The use of cultured human alveolar basal cells to mimic honeycomb formation in idiopathic pulmonary fibrosis.

BACKGROUND: Honeycomb cysts (HC) within the alveolar region are distinct histopathological features in the lungs of idiopathic pulmonary fibrosis (IPF) patients. HC are lined with a single-or stratified layer of basal cells (BC), or with a bronchiolar-like epithelium composed of basal-, ciliated- and secretory epithelial cells. By using cultured IPF patient-derived alveolar BC, we aimed to establish an in vitro- and in vivo model to mimic HC formation in IPF. We (1) optimized conditions to culture and propagate IPF patient-derived alveolar BC, (2) cultured the cells on an air liquid interface (ALI) or in a three dimensional (3D) organoid model, and (3) investigated the cells` behavior after instillation into bleomycin-challenged mice. METHODS: Alveolar BC were cultured from peripheral IPF lung tissue and grown on tissue-culture treated plastic, an ALI, or in a 3D organoid model. Furthermore, cells were instilled into bleomycin-challenged NRG mice. Samples were analyzed by TaqMan RT-PCR, immunoblotting, immunocytochemistry/immunofluorescence (ICC/IF), or immunohistochemistry (IHC)/IF. Mann-Whitney tests were performed using GraphPad Prism software. RESULTS: Cultured alveolar BC showed high expression of canonical basal cell markers (TP63, keratin (KRT)5, KRT14, KRT17), robust proliferation, and wound closure capacity. The cells could be cryopreserved and propagated for up to four passages without a significant loss of basal cell markers. When cultured on an ALI or in a 3D organoid model, alveolar BC differentiated to ciliated- and secretory epithelial cells. When instilled into bleomycin-challenged mice, human alveolar BC cells formed HC-like structures composed of human basal-, and secretory epithelial cells within the mouse parenchyma. CONCLUSION: IPF patient-derived alveolar BC on an ALI, in 3D organoids or after instillation into bleomycin-challenged mice form HC-like structures that closely resemble HC within the IPF lung. These models therefore represent powerful tools to study honeycomb formation, and its potential therapeutic inhibition in IPF.

Transcriptomic profiling reveals disease-specific characteristics of epithelial cells in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an incurable disease characterized by progressive lung fibrosis ultimately resulting in respiratory failure and death. Recurrent micro-injuries to the alveolar epithelium and aberrant alveolar wound healing with impaired re-epithelialization define the initial steps of the pathogenic trajectory. Failure of timely alveolar epithelial repair triggers hyper-proliferation of mesenchymal cells accompanied by increased deposition of extracellular matrix into the lung interstitium. METHODS: We previously isolated fibrosis-specific mesenchymal stem cell (MSC)-like cells from lung tissue of patients with interstitial lung diseases. These cells produced factors bearing anti-fibrotic potential and changed their morphology from mesenchymal to epithelial upon culture in an epithelial cell (EC)-specific growth medium. Here, we set out to molecularly characterize these MSC-like cell-derived ECs using global gene expression profiling by RNA-sequencing. Moreover, we aimed at characterizing disease-specific differences by comparing the transcriptomes of ECs from IPF and non-IPF sources. RESULTS: Our results suggest that differentially expressed genes are enriched for factors related to fibrosis, hypoxia, bacterial colonization and metabolism, thus reflecting many of the hallmark characteristics of pulmonary fibrosis. IPF-ECs showed enrichment of both pro- and anti-fibrotic genes, consistent with the notion of adaptive, compensatory regulation. CONCLUSIONS: Our findings support the hypothesis of a functional impairment of IPF-ECs, which could possibly explain the poor clinical outcome of IPF that roughly compares to those of advanced-stage cancers. Our study provides a valuable resource for downstream mechanistic investigation and the quest for novel therapeutic IPF targets.

Culture of human alveolar epithelial type II cells by sprouting.

