Biology subjects

Bellusci, S.

Publications and source records attributed to Bellusci, S..

At least 19 recordsLinked to original sources

bioRxiv2024

Exploring the Antifibrotic Potential of the heparan sulfate mimetic OTR4120: Insights from Preclinical Models

RationaleIdiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive deposition of extracellular matrix (ECM), resulting in lung function impairment. Heparan sulfate mimetics (HSm) have been suggested to have potential antifibrotic effects by regulating ECM. This study aims to investigate the impact of a specific HSm named OTR4120 on fibrotic processes in ex vivo, in vitro and in vivo models. MethodsHuman Precision Cut Lung Slices (hPCLS) treated with a fibrotic cocktail alone or with OTR4120 were evaluated using second-harmonic imaging microscopy (SHIM) to assess collagen deposition. Human embryonic fibroblast WI-38 cell line and primary fibroblasts obtained from human donors were differentiated into myofibroblasts (MYF) using TGF-{beta}1 and treated with OTR4120 or a control vehicle. Gene expression analysis for MYF markers was performed using quantitative PCR (qPCR). Protein expression of MYF markers was evaluated using immunofluorescence techniques. Bulk-RNA sequencing analysis on WI-38 cells cultured under different experimental conditions was conducted. Finally, the therapeutic effects of OTR4120 on a bleomycin-induced fibrosis mouse model were investigated. ResultsSHIM analysis on OTR4120-treated hPCLS showed a decrease in collagen deposition. OTR4120 treatment of primary fibroblasts and WI-38 cells exposed to TGF-{beta}1 significantly reduced the expression of MYF markers. Bulk-RNA sequencing analysis on OTR4120-treated WI-38 cells showed significant impacts on fibrosis-related processes. Therapeutic application of OTR4120 in vivo to bleomycin-induced fibrosis mice resulted in enhanced fibrosis resolution. ConclusionOTR4120 has potential therapeutic benefits as an antifibrotic agent in the context of lung fibrosis. Further investigations are necessary to understand the precise mechanism through which OTR4120 exerts its antifibrotic effects.

cell biology↗

bioRxiv2024

Lineage-tracing of Acta2+ cells in aged mice during lung fibrosis formation and resolution supports the lipofibroblasts to myofibroblast reversible switch

Idiopathic pulmonary fibrosis (IPF) develops mostly in old man and is characterized by the irreversible accumulation of excessive extracellular matrix components by activated myofibroblasts (aMYFs) leading to lung failure. Following bleomycin administration in young mice, fibrosis formation associated with efficient resolution takes place, the later limiting the clinical relevance of this model for IPF. In young mice, we previously reported that aMYFs captured during fibrosis formation differentiate towards a lipofibroblast (LIF)-like phenotype during resolution. In this study, we used aged mice in combination with bleomycin administration to trigger enhanced fibrosis formation and delayed resolution, as a more relevant model for IPF and examined the heterogeneity and fate of aMYFs at different time points. Alveolosphere assay were carried out to compare the alveolar resident mesenchymal niche activity for AT2 stem cells in young versus old mice. Lineage tracing of the Acta2+ aMYFs in old mice exposed to bleomycin followed by scRNAseq of the lineage-traced cells isolated during fibrosis formation and resolution was performed to delineate the heterogeneity of aMYFs during fibrosis formation and their fate during resolution. Data mining of human mesenchymal cells from IPF and control datasets were also performed to decipher the heterogeneity of aMYFs and investigate differentiation trajectories during fibrosis formation. Our results show that alveolar resident mesenchymal cells from old mice display decreased supporting activity for AT2 stem cells. We report that aMYFs consist of four subclusters displaying unique pro-alveologenic versus pro-fibrotic profiles. Alveolar fibroblasts displaying a high LIF-like signature largely constitute both the origin and fate of aMYFs during fibrosis formation and resolution, respectively. The heterogeneity of aMYFs is conserved in humans and a significant proportion of human aMYFs displays a high LIF signature. In conclusion, our data indicate that the cellular and molecular bases of aMYFs formation and differentiation towards the LIF phenotype are conserved between young and old mice. Importantly, our work identifies a subcluster of aMYFs that is potentially relevant for future management of IPF.

pathology↗

bioRxiv2023

Highlighting fibroblast plasticity in lung fibrosis: the WI-38 cell line as a model for investigating the myofibroblast and lipofibroblast switch

BackgroundMyofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF). Methodsusing WI-38 cells, MYF differentiation was triggered using TGF-{beta}1 treatment and LIF differentiation using Metformin treatment. We analyzed the LIF to MYF and MYF to LIF differentiation by pre-treating the WI-38 cells with TGF-{beta}1 or Metformin first, followed by treatment with Metformin and TGF-{beta}1, respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IFP patients-derived lung fibroblasts (LF-IPF) cultured in vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells. ResultsWI-38 and LF-IPF display similar phenotypical and gene expression responses to TGF-{beta}1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 and LF-IPF treated with TGF-{beta}1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-{beta}1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the LIF to MYF reversible switch using WI-38 cells. ConclusionsWI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/572972v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@9a5c19org.highwire.dtl.DTLVardef@149edborg.highwire.dtl.DTLVardef@1c9579aorg.highwire.dtl.DTLVardef@1964167_HPS_FORMAT_FIGEXP M_FIG in vitro approach using WI-38 cells as a versatile and reliable model to study the MYF or LIF phenotype associated with lung fibrosis formation and resolution observed in vivo. WI-38 are providing valuable insights to drive future research on lung fibrosis. C_FIG

cell biology↗

bioRxiv2023

Postnatal FGF-signalling establishes gradients of cell-identities along the proximal-distal axis of the lung airways

Secretory cells are major structural and functional constituents of the lung airways. Their spatial organization and specification mechanisms are partially understood. Here, we labelled major secretory airway cell types and analysed them at single-cell resolution. We found opposing, partially overlapping gene-expression gradients along the proximal-distal airway axis superimposed on a general gene program encoding detoxification. One graded program is elevated proximally and relates to innate immunity, whereas the other is enriched distally, encoding lipid metabolism and antigen presentation. Intermediately positioned cells express low levels of both graded programs and show increased clonogenic potency in vitro, relating cell-plasticity to location in each branch. Single-cell RNA-sequencing following lineage-tracing revealed the sequential and postnatal establishment of the gradients in common epithelial progenitors. Fgfr2b is distally enriched and its postnatal inactivation reduces distal gene expression and expands proximal genes into distally located cells. This suggests a central role of FGFR-signaling in tissue-scale airway patterning.

cell biology↗

bioRxiv2023

Lung injury shifts pulmonary capillary endothelial cells towards regeneration-associated Lrg1+ subpopulations with delayed dynamics in aged mice

Aging increases the risk of developing fibrotic diseases by hampering tissue regeneration after injury. Using longitudinal single-cell RNA-seq and spatial transcriptomics, here we compare the transcriptome of bleomycin-induced fibrotic lungs of young and aged mice, at 3 time points corresponding to the peak of fibrosis, regeneration and resolution. We find that lung injury shifts the transcriptomic profiles of three pulmonary capillary endothelial cells (PCEC) subpopulations. The associated signatures are linked to pro-angiogenic signaling with strong Lrg1 expression and do not progress similarly throughout the resolution process between young and old animals. Moreover, part of this set of resolution-associated markers is also detected in PCEC from samples of patients with idiopathic pulmonary fibrosis. Finally, we find that aging also alters the transcriptome of PCEC which display typical pro-fibrotic and pro-inflammatory features. We propose that age-associated alterations in specific PCEC subpopulations may interfere with the process of lung progenitor differentiation, thus contributing to the persistent fibrotic process typical of human pathology.

pathology↗

bioRxiv2022

A dominant negative mutation uncovers cooperative control of caudal Wolffian Duct development by Sprouty genes

The Wolffian Ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting on ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1+/- mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant-negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.

developmental biology↗

bioRxiv2022

FGF10 triggers de novo alveologenesis in a BPD model: impact on the resident mesenchymal niche cells

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 were 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.

cell biology↗

bioRxiv2022

Cell-surface PD-L1 expression identifies a sub-population of distal epithelial cells enriched in idiopathic pulmonary fibrosis

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.

cell biology↗

bioRxiv2022

Fgfr2b signaling is essential for the maintenance of the alveolar epithelial type 2 lineage during lung homeostasis in mice

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; 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 one or two weeks to delete Fgfr2b, a shrinking of the AT2s 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 one-week tamoxifen exposure followed by an eight-week chase period. While 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 and suggest that the IAAPs could represent a new population of AT2 progenitors.

