P152 Idiopathic pulmonary fibrosis related expression of cell extrusion genes within epithelial cell types

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Background Idiopathic pulmonary fibrosis (IPF) is characterised by scarring of lung interstitium and destruction of epithelial basement membrane. Cell extrusion maintains tissue architecture, whilst dysfunction may promote persistence of damaged and apoptotic cells, compromising barrier integrity, potentially resulting in fibrotic remodelling. Cell extrusion is a difficult process to capture experimentally, and it’s not known whether it occurs in the alveolus. PIEZO1 is a stretch-activated ion channel and mechanosensor described as an upstream factor in live cell extrusion. It is a key mediator of extrusion therefore, to help validate whether extrusion is happening in alveolus an extrusion gene signature was needed. Hence, we created a gene panel based on network interactions with PIEZO1 to investigate dysfunctional extrusion in IPF. We hypothesised that dysregulated extrusion-related genes are differentially expressed in IPF lung cells compared to control. Methods Using PIEZO1 as a hub gene, a panel of extrusion-related genes was curated with STRING database, PANTHER classification and KEGG pathway analysis. Differential expression was evaluated with Seurat using IPF and control single cell RNA sequencing (scRNAseq), integrated from three publicly available datasets (GSE136831; GSE135893; GSE128033). Cell types of focus included AT2, pan-endothelial, basal and dividing cells annotated with CellMarker2.0. Results A total of 74 cell extrusion-related genes that interacted with PIEZO1 were selected using STRING database for evaluation in scRNAseq data from 52 IPF and 48 control lung tissues. Epithelial cell types showed six upregulated genes (RHOB, TRPV4, ACTN1, EGFR, PLXNB1, IL18) and seven downregulated genes (NAIP, CAV1, ARHGEF1, EFNA1, TSPAN5, PIEZO2, TRAF6). AT2 cells showed robust upregulation of RHOB (p=2.19E-07) and TRPV4 (p=4.36E-05), downregulation in CAV1 (p=1.75E-06), ARHGEF1 (p=1.349E-3) and EFNA1 (p=2.017E-03). Basal cells showed upregulation in TRPV4 (p=3.767E-03), ACTN1 (p=8.22E-05), EGFR (p=4.26E-07), PLXN1 (p=1.31E-05) and IL18 (p=1.002E-03). A downregulation of TRAF6 was observed in dividing cells (p=4.742E-03). Conclusion Using PIEZO1 as a hub gene, a restricted set of extrusion-related genes were found to be differentially expressed in IPF lung epithelial cells. Expression changes in the gene panel can be further utilised in the design of mechanistic studies to assess and validate the role of epithelial cell extrusion in IPF pathology.