Non-neuronal cells are key to the complex cellular interplay that follows central nervous system insult. To understand this interplay, we generated a single-cell atlas of immune, glial and retinal pigment epithelial cells from adult mouse retina before and at multiple time points after axonal transection. We identified rare subsets in naive retina, including interferon (IFN)-response glia and border-associated macrophages, and delineated injury-induced changes in cell composition, expression programs and interactions. Computational analysis charted a three-phase multicellular inflammatory cascade after injury. In the early phase, retinal macroglia and microglia were reactivated, providing chemotactic signals concurrent with infiltration of CCR2+ monocytes from the circulation. These cells differentiated into macrophages in the intermediate phase, while an IFN-response program, likely driven by microglia-derived type I IFN, was activated across resident glia. The late phase indicated inflammatory resolution. Our findings provide a framework to decipher cellular circuitry, spatial relationships and molecular interactions following tissue injury.
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Data generated during this study have been deposited in Gene Expression Omnibus under accession number GSE199317. The data can be visualized in the Broad Institute Single Cell Portal (https://singlecell.broadinstitute.org/single_cell/study/SCP1785).
Scripts have been deposited to https://bitbucket.org/jerry00/onc-retina-script/src/master/.
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We acknowledge the authors whose work could not be cited due to space limitations. We thank I. Avraham-Davidi, T. van Zyl, R. Kedmi, I. Shachar and M. Schwartz for helpful discussions, M. Schwartz and M. Mack for kindly providing the MC-21 antibody, Y. Okunuki and K. Connor for their help with the PLX experiment, L. Jerby-Arnon for assistance with the DIALOGUE analysis, K. Dey and K. Jagadeesh for help with genome-wide association study analysis, R. Harpaz for help with RPE image analysis and Z. Niziolek, S. Turney, Y. Li, R. Schaffer, M. Laboulaye, E. Martersteck, T. Delorey, D. Phillips and staff members of the Harvard University Bauer Core Facility and the Koch Institute Histology Core for technical assistance. We thank L. Gaffney and A. Hupalowska for help with figure preparation and the Richter family for support. This study was supported by the Human Frontier Science Program (I.B.), the Center for Integration in Science of the Israeli Ministry of Absorption (I.B.), HHMI (A.R.), the Klarman Family Foundation (A.R.), Wings for Life Spinal Cord Research Foundation (A.J.) and NIH grants EY028633 and MH105960 (J.R.S.). The funders had no role in the study design, experiments performed, data collection, data analysis and interpretation or preparation of the manuscript.
A.R. is a founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and, until 31 August 2020, was a SAB member of Syros Pharmaceuticals, Neogene Therapeutics, Asimov and Thermo Fisher Scientific. From 1 August 2020, A.R. is an employee of Genentech and has equity in Roche. J.R.S. is a consultant for Biogen. Z.H. is an advisor of Myro Therapeutics, Axonis and Rugen Therapeutics. The remaining authors declare no competing interests.
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Extended Data Fig. 1 Experimental strategy and reproducibility.
a, Experimental overview. b, Flow cytometry gating strategy used to enrich for retinal CD45+ immune cells, CD140a+ (Pdgfra+) astrocytes and GLAST+ Müller glia (MG). c, Distribution of expression levels of RNAs for the markers used to sort immune (Ptprc = CD45), Müller glia (Slc1a3 = GLAST), and astrocyte (Pdgfra = CD140a) subsets. d-f, Variation of cell composition across experiments. Fraction (y axis) of immune (d), MG (e) and astrocyte (f) subsets across 10x channels (x axis; legend).
Extended Data Fig. 2 Changes in cell composition and stability in cell marker expression throughout the time course.
a, 2D Uniform Manifold Approximation and Projection (UMAP) for 121,309 single cells profiled from the retina across 0, 0.5, 1, 2, 4, 7 and 14dpc, colored by time point (legend). b, ScPhere embedding of 121,309 single cell profiles (dots) from the retina, projected to 2D by the Equal Earth map projection method, colored by time point (legend). c, Fraction of expressing cells (dot size) and mean expression levels in expressing cells (dot color) of selected marker genes (columns) across 14 non-neuronal cell types (rows), plotted at each time point. d, UMAP for 21,275 cells profiled from mouse posterior eyecup at 0, 0.5, 1, 2dpc, colored by time point. e, Fraction of expressing cells (dot size) and normalized expression in expressing cells (dot color) of selected marker genes (columns) across 12 identified cell types in the eyecup (rows). f, Endothelial cells collected from mouse eyecup, colored by expression of genes associated with choroidal Kdrhi (left) and Kdrlo (right) cells20.
