s)
Alveolar Epithelial Cells/drug effects , Endoribonucleases/antagonists & inhibitors , Influenza, Human/complications , Molecular Targeted Therapy , Orthomyxoviridae Infections/complications , Pneumonia, Viral/drug therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Salicylanilides/therapeutic use , Alveolar Epithelial Cells/enzymology , Animals , Apoptosis/drug effects , Cells, Cultured , Endoplasmic Reticulum Stress/drug effects , Endoribonucleases/physiology , Gene Expression Regulation, Viral/drug effects , Humans , Hymecromone/analogs & derivatives , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/physiology , Influenza, Human/drug therapy , Mice , Mice, Inbred C57BL , Orthomyxoviridae Infections/drug therapy , Pneumonia, Viral/enzymology , Pneumonia, Viral/etiology , Protein Serine-Threonine Kinases/physiology , Transcription, Genetic , Unfolded Protein Response/drug effects , Viral Proteins/biosynthesis , Viral Proteins/genetics , Virus Replication/drug effectsABSTRACT
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 TGFß1 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.
Subject(s)
Idiopathic Pulmonary Fibrosis/drug therapy , Lipogenesis/drug effects , Lung/drug effects , Metformin/pharmacology , Myofibroblasts/drug effects , AMP-Activated Protein Kinases/metabolism , Animals , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 2/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Collagen/biosynthesis , Disease Models, Animal , Humans , Idiopathic Pulmonary Fibrosis/etiology , Idiopathic Pulmonary Fibrosis/pathology , Lung/cytology , Lung/pathology , Male , Metformin/therapeutic use , Mice , Myofibroblasts/metabolism , PPAR gamma/genetics , PPAR gamma/metabolism , Primary Cell Culture , RNA, Small Interfering/metabolism , Signal Transduction/drug effects , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism , Treatment Outcome , Up-Regulation/drug effectsABSTRACT
Bronchioalveolar stem cells (BASCs) are a potential source for lung regeneration, but direct in vivo evidence for a multipotential lineage contribution during homeostasis and disease is critically missing, since specific genetic labeling of BASCs has not been possible. We developed a novel cell tracing approach based on intein-mediated assembly of newly engineered split-effectors, allowing selective targeting of dual-marker expressing BASCs in the mouse lung. RNA sequencing of isolated BASCs demonstrates that BASCs show a distinct transcriptional profile, characterized by co-expression of bronchiolar and alveolar epithelial genes. We found that BASCs generate the majority of distal lung airway cells after bronchiolar damage but only moderately contribute to cellular turnover under homeostatic conditions. Importantly, DTA-mediated ablation of BASCs compromised proper regeneration of distal airways. The study defines BASCs as crucial components of the lung repair machinery and provides a paradigmatic example for the detection and manipulation of stem cells that cannot be recognized by a single marker alone.