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.

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.

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.