BACKGROUND: Type II alveolar epithelial cells (AT2) play a pivotal role in maintaining the integrity and function of the alveoli. Only recently, the role of impaired epithelial repair mechanisms after injury in the pathogenesis of idiopathic pulmonary fibrosis has been demonstrated, and has shifted the AT2 cell in the focus of interest. Therefore, using primary human AT2 cells instead of cell lines for in vitro experiments has become desirable. Several groups have developed methods to isolate human AT2 cells applying tissue digestion and consecutive filtration in their protocols. Here we present a technique to isolate primary human AT2 cells by sprouting directly from peripheral human lung tissue. METHODS: Epithelial cell cultures were established from lung tissue obtained from patients undergoing diagnostic or therapeutic video-assisted thoracoscopic surgery or undergoing flexible bronchoscopy with transbronchial biopsy. Lung tissue was cut into small pieces and those were placed into cell culture flasks containing supplemented epithelial growth medium for cell sprouting. Cells were characterized by immunofluorescence stainings for E-cadherin, pan-cytokeratin, surfactant protein C (SP-C), and for lysotracker; fluorescent surfactant associated protein B (SP-B) uptake and secretion was assessed by live cell imaging; RNA levels of SP-A, SP-B, SP-C, and SP-D were determined by real-time PCR; Electron microscopy was used to search for the presence of lamellar bodies. RESULTS: Sprouting of cells started two to four days after the start of culture. Epithelial differentiation was confirmed by positive staining for E-cadherin and pan-cytokeratin. Further characterization demonstrated positivity for the AT2 cell marker SP-C and for lysotracker which selectively labels lamellar bodies in cultured AT2 cells. The up-take and release of SP-B, a mechanism described for AT2 cells only, was demonstrated by live cell imaging. Real-time RT-PCR showed mRNA expression of all four surfactant proteins with highest levels for SP-B. The presence of lamellar bodies was demonstrated by electron microscopy. CONCLUSIONS: This study describes a novel method for isolating AT2 cells from human adult lung tissue by sprouting. The characterization of the cultured AT2 cells complies with current criteria for an alveolar type 2 cell phenotype. Compared to current protocols for the culture of AT2 cells, isolating the cells by sprouting is simple, avoids proteolytic tissue digestion, and has the advantage to be successful even from as few tissue as attained from a transbronchial forceps biopsy.

Constitutive high expression of protein arginine methyltransferase 1 in asthmatic airway smooth muscle cells is caused by reduced microRNA-19a expression and leads to enhanced remodeling.

BACKGROUND: In asthma remodeling airway smooth muscle cells (ASMCs) contribute to airway wall thickness through increased proliferation, migration, and extracellular matrix deposition. Previously, we described that protein arginine methyltransferase 1 (PRMT1) participates in airway remodeling in pulmonary inflammation in E3 rats. OBJECTIVE: We sought to define the asthma-specific regulatory mechanism of PRMT1 in human ASMCs. METHODS: ASMCs from healthy subjects and asthmatic patients were activated with platelet-derived growth factor (PDGF)-BB. PRMT1 was localized by means of immunohistochemistry in human lung tissue sections and by means of immunofluorescence in isolated ASMCs. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI-1, signal transducer and activator of transcription 1 (STAT1) was suppressed by small interfering RNA, and extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) was suppressed by PD98059. MicroRNAs (miRs) were assessed by using real-time quantitative PCR and regulated by miR mimics or inhibitors. RESULTS: PRMT1 expression was significantly increased in lung tissue sections and in isolated ASMCs of patients with severe asthma. PDGF-BB significantly increased PRMT1 expression through ERK1/2 MAPK and STAT1 signaling in control ASMCs, whereas in ASMCs from asthmatic patients, these proteins were constitutively expressed. ASMCs from asthmatic patients had reduced miR-19a expression, causing upregulation of ERK1/2 MAPK, STAT1, and PRMT1. Inhibition of PRMT1 abrogated collagen type I and fibronectin deposition, cell proliferation, and migration of ASMCs from asthmatic patients. CONCLUSIONS: PRMT1 is a central regulator of tissue remodeling in ASMCs from asthmatic patients through the pathway: PDGF-BB-miR-19a-ERK1/2 MAPK and STAT1. Low miR-19a expression in ASMCs from asthmatic patients is the key event that results in constitutive increased PRMT1 expression and remodeling. Therefore PRMT1 is an attractive target to limit airway wall remodeling in asthmatic patients.

MicroRNA-101-3p Suppresses mTOR and Causes Mitochondrial Fragmentation and Cell Degeneration in COPD.

