Disparate Interferon Signaling and Shared Aberrant Basaloid Cells in Single-Cell Profiling of Idiopathic Pulmonary Fibrosis and Systemic Sclerosis-Associated Interstitial Lung Disease

Abstract

Idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) differ in the predominant demographics and identified genetic risk alleles of effected patients, however both diseases frequently progress to respiratory failure and death. Contrasting advanced SSc-ILD to IPF provides insight to the role dysregulated immunity may play in pulmonary fibrosis. To analyze cell-type specific transcriptome commonalities and differences between IPF and SSc-ILD, we compared single-cell RNA-sequencing (scRNA-seq) of 21 explanted lung tissue specimens from patients with advanced IPF, SSc-ILD, and organ donor controls. Comparison of IPF and SSc-ILD tissue identified divergent patterns of interferon signaling, with interferon-gamma signaling upregulated in the SPP1^hi and FABP4^hi macrophages, cytotoxic T cells, and natural kill cells of IPF, while type I interferon signaling and production was upregulated in the corresponding SSc-ILD populations. Plasmacytoid dendritic cells were found in diseased lungs only, and exhibited upregulated cellular stress pathways in SSc-ILD compared to IPF. Alveolar type I cells were dramatically decreased in both IPF and SSc-ILD, with a distinct transcriptome signature separating these cells by disease. KRT5^-/KRT17⁺ aberrant basaloid cells exhibiting markers of cellular senescence and epithelial-mesenchymal transition were identified in SSc-ILD for the first time. In summary, our study utilizes the enriched capabilities of scRNA-seq to identify key divergent cell types and pathways between IPF and SSc-ILD, providing new insights into the shared and distinct mechanisms between idiopathic and autoimmune interstitial lung diseases.

Keywords: idiopathic pulmonary fibrosis; interstitial lung disease (ILD); single-cell RNA-sequencing (scRNA-seq); systemic sclerosis; systemic sclerosis (scleroderma).

Figure 1

ScRNA-seq analysis of 8 IPF, 8 SSc-ILD, and 5 organ donor control lung samples. (A) UMAP plot of all 21 integrated samples, identified by cell type. (B) UMAP plots of all samples, divided by disease status. (C) Mean percentage of total cells comprised of each cell type, comparing, control, upper lobe IPF, lower lobe IPF, upper lobe SSc-ILD, and lower lobe SSc-ILD. Bars represent the mean percentage of total cells, with error bars representing the standard error of the mean. *=p-value<0.05. scRNA-seq, single-cell RNA-sequencing; IPF, idiopathic pulmonary fibrosis; SSc-ILD, systemic sclerosis-associated interstitial lung disease; UMAP, uniform manifold approximation and projection.

Figure 2

ScRNA-seq analysis of IPF, SSc-ILD, and control myeloid populations. (A) UMAP plot of macrophage and dendritic cell populations, identified by cell type. (B) UMAP plot of macrophage and dendritic cell populations, identified by disease status. (C) Pathways upregulated in IPF SPP1^hi macrophages compared to SSc-ILD SPP1^hi macrophages (D) Pathways upregulated in SSc-ILD SPP1^hi macrophages compared to IPF SPP1^hi macrophages (E) Expression of selected genes and pathways in SPP1^hi macrophages, separated by disease state. Pathway expression depicted as a module score of all genes in the specific GO biological process. (F) Pathways upregulated in IPF FABP4^hi macrophages compared to SSc-ILD FABP4^hi macrophages. (G) Pathways upregulated in SSc-ILD FABP4^hi macrophages compared to IPF FABP4^hi macrophages. (H) Expression of selected genes and pathways in FABP4^hi macrophages, separated by disease state. scRNA-seq, single-cell RNA-sequencing; IPF, idiopathic pulmonary fibrosis; SSc-ILD, systemic sclerosis-associated interstitial lung disease; UMAP, uniform manifold approximation and projection; GO, gene ontology.