developmental biology↗

bioRxiv2022

Characterization of alveolar epithelial lineage heterogeneity during the late pseudoglandular stage of mouse lung development

The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and 2) progenitors during embryonic lung development remains mostly elusive. 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 lung development. Here, we looked at the regulation by Fgfr2b signalling on alveolar progenitors during late pseudoglandular/early canalicular (E14.5-E16.5) development. Using a dominant negative mouse model to conditionally inhibit Fgfr2b ligands at E16.5, we used gene array analyses to characterize a set of potential direct targets of Fgfr2b signalling. By mining published single-cell RNA sequence (scRNAseq) datasets, we showed that these Fgfr2b signature genes narrow on a discreet subset of AT2 cells at E17.5 and in adult lungs. Furthermore, we demonstrated that Fgfr2b signalling is lost in AT2 cells in their transition to AT1 cells during repair after injury. We also used CreERT2-based mouse models to conditionally knock-out the Fgfr2b gene in AT2 and in AT1 progenitors, as well as lineage label these cells. We found, using immunofluorescence, that in wildtype controls AT1 progenitors labeled at E14.5-E15.5 contribute a significant proportion to AT2 cells at E18.5; while AT2 progenitors labeled at the same time contribute significantly to the AT1 lineage. We show, using immunofluorescence and FACS-based analysis, that knocking out of Fgfr2b at E14.5-E15.5 in AT2 progenitors leads to an increase in lineage-labeled AT1 cells at E18.5; while the reverse is true in AT1 progenitors. Furthermore, we demonstrate that increased Fgfr signalling in AT2 progenitors reduces their contribution to the AT1 pool. Taken together, our results suggest that a significant proportion of AT2 and AT1 progenitors are cross-lineage committed during late pseudoglandular 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 concentrate 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 alveolar cells by elucidating the role of Fgfr2b signalling in these cells during early alveolar lineage formation, as well as during repair after injury.

developmental biology↗

bioRxiv2021

Characterization of a novel Fgf10CreERT2 knock-in mouse line targeting postnatal lung Fgf10 lineages

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a Fgf10CreERT2 line (with the CreERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki-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 on our research. We report here the generation and validation of a new Fgf10CreERT2 (called thereafter Fgf10Ki-v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. This new Fgf10Ki-v2 line exhibited comparable Fgf10 expression level to their wild type counterpart. However, a disconnection between the Fgf10 and the Cre expression was observed in Fgf10Ki-v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10Ki-v2 mice. Bio-informatics analysis shows that the 3UTR, where the CreERT2 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. Significantly, Fgf10Ki-v2 mouse is capable of significantly targeting Fgf10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10Ki-v2 line opens the way for future mechanistic experiments involving the postnatal lung.

developmental biology↗

bioRxiv2021

Characterization in mice of the stromal niche maintaining AT2 stem cell self-renewal in homeostasis and disease

Resident mesenchymal cells (rMCs defined as Cd31NegCd45NegEpcamNeg) control the self-renewal 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-Sca1PosFgf10Pos) are equally important to maintain AT2 stem cell self-renewal. 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 self-renewal and differentiation of AT2 stem cells. We demonstrate that rMC-Sca1PosFgf10Pos cells are efficient to promote the self-renewal 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-Sca1PosAxin2Pos and rMC-Sca1PosFgf10Pos support that these two cell subsets express differential markers. In addition, rMC function is decreased in diabetic and obese ob/ob mutant compared to WT mice with a much stronger loss of function in males compared to females. In conclusion, rMC-Sca1PosFgf10Pos cells play important role in supporting AT2 stem cells self-renewal 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 COVID-19 pathogenesis. Key messageO_ST_ABSWhat is already known about the subject?C_ST_ABSResident mesenchymal cells (rMCs defined as Cd31NegCd45NegEpcamNeg) control the self-renewal 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. What does this study add?Our study shows that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1PosFgf10Pos) is equally important to maintain AT2 stem cell self-renewal. rMC-Sca1PosFgf10Pos are LipidToxHigh and are located close to AT2s. In addition, rMC-Sca1PosFgf10Pos cells support AT2 stem cell self-renewal and differentiation thereby identifying these cells as bone fide functional lipofibroblasts (LIFs). We have previously reported that LIF can transdifferentiate into activated MYF in the context of bleomycin-induced fibrosis in mice [1] and that activated MYF isolated from the lungs of end stage idiopathic fibrosis human patients can respond to Metformin to undergo transdifferentiation back to the LIF phenotype [2]. We also show that the function of rMCs-Sca1Pos is negatively impacted by gender and obesity, which represent two major aggravating factors for COVID-19 pathogenesis, leading to either death or major complications after infection recovery such as lung fibrosis. How might this impact on clinical practice and future development?By establishing that rMC-Sca1PosFgf10Pos are different from the MANCs, our study opens the way for a new key mesenchymal cell population that should be targeted to either prevent or reverse fibrosis. In addition, as this population maintains the AT2 stem cells self-renewal and differentiation, such targeting will also allow to progressively recover the loss in respiratory function.