Background: Cigarette smoke is assumed to cause the loss of airway wall structure in chronic obstructive pulmonary disease (COPD) by reducing airway smooth muscle cell (ASMC) function. It also modifies mTOR activity, microRNA (miR)-101-3p expression, and mitochondria function. Here, the link between miR-101-3p and mTOR-regulated mitochondria integrity and ASMC deterioration was assessed. Methods: Disease-specific miR-101-3p expression was determined by RT-PCR in primary ASMC (non-COPD smokers: n = 6; COPD: n = 8; healthy: n = 6). The regulatory effect of miR-101-3p modification on mTOR expression, mitochondrial fragmentation, and remodeling properties (α-SMA, fibronectin, MTCO2, and p70S6 kinase) was assessed in ASMC (healthy nonsmokers: n = 3; COPD: n = 3) by Western blotting and immunofluorescence microscopy. MiR-101-3p was modified by specific mimics or inhibitors, in ASMC stimulated with TNF-α (10 ng/ml) or cigarette smoke extract (CSE). Results: MiR-101-3p expression was significantly higher in ASMC of COPD patients, compared to ASMC of healthy or active smokers. MiR-101-3p expression was increased by TNF-α or CSE. TNF-α or miR-101-3p deteriorated ASMC and mitochondria, while decreasing mTOR signaling, α-SMA, fibronectin, and MTCO2. MiR-101-3p inhibition reduced ASMC deterioration and mitochondrial fragmentation. Conclusion: Constitutive high miR-101-3p expression characterizes COPD-ASMC, causing increased mitochondrial fragmentation and ASMC deterioration. Thus, reactivation mTOR or blocking miR-101-3p presents a potential new strategy for COPD therapy.

Alveolar Basal Cells Differentiate towards Secretory Epithelial- and Aberrant Basaloid-like Cells In Vitro.

In idiopathic pulmonary fibrosis (IPF), keratin (KRT)17+/KRT5+ basal and KRT17+/KRT5- aberrant basaloid cells are atypically present within the alveolar space. We previously described the fibrosis-enriched outgrowth of alveolar basal cells from peripheral fibrotic lung tissue. Using single cell RNA sequencing (scRNA-seq), we here characterize the transcriptome of these cultured alveolar basal cells under different culture conditions. METHODS: Fibrotic peripheral lung tissue pieces were placed in DMEM growth medium. Outgrown cells were analysed by scRNA-seq, TaqMan-PCR or immunofluorescence (IF) either directly or after medium change to an epithelial cell specific medium (Cnt-PR-A). RESULTS: A fraction of alveolar basal cells cultured in DMEM growth medium showed close transcriptomic similarities to IPF basal cells. However, although they expressed KRT5, the transcriptome of the majority of cells matched best to the transcriptome of recently described KRT17+/KRT5- aberrant basaloid cells, co-expressing the canonical basal cell marker KRT17 and mesenchymal cell marker (VIM, FN1). A smaller fraction of cells matched best to secretory epithelial cells. Two differentiation gradients from basal to aberrant basaloid-like cells and basal to secretory epithelial-like cells were apparent. Interestingly, these differentiation paths seemed reversed when the cell culture medium was changed to Cnt-PR-A. CONCLUSIONS: Our results suggest that cultured alveolar basal cells have the capacity to differentiate towards secretory epithelial-like cells and to aberrant basaloid-like cells. However, due to the persistent expression of KRT5, a complete differentiation towards aberrant basaloid cells did not seem to be achieved in our culture conditions. Importantly, differentiation seemed reversible by changing the cells microenvironment. Determining specific factors influencing these differentiation paths may help to define novel drug targets for IPF therapy.

Anti-Fibrotic Effect of SDF-1ß Overexpression in Bleomycin-Injured Rat Lung.

Rational: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease and is associated with high mortality due to a lack of effective treatment. Excessive deposition of the extracellular matrix by activated myofibroblasts in the alveolar space leads to scar formation that hinders gas exchange. Therefore, selectively removing activated myofibroblasts with the aim to repair and remodel fibrotic lungs is a promising approach. Stromal-derived growth factor (SDF-1) is known to stimulate cellular signals which attract stem cells to the site of injury for tissue repair and remodeling. Here, we investigate the effect of overexpression of SDF-1ß on lung structure using the bleomycin-injured rat lung model. Methods: Intratracheal administration of bleomycin was performed in adult male rats (F344). Seven days later, in vivo electroporation-mediated gene transfer of either SDF-1ß or the empty vector was performed. Animals were sacrificed seven days after gene transfer and histology, design-based stereology, flow cytometry, and collagen measurement were performed on the tissue collected. For in vitro experiments, lung fibroblasts obtained from IPF patients were used. Results: Seven days after SDF-1ß gene transfer to bleomycin-injured rat lungs, reduced total collagen, reduced collagen fibrils, improved histology and induced apoptosis of myofibroblasts were observed. Furthermore, it was revealed that TNF-α mediates SDF-1ß-induced apoptosis of myofibroblasts; moreover, SDF-1ß overexpression increased alveolar epithelial cell numbers and proliferation in vivo and also induced their migration in vitro. Conclusions: Our study demonstrates a new antifibrotic mechanism of SDF-1ß overexpression and suggests SDF-1ß as a potential new approach for the treatment of lung fibrosis.

Basal-Like Cell-Conditioned Medium Exerts Anti-Fibrotic Effects In Vitro and In Vivo.