Figure 3

ScRNA-seq analysis of IPF, SSc-ILD, and control lymphoid populations. (A) UMAP plot of lymphoid subpopulations, identified by cell type. (B) UMAP plot of lymphoid subpopulations, identified by disease status. (C) Pathways upregulated in IPF cytotoxic T cells compared to SSc-ILD cytotoxic T cells. If fold enrichment was calculated as >100 it is depicted as 100 for visualization. (D) Pathways upregulated in SSc-ILD cytotoxic T cells compared to IPF cytotoxic T cells (E) Expression of selected genes and pathways in cytotoxic T cells, separated by disease state. Pathway expression depicted as a module score of all genes in the specific GO biological process. (F) Pathways upregulated in IPF NK cells compared to SSc-ILD NK cells (G) Pathways upregulated in SSc-ILD NK cells compared to IPF NK cells (H) Expression of selected genes and pathways in NK cells, separated by disease state. scRNA-seq, single-cell RNA-sequencing; IPF, idiopathic pulmonary fibrosis; SSc-ILD, systemic sclerosis-associated interstitial lung disease; UMAP, uniform manifold approximation and projection; NK, natural killer.

Figure 4

ScRNA-seq analysis of IPF, SSc-ILD, and control plasmacytoid dendritic cell populations. (A) UMAP plot of the reclustered original B lymphocyte and plasma cell cluster, identified by cell type. (B) UMAP plot of the reclustered original B lymphocyte and plasma cell cluster, identified by disease status. (C) Expression of IL3RA, CLEC4C, CLIC3, and NRP1, identifying pDCs. (D) Expression of IFNAR1and IFNAR2 in IPF versus SSc-ILD pDCs. (E) Pathways upregulated in SSc-ILD pDCs compared to IPF pDCs. scRNA-seq, single-cell RNA-sequencing; IPF, idiopathic pulmonary fibrosis; SSc-ILD, systemic sclerosis-associated interstitial lung disease; UMAP, uniform manifold approximation and projection; pDCs, plasmacytoid dendritic cells.

Figure 5

ScRNA-seq analysis of IPF, SSc-ILD, and control epithelial cell populations. (A) UMAP plot of epithelial cell populations, identified by cell type (B) UMAP plot of epithelial cell populations, identified by disease status. (C) Expression of ITGAV, ITGB6, IL32, and MMP7, highlighting the aberrant basaloid population. (D) Pathways upregulated in IPF AT1 cell gene signature compared to SSc-ILD AT1 cell gene signature. (E) Pathways upregulated in SSc-ILD AT1 cell gene signature compared to IPF AT1 cell gene signature. scRNA-seq, single-cell RNA-sequencing; IPF, idiopathic pulmonary fibrosis; SSc-ILD, systemic sclerosis-associated interstitial lung disease; UMAP, uniform manifold approximation and projection; AT1, alveolar type 1.

Figure 6

Hierarchical clustering on gene average expression levels of epithelial cell subtypes divided by disease status, demonstrating distinct gene signatures of IPF and SSc-ILD AT1 cells. Yellow indicates positive and blue indicates negative scaled expression. (A) Partial gene signature of IPF AT1 cells (B) Partial gene signature of SSc-ILD AT1 cells. IPF, idiopathic pulmonary fibrosis; SSc-ILD, systemic sclerosis-associated interstitial lung disease; AT1, alveolar type 1; AT2, alveolar type 2.

Figure 7

ScRNA-seq analysis of IPF, SSc-ILD, and control stromal cell populations. (A) UMAP plot of stromal cell populations, identified by cell type. (B) UMAP plot of stromal cell populations, identified by disease status. (C) Pathways upregulated in IPF endothelial cells compared to SSc-ILD endothelial cells (D) Pathways upregulated in SSc-ILD endothelial cells compared to IPF endothelial cells (E) Expression of selected genes in all non-lymphatic endothelial cells, separated by disease state. scRNA-seq, single-cell RNA-sequencing; IPF, idiopathic pulmonary fibrosis; SSc-ILD, systemic sclerosis-associated interstitial lung disease; UMAP, uniform manifold approximation and projection.