Extended Data Fig. 3 Visualizing immune cells in the retina and eyecup.
a, Fraction of expressing cells (dot size) and mean expression levels in expressing cells (dot color) of selected marker genes (columns) across 18 identified immune cell subsets (rows). b,c, Representative IHC on 0 and 0.5dpc retinal sections showing LY6G+ neutrophils that express MMP9 (red) and localize proximal to the optic nerve head (ONH) (n = 3 mice per time point) (b), and on 2dpc retinal whole-mounts from CCR2RFP/+ mice showing CCR2-RFP+ cells (red) in the GCL and IPL (c). Higher magnification inset of the outlined region in c is shown in Fig. 2d (n = 2). Scale bar, 50 μm. d, Representative IHC on 0dpc eyecup sections for IA-IE (MHC-II, magenta) and CD206 (green). Scale bar, 50 μm. Inset shows double positive cells, scale bar, 10 μm. BF, brightfield (n = 2-3 mice, representative of two independent experiments). e, Representative image of smFISH on 2dpc sections showing Ms4a7+Ccr2- cells (green) in the eyecup. This is a subset of the image presented in Fig. 5h. Scale bar, 50 μm. f,g, Representative images of IHC on eyecup whole-mounts (f) from CCR2RFP/+ mice showing that the majority of CCR2-RFP+ cells (red) in naïve eyecup are located posterior to the RPE, at the level of the choroid. ZO-1 (green) depicts tight junctions between individual RPE cells and nuclei are stained with Hoechst (blue) (n = 2 mice, representative of three independent experiments), and sections from 2dpc (g) showing that infiltrating CCR2-RFP+ cells (red) express the macrophage marker, IBA1 (green) (n = 2 mice). Arrowhead points to the RPE. Scale bars, 50 μm.
Extended Data Fig. 4 Glial reactivation and microglial proliferation after ONC.
a, Distribution of expression of genes differentially expressed between Müller glia (MG; n = 54,565 cells) and astrocytes (n = 18,959 cells). b, Fraction of expressing microglia cells (dot size) and mean expression levels in expressing cells (dot color) of microglia signature genes across time (0dpc (n = 4,479 cells); 0.5dpc (n = 2,136); 1dpc (n = 1,712); 2dpc (n = 3,616); 4dpc (n = 2,950), 7dpc (n = 4,433); 14dpc (n = 5,233)). c,d, Representative smFISH on 4dpc retinal sections (c) showing P2ry12+ microglia (green) expressing Mki67 (red) (n = 3 mice), and IHC on 4dpc retinal whole-mounts (d) of KI67+ (red) IBA1+ microglia (green) (n = 3 mice). Scale bars, 50 μm. e, Fraction of cells (dot size) in each MG subset, and mean expression level in expressing cells (dot color) of light and excitotoxin injury-induced genes31. f, Distribution of expression scores for signature genes of pan-reactive astrocytes35 across the astrocyte and astroIFN subsets throughout the time course (Astrocyte/Reactive astro: 0dpc (n = 1,009/17 cells); 0.5dpc (n = 586/299); 1dpc (n = 1,780/767); 2dpc (n = 2,268/439); 4dpc (n = 2,671/652), 7dpc (n = 2,051/396); 14dpc (n = 368/55)). g,h, Distribution of expression scores for Cluster 4 (‘inflammatory‘) signature from LPS-induced astrocytes33 (g) and for a tissue dissociation signature (h) on astrocytes from LPS-induced neuroinflammation33 classified to ONC astrocyte subsets (left) and across the ONC astrocyte subsets (right) (Student’s t-test, one sided). Boxplots in a and f denote medians and IQRs; whiskers are the lowest datum still within 1.5 IQR of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile.
Extended Data Fig. 5 ONC-induced expression changes in the RPE.
a, Representative image of IHC on ocular sections, depicting the RPE with specific staining (RPE65, green) in relation to the retina and optic nerve. Scale bar, 50 μm. b, Fraction of expressing cells (dot size) and normalized expression level in expressing cells (dot color) of differentially expressed genes in the RPE between 0, 0.5, 1 and 2dpc. c,d, Distribution of expression of Plin2 (c) and Nog (d) on RPE cells by time (left; 0dpc (n = 2,605 cells); 0.5dpc (n = 1,746); 1dpc (n = 631); 2dpc (n = 3,031)) and across subsets (right). Boxplots denote medians and IQRs; whiskers are the lowest datum still within 1.5 IQR of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile. e, Representative image of smFISH on 0 and 2dpc RPE for Nog (green) (n = 3 mice per time point). Scale bar, 50 μm.
