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

Abstract

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.

Figure 1.

Single-cell RNA-sequencing analysis of 5 lung samples in 3’ v3 chemistry. (A) Visualization of clustering by UMAP per cell type: 0-SPP1 macrophages, 1-FCN1 macrophages and Dendritic cells, 2-T cells, 3-FABP4 macrophages, 4-Endothelial cells, 5-Fibroblasts, 6-Goblet cells and Alveolar Type 1 cells, 7-NK cells, 8-Ciliated cells and Goblet cells, 9-Alveolar Type 2 cells, 10-Mast cells, 11-Low quality cells, 12-Basal cells, 13-Proliferating cells, 14-Goblet cells, 15-Pericytes and Smooth muscle cells, 16-B cells, 17-Lymphatic endothelial cells, 18- Ciliated cells. Macrophage subpopulations, dendritic cells and proliferating cells are located along the center of the UMAPs. (B) Visualization of clustering by UMAP per individual identity. (C) Visualization of clustering by UMAP per health status. SPP1 macrophages cluster 0 is formed primarily of macrophages from the SSc-ILD patients.

Figure 2.

Single-cell RNA-sequencing analysis of macrophage subpopulations, dendritic cells, proliferating Mφ from 5 lung samples in 3’ v3 chemistry, 1 healthy control and 4 SSc-ILD lung samples. (A) Visualization of clustering by UMAP per cell type. (B) Visualization of clustering by UMAP per health status. (C) Visualization of gene expression by FeaturePlot of macrophage subpopulations gene markers, dendritic cells, proliferating Mφ and other genes expression during analysis of only macrophages, dendritic cells, proliferating Mφ. (D) Visualization of gene expression by DotPlot of macrophages, dendritic cells, proliferating Mφ from 1 healthy control and 4 SSc-ILD lung samples in scRNA-seq 3’ v3 Chemistry. DotPlot shows upregulation of SPP1, MERTK, LGMN genes in SSc-ILD SPP1 Mφ and proliferating Mφ. MMP9 and PLA2G7 genes markers are upregulated in SSc-ILD SPP1 Mφ compared to all other macrophages.

Figure 3.

Single-cell RNA-sequencing analysis of 6 lung samples in 5’ v1 chemistry, including 1 SSc-ILD and 5 healthy control lungs. (A) Visualization of clustering by UMAP per cell type. Macrophage subpopulations, dendritic cells and proliferating cells are located along the left upper part of the UMAPs. Cell clusters were identified as follows: 0-FCN1 macrophages and Dendritic cells, 1-T cells and NK cells, 2-Endothelial cells, 3-Epithelial cells, 4-FABP4 macrophages, 5-SPP1 macrophages, 6-Fibroblasts, 7-Lymphatic endothelial cells, 8-B cells, 9-Pericytes and Smooth muscle cells, 10-Mitochondrial related genes, 11-Mast cells, 12-Proliferating cells, 13-Dendritic cells, 14-Hemoglobin related cells. (B) Visualization of clustering by UMAP per health status. SPP1 macrophages cluster 5 is formed primarily of macrophages from the SSc-ILD patient SC335. (C) Visualization of clustering by UMAP per individual identity.

Figure 4.

Regulons and Transcription Factors predicted to be important for SPP1 Mφ during analysis of macrophages, dendritic cells, proliferating Mφ from 5 lung samples in scRNA-seq 3’ v3 chemistry by SCENIC. (A) Cell clusters are identified as follows: 0- SSc-ILD FABP4 Mφ (blue), 1- SSc-ILD SPP1 Mφ (green), 2- HC SPP1 and FABP4 Mφ (purple), 3- SSc-ILD and HC FCN1 Mφ (pink), 4- Proliferating Mφ (yellow), 5- Dendritic cells (light blue). (B) SPP1 is a target gene for ATF5 and TFEB transcription factors and regulons. (C) Transcription factor DotPlot predicted to be important for SPP1 Mφ by SCENIC including transcription factors ATF5 and TFEB indicated in red box. (D) Regulon DotPlot predicted to be important for SPP1 Mφ by SCENIC including regulons ATF5 and TFEB indicated in red box.

Figure 5.

ScATAC-sequencing analysis of 2 SSc-ILD lung samples by Signac showing chromatin pattern changes for SPP1 macrophages compared to FABP4 macrophages. The red arrow indicates the direction of transcription. Exons are shown in blocks and introns flank exons. (A, B) SPP1 gene showed more accessible chromatin for the SSc-ILD SPP1 Mφ compared to FABP4 Mφ in the region proximal to the transcriptional start site for a SSc-ILD lung, as well as regions further 5’ of the promoter and in intron 4. (C, D) MMP9 gene also showed more accessible chromatin in SPP1 Mφ, but in this case increased accessibility was not seen around the promoter but rather in regions around exon 6, exons 9–12 and introns. (E, F) FABP4 gene showed strikingly more accessible chromatin in a region proximal to the transcriptional start site and in a broad second region 3’ from the gene in FABP4 Mφ compared to SPP1 Mφ.