FIGURE SUMMARY
Title

The immune response is a critical regulator of zebrafish retinal pigment epithelium regeneration

Authors
Leach, L.L., Hanovice, N.J., George, S.M., Gabriel, A.E., Gross, J.M.
Source
Full text @ Proc. Natl. Acad. Sci. USA

Enrichment of immune system genes during RPE regeneration. (A) Experimental workflow showing steps for tissue processing (i, ii) and isolation of eGFP+ RPE (iii, iv). Example 9 dpf and 4 dpi FACS plots showing cell sorting gate (iii; green). (B and C) Top Reactome pathways enriched from groups of significant DEGs at 2 dpi/7 dpf (B) and 4 dpi/9 dpf (C). Numbers in parentheses indicate quantities of significantly enriched DEGs. (D) Heatmap showing hierarchical clustering of immune-related genes in the top 50 up-regulated DEG sets from 2 dpi/7 dpf and 4 dpi/9 dpf (SI Appendix, Tables S1 and S2). Heatmap legend represents log2 (transcripts per million +1).

Leukocyte infiltration into the RPE injury site during regeneration. (A–H) Confocal micrographs of MTZ− (A–D) and MTZ+ (E–H) Tg(lyz:TagRFP;rpe65a:nfsB-eGFP) whole-mount eyes. Ratios at top right (A–D) indicate number of eyes lacking lyz:TagRFP+ neutrophils (white arrowheads in E–H over total number of eyes). (I–P) Fluorescent confocal micrographs of MTZ− (I–L) and MTZ+ (M–P) Tg(rpe65a:nfsB-eGFP) whole-mount eyes labeled with 4C4 to mark MΦs/μglia. (I–P) Insets show digital zooms of single cells or cell clusters to highlight 4C4+ cell morphologies. (A–P) White dashed lines designate edges of eyes. Magenta labels endogenous TagRFP or 4C4 and green labels endogenous eGFP. (Scale bars, 100 μm.) (Q) Violin plots showing a significant increase in the number of lyz:TagRFP+ neutrophils at 2 dpi when compared with 7 dpf controls. A maximum of six infiltrating cells were present at 2 dpi (datapoint in F). (R) Violin plots showing significant increases in 4C4+ staining from 2 to 4 dpi (MTZ+) when compared with MTZ− controls. (Q and R) Dashed black lines represent the median, and dotted black lines represent quartiles. SI Appendix, Table S12 contains statistical information. Dorsal is up; **P value ≤0.01.

Macrophages/microglia show increased sphericity and association with RPE debris at 3 dpi. In vivo light-sheet micrographs from 8 dpf MTZ− (A and A’) and 3 dpi MTZ+ (B and B’) Tg(mpeg1.1:Dendra2;rpe65a:nfsB-eGFP) larvae. (A’ and B’) Digital zooms of cropped 100 μm z-stacks (z-step = 100 to 200) showing mpeg1.1:Dendra2 (red)+ cell localization (magenta) in/adjacent to the back of the eye (green dashed line). (A and B’) White labels endogenous eGFP. Yellow boxes correspond to areas shown in Movies S2 and S3 (A) and in Movies S5 and S6 (B). Anterior is up. (Scale bars, 100 μm.) (C) Violin plots showing a significant increase in sphericity of anterior mpeg1.1:Dendra2 (red)+ cells in 3 dpi MTZ+ larvae when compared with 8 dpf MTZ− controls. SI Appendix, Table S12 contains statistical information. Dashed black lines represent the median, and dotted black lines represent quartiles; *P value ≤ 0.05. (D–G) Confocal micrographs of transverse sections from 3 dpi MTZ+ Tg(mpeg1:mCherry; rpe65a:nfsB-eGFP) larval eyes. Cyan arrowheads point to MΦs/µglia (magenta) overlapping with RPE debris (green) in central–dorsal (D and E) and central–ventral (F and G) regions. Dorsal is up; anterior is left. (Scale bars, 20 μm.)

Proliferation signatures are present in macrophages/microglia during RPE regeneration. Top 10 Reactome pathways enriched from groups of significantly up-regulated DEGs at 2 dpi/7 dpf (A) and 4 dpi/9 dpf (B) in FACS-isolated mCherry+ MΦs/μglia. Numbers in parentheses indicate quantities of significantly enriched DEGs. (C) Heatmap showing hierarchical clustering of cell cycle– and mitosis-related genes selected for representation based on presence in the top 50 up-regulated gene sets from 2 dpi/7 dpf and 4 dpi/9 dpf DEG analyses (SI Appendix, Tables S7 and S8). Heatmap legend represents log2 (transcripts per million +1). Confocal micrographs of transverse sections from MTZ− and MTZ+ Tg(mpeg1:mCherry; rpe65a:nfsB-eGFP) eyes at 7 dpf/2 dpi (D and E) and 9 dpf/4 dpi (F and G). Digital zooms (D–E” and G) highlight proliferating (EdU+; cyan) mCherry+ MΦs/μglia (magenta). (Scale bar, 40 μm.) (H and I) Violin plots showing significant increases in EdU+ mCherry+ costaining in the RPE (H) and in the RPE and retina (I) of 2 dpi MTZ+ larvae. Dashed black lines represent the median, and dotted black lines represent quartiles. SI Appendix, Table S12 contains statistical information. Dorsal is up; *P value ≤ 0.05; and **P value ≤ 0.01.

Suppression of inflammation with dexamethasone impairs RPE regeneration. (A) Schematic depicting treatment timeline. (B) Bar graph showing fold change in pxr gene expression from larvae treated with dexamethasone or DMSO for 24 h (4 to 5 dpf). Error bar represents 95% CI. (C–F) Confocal micrographs of transverse sections from 4 dpi MTZ+ DMSO- (C and D) and dexamethasone-treated (E and F) Tg(rpe65a:nfsB-eGFP) eyes. (C and E) White arrowheads highlight BrdU+ cells (magenta) in the RPE layer. White (DAPI) labels nuclei. (F) Magenta arrowheads designate edges of the regenerating RPE monolayer. (Scale bars, 40 μm.) (G and H) Violin plots showing a significant decrease in the number of BrdU+ cells in the RPE (G) and the percent recovery of a pigmented monolayer (H) in MTZ+ dexamethasone-treated larvae. Dashed black lines represent the median, and dotted black lines represent quartiles. Statistical information can be found in SI Appendix, Table S12. Dorsal is up; *P value ≤ 0.05; and ***P value ≤ 0.001. PTU = n-phenylthiourea.

irf8 mutant larvae show impaired RPE regeneration. (A and B’) Confocal micrographs of transverse sections from 4 dpi MTZ+ irf8+/+ (wild-type; A) and irf8−/− (mutant; B) eyes. Digital zooms highlight clusters of pyknotic nuclei retained between the outer plexiform layer and basal RPE in irf8 mutants (B; white dashed lines). Yellow arrowheads mark BrdU+ cells (cyan) within the RPE layer. (C) Violin plots showing a significant increase in proliferative cells in the RPE of 4 dpi MTZ+ irf8 mutants when compared with MTZ+ wild-type siblings. (D–G) Confocal micrographs showing TUNEL staining (red) on transverse sections from 4 dpi MTZ+ irf8 wild-type (D and F) and irf8 mutant (E and G) eyes. Images depict representative larvae (D and E) alongside larvae with the most TUNEL+ puncta (F and G). (H) Violin plots showing a significant increase in TUNEL labeling in 4 dpi MTZ+ irf8 mutants when compared with 4 dpi MTZ+ wild-type siblings. (C and H) There was no significant difference between 9 dpf MTZ− irf8 wild-type and mutant siblings. (I–L) Confocal micrographs of representative larvae (D and E) showing endogenous eGFP expression (I and J; green) and pigmentation relative to eGFP expansion (K and L). Magenta arrowheads delineate where continuous peripheral-to-central eGFP expression ends. (M) Schematic showing method of quantifying RPE regeneration. (N) Violin plots showing a significant decrease in RPE regeneration in 4 dpi MTZ+ irf8 mutants. In all violin plots, dashed black lines represent the median, and dotted black lines represent quartiles. (Scale bars, 40 μm.) SI Appendix, Table S12 contains statistical information. Dorsal is up; *P value ≤ 0.05; **P value ≤ 0.01; and ***P value ≤ 0.001.

Treatment with the CSF-1R inhibitor, PLX3397, impairs RPE regeneration. Confocal micrographs of transverse sections showing BrdU (A and B; cyan) and TUNEL (D and E; red) staining and endogenous eGFP (G–J; green) from 4 dpi MTZ+ DMSO- and PLX3397-treated larvae. White (DAPI) labels nuclei. Violin plots showing quantification of BrdU (C) and TUNEL (F) in 9 dpf MTZ− and 4 dpi MTZ+ larval treatment groups. (C) Although not significant, the number of BrdU+ cells in the RPE (A and B; yellow arrowheads) trends upward in 4 dpi MTZ+ PLX3397-treated larvae when compared with 4 dpi MTZ+ DMSO controls. (F) A significant increase was observed in the number of TUNEL+ puncta between the outer plexiform layer and basal RPE (D and E; cyan line) of 4 dpi MTZ+ PLX3397-treated larvae when compared with 4 dpi MTZ+ DMSO controls. In G and H, magenta arrowheads delineate where continuous peripheral-to-central eGFP expression ends, and brightfield confocal micrographs show pigmentation relative to eGFP expansion (I and J; magenta arrowheads). (K) Violin plots showing a significant decrease in RPE regeneration in 4 dpi MTZ+ PLX3397-treated larvae. (Scale bars, 40 μm.) In all violin plots, dashed black lines represent the median, and dotted black lines represent quartiles. SI Appendix, Table S12 contains statistical information. Dorsal is up; *P value ≤ 0.05; and **P value ≤ 0.01.

Phases of immune involvement during RPE regeneration. Schematic showing few ramified MΦs/μglia (magenta) present in the RPE (green) of unablated larvae. Infiltration of MΦs/μglia to the central RPE injury site after ablation begins at 2 dpi, peaks at 3 dpi, and wanes by 4 dpi, representing a time window when inflammation is likely resolved (2 to 4 dpi; yellow). During resolution, MΦs/μglia appear amoeboid in morphology, proliferate and express phagocytosis markers (e.g., anxa1a), and RPE express il34 and other cytokines. Peak RPE layer proliferation and recovery of pigment occurs between 3 to 4 dpi (18). This coupled with the decreased presence of MΦs/μglia in the RPE by 4 dpi may hint to a time window after ablation (3 to 4 dpi; green) when inflammation has been resolved, enabling peak RPE regeneration.

Acknowledgments
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