In idiopathic pulmonary fibrosis (IPF), basal-like cells are atypically present in the alveolar region, where they may affect adjacent stromal cells by paracrine mechanisms. We here aimed to confirm the presence of basal-like cells in peripheral IPF lung tissue in vivo, to culture and characterize the cells in vitro, and to investigate their paracrine effects on IPF fibroblasts in vitro and in bleomycin-injured rats in vivo. Basal-like cells are mainly localized in areas of pathological bronchiolization or honeycomb cysts in peripheral IPF lung tissue. Single-cell RNA sequencing (scRNA-seq) demonstrated an overall homogeneity, the expression of the basal cell markers cytokeratin KRT5 and KRT17, and close transcriptomic similarities to basal cells in the majority of cells cultured in vitro. Basal-like cells secreted significant levels of prostaglandin E2 (PGE2), and their conditioned medium (CM) inhibited alpha-smooth muscle actin (α-SMA) and collagen 1A1 (Col1A1) and upregulated matrix metalloproteinase-1 (MMP-1) and hepatocyte growth factor (HGF) by IPF fibroblasts in vitro. The instillation of CM in bleomycin-injured rat lungs resulted in reduced collagen content, improved lung architecture, and reduced α-SMA-positive cells. Our data suggested that basal-like cells may limit aberrant fibroblast activation and differentiation in IPF through paracrine mechanisms.

IPF-Fibroblast Erk1/2 Activity Is Independent from microRNA Cluster 17-92 but Can Be Inhibited by Treprostinil through DUSP1.

Idiopathic pulmonary fibrosis (IPF) is a progressive terminal lung disease, and therapies aim to block fibrosis. Fibroblast proliferation is controlled by C/EBP-ß, microRNA cluster 17-92 (miR17-92), and Erk1/2 mitogen-activated protein kinase. This study assessed the role of miR17-92 in IPF-fibroblast proliferation and its modification by treprostinil. Fibroblasts were isolated from eight IPF patients, five interstitial lung fibrosis patients, and seven control lungs. Fibroblasts were stimulated with TGF-ß1 over 24 h. The miR17-92 expression was analyzed by RT-qPCR, and protein expression by Western blotting. TGF-ß1 upregulated C/EBP-ß in all fibroblasts, which was reduced by treprostinil in control-fibroblasts, but not in IPF-fibroblasts. Compared to controls, the guide strands miR-19a-3p, miR-19b-3p, miR-20a-5p, and miR-92a-3p, as well as the passenger strands miR-17-3p, miR-18-3p, miR-19a-1-5p, and miR-92a-5p were significantly increased in IPF-fibroblasts. In controls, TGF-ß1 and treprostinil significantly reduced specific miR17-92 members. IPF-fibroblast proliferation was inhibited by treprostinil through increased expression of the Erk1/2 inhibitor DUSP1. These data suggest that proliferation control via miR17-92 and C/EBP-ß is disrupted in IPF-fibroblasts. Therefore, the inhibition of early stages of signaling cascades or specific mitogen receptors might be less effective. However, the increased proliferation is sensitive to Erk1/2 inhibition by treprostinil-induced DUSP1.

Multipotent mesenchymal stem cells in lung fibrosis.

RATIONALE: Stem cells have been identified in the human lung; however, their role in lung disease is not clear. We aimed to isolate mesenchymal stem cells (MSC) from human lung tissue and to study their in vitro properties. METHODS: MSC were cultured from lung tissue obtained from patients with fibrotic lung diseases (n = 17), from emphysema (n = 12), and normal lungs (n = 3). Immunofluorescence stainings were used to characterize MSC. The effect of MSC-conditioned media (MSC-CM) on fibroblast proliferation and on lung epithelial wound repair was studied. RESULTS: Expression of CD44, CD90, and CD105 characterized the cells as MSC. Moreover, the cells stained positive for the pluripotency markers Oct3/4 and Nanog. Positive co-stainings of chemokine receptor type 4 (CXCR4) with CD44, CD90 or CD105 indicated the cells are of bone marrow origin. MSC-CM significantly inhibited the proliferation of lung fibroblasts by 29% (p = 0.0001). Lung epithelial repair was markedly increased in the presence of MSC-CM (+ 32%). Significantly more MSC were obtained from fibrotic lungs than from emphysema or control lungs. CONCLUSIONS: Our study demonstrates enhanced numbers of MSC in fibrotic lung tissue as compared to emphysema and normal lung. The cells inhibit the proliferation of fibroblasts and enhance epithelial repair in vitro. Further in vivo studies are needed to elucidate their potential role in the treatment of lung fibrosis.