Single-cell RNA sequencing depicts the local cell landscape in thyroid-associated ophthalmopathy

Abstract

There is a specific reactivity and characteristic remodeling of the periocular tissue in thyroid-associated ophthalmopathy (TAO). However, local cell changes responsible for these pathological processes have not been sufficiently identified. Here, single-cell RNA sequencing is performed to characterize the transcriptional changes of cellular components in the orbital connective tissue in individuals with TAO. Our study shows that lipofibroblasts with RASD1 expression are highly involved in inflammation and adipogenesis during TAO. ACKR1⁺ endothelial cells and adipose tissue macrophages may engage in TAO pathogenesis. We find CD8⁺CD57⁺ cytotoxic T lymphocytes with the terminal differentiation phenotype to be another source of interferon-γ, a molecule actively engaging in TAO pathogenesis. Cell-cell communication analysis reveals increased activity of CXCL8/ACKR1 and TNFSF4/TNFRSF4 interactions in TAO. This study provides a comprehensive local cell landscape of TAO and may be valuable for future therapy investigation.

Keywords: autoimmune disease; orbital fibroblast; single-cell RNA sequencing; thyroid-associated ophthalmopathy.

Graphical abstract

Figure 1

Study design and unbiased classification of cells (A) Schematic of the experimental design for single-cell RNA sequencing (scRNA-seq). The orbital connective tissue (OCT) was isolated from healthy controls (HCs) and individuals with thyroid-associated ophthalmopathy (TAO) and subsequently processed through scRNA-seq using the 10X Genomics platform. (B) t-SNE plot showing clusters of major cell types. (C) t-SNE plots of canonical markers for major cell types in the OCT. (D) Heatmap showing scaled expression of discriminative gene sets for major cell types in the OCT. The color scheme is based on Z score distribution from −2 (blue) to 2 (red). (E) Bar chart showing the relative proportion of major cell types in OCT from HCs and individuals with TAO derived from scRNA-seq data.

Figure 2

Characterization of OF subsets in OCT in TAO (A) Uniform manifold approximation and projection (UMAP) plot showing clusters of orbital fibroblast (OF) subsets. (B) The expression levels of selected functional genes in OF subsets. (C) Heatmap showing scaled expression of discriminative gene sets in each OF subset. The color scheme is based on Z score distribution from −2 (blue) to 2 (red). (D) Representative GO terms and KEGG pathway enriched by upregulated DEGs of each OF subset. (E) Bar chart showing the relative proportion of each OF subset in OCT from HCs and individuals with TAO. (F) Pseudotime analysis of OF subsets. Left: distribution of the three subpopulations on each of the branches. Center: distribution of the cells from HCs or individuals with TAO on each of the branches. Right: the relative proportion of cells in each state in HCs and individuals with TAO.

Figure 3

Characterization of LPF subsets in OCT in TAO (A) Volcano plot showing DEGs of LPF subsets in TAO/HC comparison. (B) Venn diagram showing comparative analysis of upregulated DEGs of LPF subsets in three fibroblast subtypes and upregulated DEGs of LPF subsets in TAO/HC comparison. The count shows the number of DEGs. (C) Heatmap of the relative expression of selected LPF-specific upregulated DEGs in HC and TAO groups in different fibroblast subtypes. (D) Bar chart showing representative GO terms and KEGG pathways enriched in upregulated DEGs of LPF subtypes in TAO/HC comparison. (E) UMAP plot showing the expression of ACKR1 and CXCR2 of OCT cells from HCs and individuals with TAO. (F) Pseudotime-ordered single-cell expression trajectories for FABP4, FABP5, CEBPB, CEBPD, PLIN2, PPARG, RASD1, and PIM1. (G) Representative images of immunostaining of cross-sections of OCTs from HCs and individuals with TAO for PIM1 (green) and nuclei (4′,6-diamidino-2-phenylindole [DAPI]; blue). Each group contains six samples. Data are represented as mean ± SEM. Significance was determined using unpaired Student’s t test. ∗p < 0.05.

Figure 4

Characterization of EC subsets in OCT in TAO (A) UMAP plot showing clusters of endothelial cell (EC) subsets. (B) Heatmap showing scaled expression of discriminative gene sets for ACKR1⁺ and ACKR1⁻ ECs in OCT. The color scheme is based on Z score distribution from −2 (blue) to 2 (red). (C) Bar chart showing the relative proportion of ACKR1⁺ and ACKR1⁻ ECs in OCT from HCs and individuals with TAO. (D) Representative GO terms and KEGG pathway enriched by upregulated DEGs of each EC subtype. (E and F) Volcano plots showing DEGs of ACKR1⁺ EC (E) and ACKR1⁻ EC (F) subsets in TAO/HC comparison. (G) Violin plot showing the expression of CXCL2, CXCL8, CXCL14, and IL-6 in ACKR1⁺ ECs and ACKR1⁻ ECs from HCs and individuals with TAO. (H) Violin plot showing the expression of CXCL2, CSF3, IL-1R1, ITGAV, SELE, SELP, and VCAM1 in ACKR1⁺ ECs and ACKR1⁻ ECs from HCs and individuals with TAO. (I) Representative GO terms and KEGG pathway enriched by upregulated DEGs of ACKR1⁺ ECs and ACKR1⁻ ECs in TAO/HC comparison.

Figure 5

Characterization of NK&TC subsets in OCT in TAO (A) UMAP plot showing clusters of NK&TC subsets. (B) Heatmap showing scaled expression of discriminative gene sets for NK&TC subsets in OCT. The color scheme is based on Z score distribution from −2 (blue) to 2 (red). (C) UMAP plot showing the expression of selected genes in NK&TC subsets. (D and E) Volcano plots showing DEGs of CD4⁺ Tm cell (D) and CD8⁺ CTL (E) subsets in TAO/HC comparison. (F) Representative GO terms and KEGG pathway enriched by upregulated DEGs of each NK&TC subtype in OCT in TAO/HC comparison.

Figure 6

Characterization of MC subsets in OCT in TAO (A) UMAP plot showing clusters of mononuclear phagocyte subsets. (B) Heatmap showing scaled expression of discriminative gene sets for mononuclear phagocyte subsets in OCT. The color scheme is based on Z score distribution from −2 (blue) to 2 (red). (C–F) Volcano plots showing DEGs of CD16⁺ Mos (C), CD14⁺ Mos (D), DCs (E), and Macs (F) in TAO/HC comparison. (G) The expression of CD36, CD163, and MRC1 by mononuclear phagocyte subsets from HCs and individuals with TAO patients by UMAP plot (left) and violin plot (right). (H) Representative GO terms and KEGG pathway enriched by upregulated DEGs of Macs in OCT in TAO/HC comparison.

Figure 7

Development of an OF-based regulatory network for TAO (A) Heatmap showing the interaction intensity among major cell subsets in OCT from HCs. (B) Heatmap showing the interaction intensity among major cell subsets in OCT from TAO. (C) The interaction of ACKR1⁺ ECs with COFs, LPFs, and MYFs in individuals with TAO and HCs. (D) The interaction of ACKR1⁺ ECs with other major cell subsets in OCT from individuals with TAO and HCs via the CXCL8/ACKR1 interaction pair. (E) UMAP plots showing the expression of CXCL8 and ACKR1 by OCT cells from HCs and individuals with TAO. (F) The interaction of OF subsets with other major cell subsets in OCT from individuals with TAO and HCs via the TNFSF4/TNFRSF4 interaction pair. (G) UMAP plots showing the expression of TNFRSF4 and TNFSF4 by OCT cells from HCs and individuals with TAO.