developmental biology↗

bioRxiv2020

Identification a novel subset of alveolar type 2 cells expanding following pneumonectomy and enriched in PD-L1

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.

cell biology↗

bioRxiv2020

Positioning of nucleosomes containing γ-H2AXprecedes active DNA demethylation and transcription initiation

In addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group (HMG) proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of HMG proteins during transcription initiation remain unclear. Here we show that HMG AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT (facilitates chromatin transcription) complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 ({gamma}-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure the integrity of the genome.

molecular biology↗

bioRxiv2019

Restored alveolar epithelial differentiation and reversed human lung fibrosis upon Notch inhibition

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.\n\nOne sentence summaryNotch1 inhibition restores alveolar epithelial differentiation and surface tension and reverses matrix deposition in lung fibrosis

cell biology↗

bioRxiv2019

A smooth muscle-like niche facilitates lung epithelial regeneration

The mammalian lung is a highly complex organ due to its branched, tree-like structure and diverse cellular composition. Recent efforts using state-of-the-art genetic lineage tracing and single-cell transcriptomics have helped reduce this complexity and delineate the ancestry and fate of various cell subpopulations during organogenesis, homeostasis and repair after injury. However, mesenchymal cell heterogeneity and function in development and disease remain a longstanding issue in the lung field. In this study, we break down smooth muscle heterogeneity into the constituent subpopulations by combining in vivo lineage tracing, single-cell RNA sequencing and in vitro organoid cultures. We identify a repair-supportive mesenchymal cell (RSMC) population that is distinct from pre-existing airway smooth muscle cells (ASMC) and is critical for regenerating the conducting airway epithelium. Progenitors of RSMCs are intertwined with airway smooth muscle, undergo active WNT signaling, transiently acquire the expression of the smooth muscle marker ACTA2 in response to epithelial injury and are marked by PDGFR expression. Our data simplify the cellular complexity of the peribronchiolar domain of the adult lung and represent a forward step towards unraveling the role of mesenchymal cell subpopulations in instructing epithelial behavior during repair processes.

developmental biology↗

bioRxiv2018

Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

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 TGF{beta}1 action, suppressing collagen formation, activating PPAR{gamma} 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-PPAR{gamma}-dependent fashion and was largely AMPK-independent. Our data report an unprecedented role for metformin in lung fibrosis, thus warranting further therapeutic evaluation.

cell biology↗

bioRxiv2018

A critical role for miR-142 in alveolar epithelial lineage formation

The development of a functional lung, capable of gas exchange, requires proper alveologenesis. Mechanisms regulating AT1 and AT2 cell maturation are poorly defined. We report the activation of the glucocorticoid pathway in an in vitro alveolar epithelial lineage differentiation assay led to increased AT2 marker Sftpc and decreased miR-142 expression. Using a constitutive KO mouse model, we further demonstrate a relative increase of AT2 and a decrease in AT1 cell number with a global decrease of AT2 gene profile signature in miR-142 KO AT2 cells. Over-expression of miR-142 in alveolar progenitor cells in vivo led to an opposite effect. Examination of the KO lungs at E18.5, revealed enhanced expression miR-142 targets like Apc, Ep300 and Kras associated with increased Ctnnb1 and p-Erk signaling. Pharmacological inhibition of Ep300-Ctnnb1 in vitro prevented an increase in Sftpc expression triggered by loss of miR-142. These results together suggest glucocorticoid-miR-142-p300 signaling axis controls pneumocyte maturation.

developmental biology↗