Extended Data Fig. 6 Dynamics of resident and infiltrating mononuclear phagocytes in the retina, and conserved signature of Gpnmb+ macrophages.
a, Changes in frequency of Ms4a7+MHC-IIhi, Ms4a7+MHC-IIlo and Gpnmb+ macrophages across 0, 0.5, 1, 2, 4, 7, and 14dpc. b, Distribution of expression of genes associated with MHC-IIhi and MHC-IIlo BAMs23 on retinal Ms4a7+MHC-IIhi (n = 741) and Ms4a7+MHC-IIlo macrophages (n = 1,406). Boxplots denote medians and IQRs; whiskers are the lowest datum still within 1.5 IQR of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile. c, Representative image of smFISH on 7dpc retinal for Gpnmb (blue), Ms4a7 (red) and Ptprc (green) (n = 3 mice). Scale bar, 25 μm. d, Distribution of expression of Gpnmb+ macrophage marker gene orthologs in immune cell scRNA-seq data from chronic lesions in human patients with multiple sclerosis47. e, Fraction of expressing cells (dot size) and normalized expression level in expressing cells (dot color) of the top 30 differentially expressed genes of Gpnmb+ macrophages across the RPE cell clusters. f, Representative IHC on uninjured (0 dpc) retinal whole-mounts showing perivascular and CB-adjacent IA-IE+ (green) cells. Blood vessels are labeled with IB4 (red) (representative of five independent experiments with n = 1-3 mice each). The middle panel shows a zoomed-in image of the perivascular macrophage in the inset of the left image. Scale bars, 50 μm g, Force-directed layout view of monocyte and macrophage subsets with optimal transport analysis. Each dot is a cell, color-coded by ancestor probabilities for Ms4a7+MHC-IIhi (top row) and Ms4a7+MHC-IIlo macrophages (bottom row), as estimated by Waddington-OT88.
Extended Data Fig. 7 A co-regulated IFN-response program in the injured retina.
a, Distribution of expression of genes differentially expressed in microgliaIFN by microglia repopulating the brain after depletion (Repop) compared to microglia from control brain55 (Mann-Whitney U test, two sided). b, Distribution of Ifit3 expression across time in MG (top) and astrocytes (bottom) across subsets. c, Distribution of a signature score of Cluster 8 (‘ISG‘)astrocytes from LPS-induced neuroinflammation33 in the LPS-induced astrocytes as classified to the ONC astrocyte subsets (left) and across the ONC astrocyte subsets (right) (Student’s t-test, one sided). d, Off-diagonal panels: Comparison of overall expression scores (y and x axes) for each cell component of MCP1 (rows and columns, labels on diagonal) across the samples; lines correspond to linear fit. Pearson correlation (r) and significance (***p < 0.001; Pearson correlation t-test, one-sided) are shown in the panels above the diagonal. Diagonal panels: Distribution of overall expression scores for each cell type component, with kernel density estimates91. e, Distribution of MCP1 expression scores by astrocytes (n = 18,956 cells), microglia (n = 20,000 cells) and MG (n = 19,998 cells) across 0, 0.5, 1, 2, 4, 7 and 14dpc. Numbers above violins indicate median expression scores. Boxplots in b, e denote medians and IQRs; whiskers are the lowest datum still within 1.5 IQR of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile.
Extended Data Fig. 8 Schematic model summarizing the tissue dynamics along the response to ONC.
An inflammatory cascade is initiated early after injury (phase I), prior to RGC death, with activation of resident glia involving chemokine signals for leukocyte infiltration, which is observed in the oBRB and inner retina. At intermediate time points (phase II), concurrent with the peak rate of RGC death, infiltrating monocytes differentiate into distinct macrophage subsets, including Ms4a7+MHC-IIhi, Ms4a7+MHC-IIlo and Gpnmb+ macrophages. In parallel, during this phase, a synchronous interferon response program is induced in astrocytes, Müller glia and microglia. The latter also express a disease-associated microglia signature, which overlaps with that of Gpnmb+ macrophages. Finally, at 1-2wpc (phase III), glial cell proportions begin to return to their baseline levels, with enriched interactions among them including TGFβ signaling, collectively indicating restoration of homeostasis.
Supplementary Notes 1–4, Figs. 1–5 and Table titles.
Supplementary Tables 1–4
Supplementary Tables 1–4. Titles are in the Supplementary Information file.
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Benhar, I., Ding, J., Yan, W. et al. Temporal single-cell atlas of non-neuronal retinal cells reveals dynamic, coordinated multicellular responses to central nervous system injury. Nat Immunol 24, 700–713 (2023). https://doi.org/10.1038/s41590-023-01437-w