Epigenetic Regulation of Profibrotic Macrophages in Systemic Sclerosis–Associated Interstitial Lung Disease

Objective Systemic sclerosis–associated interstitial lung disease (SSc‐ILD) is the leading cause of death in patients with SSc with unclear pathogenesis and limited treatment options. Evidence strongly supports an important role for profibrotic secreted phosphoprotein 1 (SPP1)–expressing macrophages in SSc‐ILD. This study was undertaken to define the transcriptome and chromatin structural changes of SPP1 SSc‐ILD macrophages in order to better understand their role in promoting fibrosis and to identify transcription factors associated with open chromatin driving their altered phenotype. Methods We performed single‐cell RNA sequencing (scRNA‐Seq) on 11 explanted SSc‐ILD and healthy control lung samples, as well as single‐cell assay for transposase‐accessible chromatin sequencing on 5 lung samples to define altered chromatin accessibility of SPP1 macrophages. We predicted transcription factors regulating SPP1 macrophages using single‐cell regulatory network inference and clustering (SCENIC) and determined transcription factor binding sites associated with global alterations in SPP1 chromatin accessibility using Signac/Seurat. Results We identified distinct macrophage subpopulations using scRNA‐Seq analysis in healthy and SSc‐ILD lungs and assessed gene expression changes during the change of healthy control macrophages into SPP1 macrophages. Analysis of open chromatin validated SCENIC predictions, indicating that microphthalmia‐associated transcription factor, transcription factor EB, activating transcription factor 6, sterol regulatory element binding transcription factor 1, basic helix‐loop‐helix family member E40, Kruppel‐like factor 6, ETS variant transcription factor 5, and/or members of the activator protein 1 family of transcription factors regulate SPP1 macrophage differentiation. Conclusion Our findings shed light on the underlying changes in chromatin structure and transcription factor regulation of profibrotic SPP1 macrophages in SSc‐ILD. Similar alterations in SPP1 macrophages may underpin fibrosis in other organs involved in SSc and point to novel targets for the treatment of SSc‐ILD, specifically targeting profibrotic macrophages.