Introduction Fibroblastic foci 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 unmanipulated myofibroblasts within fibrotic foci in situ by laser capture microdissection.
Methods 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.
Results WGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that transforming growth factor β1 (TGF-β1), 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-β1-induced mechanistic target of rapamycin complex 1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts.
Conclusion 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 identify the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions.
- idiopathic pulmonary fibrosis
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DG, ART and MP are joint first authors.
ADB and RCC are joint senior authors.
Twitter @DelphineGuillo6, @Manup253, @robhynds
ADB and RCC contributed equally.
DG, ART and MP contributed equally.
Contributors DG performed in vitro experiments, interpreted data, prepared figures and participated in drafting of the manuscript and reviewed and approved the final version. AT designed the bioinformatics analysis workflow, participated in drafting the manuscript and reviewed and approved the final version. MP performed in vitro experiments and analysed data and reviewed and approved the final manuscript. PFM supervised the study and identified IPF FF and reviewed and approved the final manuscript. LME designed the bioinformatics analysis workflow and approved the final manuscript. RH and GM performed laser capture microdissection (LCM) of IPF FF and microarray analysis and approved the final manuscript. RJM obtained ethical approvals, provided patient tissue, participated in the identification of IPF FF and reviewed and approved the final manuscript. AJF and TMM obtained ethical approvals, consented patients and provided IPF patient tissue, collected clinical data and reviewed and approved the final manuscript. REH isolated and characterised cancer-associated fibroblasts (CAFs) from patients with lung adenocarcinoma (TRACERx clinical study) and approved the final manuscript. MJ-H on behalf of the TRACERx consortium obtained ethics approval, provided lung adenocarcinoma patient tissue, collected clinical data and approved the final manuscript. RM conceived the study and reviewed and approved the final manuscript. RCC and AB conceived and designed the study, interpreted data, drafted the manuscript and reviewed and approved the final version.
Funding RCC acknowledges funding support via a collaborative framework agreement between University College London (UCL) and GSK; and from the National Institute for Health Research (NIHR) University College London Hospitals Biomedical Research Centre (UCLH BRC). AJF acknowledges funding support via a collaborative framework agreement between Newcastle University and GSK; and from the NIHR Newcastle Biomedical Research Centre (Newcastle BRC). TMM is supported by an NIHR Clinician Scientist Fellowship (NIHR Ref: CS-2013-13-017), holds a British Lung Foundation Chair in Respiratory Research (C17-3) and acknowledges funding support from the NIHR Royal Brompton Hospital Biomedical Research Unit (BRU). The TRACERx study is funded by Cancer Research UK (CRUK; C11496/A17786) and the derivation of TRACERx patient models was supported by the CRUK Lung Cancer Centre of Excellence. MP acknowledges funds from the British Lung Foundation.
Competing interests RCC received grants from GSK via her institution during the conduct of the study; consultancy fees from Pieris Pharmaceuticals, consultancy fees from Chiesi, consultancy fees from Theravance Biopharma outside the submitted work; and RCC spouse was an employee of GSK during the time this work was conducted. This potential COI was managed by a research framework agreement between UCL and GSK. DG and PFM received salary from this research framework agreement. RM, AT, LME and AB were employees of GlaxoSmithKline during the time this work was performed and hold GSK shares. AJF has received a research grant from GSK to support work contained in this manuscript. RH and GM were employees of Epistem with a commercial interest in the provision of services in the area of investigation. TMM has, via his institution, received industry-academic funding from GSK, R&D and UCB and has received consultancy or speakers fees from Apellis, AstraZeneca, Bayer, Blade Therapeutics, Boehringer Ingelheim, Bristol-Myers Squibb, Galapagos, GlaxoSmithKline R&D, Indalo, Novartis, Pliant, ProMetic, Respivnat, Roche, Samumed and UCB. MJH reports non-financial support from Achilles Therapeutics outside the submitted work. MP, RJM and REH have nothing to disclose.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All the relevant data are included in the article, uploaded as supplementary information, or available on the Gene Expression Omnibus (GSE98925).
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