Abstract
Background In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise.
Methods We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1-overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells.
Results TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log2 fold change (FC) 8.909, adjusted p-value 1.82×10−35) and control fibroblasts (log2FC 8.975, adjusted p-value 3.72×10−28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log2FC 3.136, adjusted p-value 1.41×10−24), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts.
Conclusions Our studies utilising human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.
Tweetable abstract
Multiomic single-cell analyses on human IPF lungs identify a global opening of TWIST1 and other E-box motifs in IPF myofibroblasts, with in vivo murine models confirming a critical regulatory function for TWIST1 in IPF myofibroblast activity https://bit.ly/423aeDl
Footnotes
This article has an editorial commentary: https://doi.org/10.1183/13993003.00881-2023
Author contributions: E. Valenzi designed research studies, conducted experiments, acquired data, analysed data and wrote the manuscript. H. Bahudhanapati conducted experiments, acquired data, analysed data and wrote the manuscript. J. Tan designed research studies, conducted experiments, acquired data, analysed data and wrote the manuscript. T. Tabib conducted experiments, acquired data and analysed data. D.I. Sullivan analysed data. M. Nouraie performed statistical analyses on in vitro and in vivo data. J. Sembrat conducted experiments and acquired data. L. Fan acquired data and provided reagents. K. Chen acquired data and provided reagents. S. Liu performed ChIP-seq data analysis. M. Rojas acquired data and provided samples. A. Lafargue acquired data and provided reagents. D.W. Felsher provided hybrid mouse and wrote the manuscript. P.T. Tran provided hybrid mouse and wrote the manuscript. D.J. Kass designed research studies, acquired data, analysed data and wrote the manuscript. R. Lafyatis designed research studies, analysed data and edited the manuscript.
Conflict of interest: R. Lafyatis reports the following conflicts of interest outside the scope of work of this manuscript: R. Lafyatis has served as a consultant for Pfizer, Bristol Myers Squibb, Boehringer Ingelheim, Formation, Sanofi, Boehringer-Mannheim, Merck and Genentech/Roche, and holds or recently had research grants from Corbus, Formation, Moderna, Regeneron, Pfizer and Kiniksa, and holds equity in Thirona. All other authors report no relevant conflicts of interest.
Support statement: Support for the studies was provided by R01 HL 126990 to D.J. Kass, P50 AR 060780-06A1 to R. Lafyatis and D.J. Kass, and from the Pulmonary Fibrosis Foundation and National Scleroderma Foundation to E. Valenzi.
- Received March 4, 2022.
- Accepted April 20, 2023.
- Copyright ©The authors 2023. For reproduction rights and permissions contact permissions{at}ersnet.org