Comprehensive analysis of a mouse model of spontaneous uveoretinitis using single-cell RNA sequencing

Abstract

Autoimmune uveoretinitis is a significant cause of visual loss, and mouse models offer unique opportunities to study its disease mechanisms. Aire^-/- mice fail to express self-antigens in the thymus, exhibit reduced central tolerance, and develop a spontaneous, chronic, and progressive uveoretinitis. Using single-cell RNA sequencing (scRNA-seq), we characterized wild-type and Aire^-/- retinas to define, in a comprehensive and unbiased manner, the cell populations and gene expression patterns associated with disease. Based on scRNA-seq, immunostaining, and in situ hybridization, we infer that 1) the dominant effector response in Aire^-/- retinas is Th1-driven, 2) a subset of monocytes convert to either a macrophage/microglia state or a dendritic cell state, 3) the development of tertiary lymphoid structures constitutes part of the Aire^-/- retinal phenotype, 4) all major resident retinal cell types respond to interferon gamma (IFNG) by changing their patterns of gene expression, and 5) Muller glia up-regulate specific genes in response to IFN gamma and may act as antigen-presenting cells.

Keywords: Aire knockout; autoimmune uveitis; mouse model; ocular immunology; single-cell RNAseq.

Fig. 1.

Characterization and scRNA-seq analysis of Aire^−/− retinas. (A) Aire^−/− retina fundus photographs (Left) and immunostaining of the corresponding cross-sections (Right) showing leukocytes (CD45; red) and nuclei (DAPI; blue). Representative examples of grade 1 to grade 4 uveoretinitis are shown. In this and other retina cross-sections, the following abbreviations are used: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Scale bar, 50 μm.) (B) Fundus photographs of 4 Aire^−/− retinas used for scRNA-seq. (C) UMAP plot showing different cell-type clusters in a merged dataset from duplicate samples of Aire^−/− retinas (shown in B) and age- and sex-matched WT control retinas. (D) UMAP plot of immune cells in the merged dataset. (E) Stacked bar plots showing the proportions of nonmicroglial immune cell types in scRNA-seq datasets from Aire^−/− and WT retinas. The numbers of immune cells in each dataset are indicated below.

Fig. 2.

Analysis of T cell diversity in Aire^−/− retinas. (A) UMAP plot of Cd3-expressing cells from Aire^−/− retinas showing different T cell and NK cell subtypes. (B) Heatmap showing, for each T and NK cell subtype (horizontal axis), the scaled mean unique molecular identifiers (UMIs) of transcripts for known cell-type markers (vertical axis). (C) Immunostaining of cross-sections of WT vs. Aire^−/− retinas showing T-BET (green), IFNG (red), CD4 (magenta), and nuclei (DAPI; blue). T-BET+ IFNG+ CD4+ Th1 cells are indicated by arrows. In this and other figures, the regions in the square Insets are enlarged below. (D) UMAP replotting of Th1 cells from the UMAP plot in A enclosed in dashed lines showing 2 distinct clusters of Th1 cells: a Cd40-ligand (Cd40Lg)–positive cluster and an IL10-positive cluster (SI Appendix, Fig. S2B). (E) Immunostaining of cross-sections of WT vs. Aire^−/− retinas showing T-BET (green), IL10 (red), CD4 (magenta), and nuclei (DAPI; blue). T-BET+ IL10+ CD4+ Tfh cells are indicated by arrows. (F) Heatmap showing 40 genes that are differentially expressed between the 2 Th1 clusters. (G, Left) Bar plots showing total UMIs for Tgfb1, Tgfb2, and Tgfb3. (G, Right) Heatmap showing mean expression per cell of Tgfb1, Tgfb2, and Tgfb3 for each cell type. (H) Retinal cross-sections showing fluorescent ISH for Tgfb2 (orange) and immunostaining for COL4A (green) and CD45 (magenta); nuclei are marked by DAPI (blue). (Scale bars in C, E, and H, 50 μm.)

Fig. 3.

Presence of tertiary lymphoid structures in Aire^−/− retinas. (A–D) Immunostaining of cross-sections of WT vs. Aire^−/− retinas. (A) CD8 (green), CD19 (red), CD4 (magenta), and nuclei (DAPI; blue). (B) BCL6 (green), CD19 (red), CD4 (magenta), and nuclei (DAPI; blue). BCL6+CD4+ cells are indicated by arrows. (C) PNAd (green), CD19 (red), CD4 (magenta), and nuclei (DAPI; blue). A PNAd+ vessel resembling an HEV is indicated by the arrows. (D) MHC class II (I-A and I-E; green), CCR7 (red), CD4 (magenta), and nuclei (DAPI; blue). (E) CD80 (green), pZAP70 (red), CD4 (magenta), and nuclei (DAPI; blue). (F) SDC1 (green), CD19 (red), CD4 (magenta), and nuclei (DAPI; blue). CD19− SDC1+ plasma cells are indicated by arrows; a CD19+ SDC1+ plasmablast is indicated by the arrowheads. (Scale bars in A–F, 50 μm.)

