Abstract

Fibroblastic foci (FF) represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple fibrotic conditions. The aim of this study was to define the major transcriptional programmes involved in fibrogenesis in IPF by profiling un-manipulated myo/fibroblasts within FF in situ by laser capture microdissection. The challenges associated with deriving gene calls from low amounts of RNA and the absence of a meaningful comparator cell type were overcome by adopting novel data mining strategies and by using weighted gene co-expression network analysis (WGCNA), as well as an eigengene-based approach to identify transcriptional signatures which correlate with fibrillar collagen gene expression. WGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that TGF-{beta}1, RhoA kinase and the TSC2/RHEB axis formed major signalling clusters associated with collagen gene expression. Functional studies using CRISPR-Cas9 gene edited cells demonstrated a key role for the TSC2/RHEB axis in regulating TGF-{beta}1-induced mTORC1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts. These data provide strong support for the human tissue-based and bioinformatics approaches adopted to identify critical transcriptional nodes associated with the key pathogenic cell responsible for fibrogenesis in situ and further identifies the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions. What is the key question?Can we identify a transcriptional signature associated with collagen gene expression in the fibrotic focus, the cardinal fibrotic lesion in IPF? What is the bottom line?We herein define the major transcriptional programmes involved in fibrogenesis in IPF by profiling myo/fibroblasts within FF in situ by laser capture microdissection. Why read on?The data provide strong support for a human tissue-based approach to identify critical transcriptional nodes associated with fibrogenesis in situ and further identifies the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions.