Fig 2: Infiltrating cells are primarily CCR2+ Ly6Chigh. a, b CD45high populations were gated on Ly6C, Cx3CR1, CCR2, and CD45-IV expression. c The number of Ly6Chigh cells increased dramatically at 24 h and remained high before falling at 72 and 96 h (red). d Ly6Clow cells were less abundant at early times, slowly increasing over the first 48 h before abruptly doubling between 72 and 96 h (blue). e CD45-IV+ populations from retinal samples were examined to determine the Ly6C status of cells trapped within the retinal vasculature (yellow). The number of Ly6Chigh cells increased sharply by 24 h and did not return to baseline by 96 h; the number of Ly6Clow cells increased briefly at 24 h before returning to baseline. *p < 0.05, **p < 0.01, ***p < 0.001; error bars are SEM, n = 6–9 retinas per time point

Fig 3: CRAMP modulates intestinal immune disorder in GIE A Il15 expression in duodenal epithelial cells ex vivo (n = 8).BThe frequency of NKG2D+ IELs among CD3+CD4+ population (n = 8).CThe frequency of CX3CR1+ macrophages in CD45+CD11b+ population and ALDH activity in CD45+CD11b+CX3CR1+ macrophages in lamina propria (n = 8).DThe percentages of three subsets of CD45+CD11c+ DCs and ALDH activity in CD45+CD11c+ DCs in lamina propria (n = 8).E–GThe percentages of (E) Foxp3+ Treg, (F) T-bet+ Th1 and (G) ROR?t+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data in A were representative and were the mean ± SD from two independent experiments. Data (B–G) were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Source data are available online for this figure.

Fig 4: scRNA-seq and translatome analysis of perivascular cells and microglia.(a) UMAP plot of 4,238 cells, color-coded based on the inferred cell type. (b) DotPlot of gene expression for a series of known cell type markers for fibroblasts (Lama1, Lum, Cdh5, Dcn), microglia (Sall1, Tmem119, P2ry12), PVMs (Pf4, Cd163), and OPC (Lhfpl3, Sox6, Bcan). Expression of genes encoding for markers used for FACS of cells prior to scRNA-seq (Pdgfra, Ptprc, Itgam, Mrc1, Cx3cr1) and for Spp1 is also included. Radius of dot is proportional to the percentage of cells expressing the gene, color is the scaled gene expression level. (c) Violin plot of Spp1 expression reanalyzed from Jung-Seok Kim et al.11. (d) RiboTag-based translatome analysis of pooled oAß and PBS-challenged brain homogenates of Cx3cr1ccre:Lyve1ncre (n = 8 mice) and Cx3cr1ccreSall1ncre:RiboTag-mice (n = 4 mice). Normalized reads of pan macrophage marker (Cx3cr1), PVM marker (Lyve1), microglia marker (Sall1) and Spp1. P Values from two-way ANOVA, Bonferroni’s multiple comparison test. Data are shown as Mean ± SEM. Data are from 1 independent experiment. (e) Gating strategy to identify CD321 (F11r) and CD29 (Itgb1) expression in microglia (CX3CR1highCD45+ CD206- CD11b+). (f) Representative image of P2Y12+ microglia expressing tgfbr1, assessed by smFISH-IHC in 6 mo AppNL-F and AppNL-F.Spp1KO/KO SLM. Scale bar represents 10 µm. Data representative of n = 3 mice per genotype examined over 1 independent experiment.

Fig 5: Microglia numbers are stable during a dramatic period of cell clearance. a, b CD45low populations were gated on Ly6C and Cx3CR1 expression; MHC-II was used to distinguish microglia from resident macrophages, and CD45-IV was used to remove cells within or associated with the vasculature. c Microglia, the resident MHC-IIlow cells, remained at constant for the first 72 h, then increased at 96 h (lavender). d The number of resident MHC-IIhigh cells was also stable until 96 h (dark purple). Together, these data show that the number of resident immune cells is stable during early retinal degeneration. *p < 0.05, **p < 0.01, ***p < 0.001; error bars are SEM, n = 6–9 retinas per time point