Fig. 4.

Divergent fates of monocyte lineage cells in Aire^−/− retinas. (A) UMAP plot from Aire^−/− retinas showing different subtypes of monocyte lineage cells. (B) Cell trajectory of monocyte lineage cells showing a bifurcation into Mo-MΦs and Mo-DCs. (C) Heatmap showing branch expression analysis modeling of the branch point between Mo-MΦs (Left) and Mo-DCs (Right). (D) Expression of Cd14, Mrc1, P2ry12, Smad7, and Zbtb46 along the cell trajectory shown in B. (E) Immunostaining of cross-sections of WT vs. Aire^−/− retinas showing P2RY12 (green), CD14 (red), IBA1 (magenta), and nuclei (DAPI; blue). A P2RY12+ CD14+ cell is indicated by the arrows. (F) Immunostaining as in E for P2RY12 (green), MRC1 (red), IBA1 (magenta), and nuclei (DAPI; blue). Most IBA1+ P2RY12+ cells are MRC1− (indicated by arrowheads) with a minority being MRC1+ (indicated by arrows). (G) Barplots showing mean log₁₀ normalized expression per cell for Cd68, Aif1, and Lgals. *P value < 0.05 and **P value < 0.01. (H) Immunostaining as in E for CD68 (green), IBA1 (red), Galectin 3 (magenta), and nuclei (DAPI; blue). (Scale bars in E, F, and H, 50 μm.)

Fig. 5.

IFN gamma response in resident cells in Aire^−/− retinas. (A and B) Statistically significant MSigDB Hallmark pathways in 3 or more cell types in GSEA reveal pathways enriched in Aire^−/− over WT retinas across multiple retinal cell types in grade 2 and grade 3 disease. *P value < 0.05. (C and D) Heatmaps showing GSEA for the IFN gamma response gene set in grade 2 (A) and grade 3 (B) Aire^−/− retinas. (Left) Single column showing normalized enrichment score (NES) for each cell type. (Right) Heatmap showing normalized regression coefficients for each cell type (rows represent cell types; columns represent genes). (E) Immunostaining of cross-sections of WT vs. Aire^−/− retinas showing IRF1 (green), CD45 (red), COL4A1 (magenta), and nuclei (DAPI; blue). (F) Immunostaining as in E for PSMB9 (green), CD45 (red), COL4A1 (magenta), and nuclei (DAPI; blue). (Scale bars in E and F, 50 μm.)

Fig. 6.

Activation of IFN gamma target genes in Muller glia in Aire^−/− retinas. (A) Immunostaining of cross-sections of WT vs. Aire^−/− retinas showing SOX9 (green), IRF1 (red), CD45 (magenta), and nuclei (DAPI; blue). SOX9 marks RPE nuclei (near the top of the image), Muller glia nuclei in the INL, and astrocyte nuclei in the GCL. (B) Heatmap showing, by cell type, IFN gamma target genes that are preferentially up-regulated in Muller glia in Aire^−/− vs. WT retinas. Individual cells are arrayed across the horizontal axis, and genes are shown along the vertical axis. Up-regulation of genes indicated by arrows was validated by fluorescent ISH in C. (C) Retinal cross-sections as in A showing fluorescent ISH of C4b transcripts (orange) and immunofluorescence of CD45 (magenta); nuclei marked by DAPI (blue). (D) Retinal cross-sections as in A showing fluorescent ISH of Cd274 transcripts (green) and immunofluorescent staining of CD45 (magenta); nuclei are marked by DAPI (blue). (E) Heatmap as in B showing, by cell type, MHC class II genes that are significantly up-regulated in Aire^−/− vs. WT retinas. MHC class II genes include the I-A and I-E alloantigens that can be detected by the anti-MHC II I-A/I-E antibody (arrows). (F) Retinal cross-sections as in A showing MHC II (I-A/I-E) (green), SOX9 (red), CD4 (magenta), and nuclei (DAPI; blue). A CD4+ T cell (indicated by arrowheads) is seen associating with an MHC class II+ process of a SOX9+ Muller glial cell (nucleus indicated by arrows). In addition, SOX9− cells with intense MHC class II staining (indicated by dashed arrows) scattered across the retina are mostly likely microglia, Mo-MΦs, or Mo-DCs. (Scale bars in A, C, D, and F, 50 μm.)