Abstract
Purpose :
Blinding diseases of the retina have few effective treatments. While non-mammalian vertebrates, such as fish, can regenerate retina and restore vision, mammals cannot. We previously found that driving the proneural transcription factor Ascl1 stimulates neurogenesis from mammalian Müller Glia (MG). We also reported that inflammation after cell death limits the neurogenic capacity of mammalian MG. In this work, we characterize the peripheral immune response to retinal injury and find that monocyte-derived signals restrict the regenerative capacity of MG in adult mice.
Methods :
To characterize the immune response after NMDA-induced injury, scRNA-sequencing was performed on FACS-purified CD45 positive cells 7 days after damage. CCR2:RFP transgenic mice were used to visualize monocyte infiltration.
To study how monocytes impact retinal regeneration, we used a mouse with inducible expression of Ascl1 in MG and deletion of the CCR2 receptor (Glast-CreER/LNL-tTA/TetO-Ascl1-GFP/CCR2RFP/RFP). To induce regeneration, intraperitoneal tamoxifen is used to induce Ascl1 in MG, followed by intravitreal injections of NMDA to elicit death of retinal ganglion cells and intravitreal Trichostatin-A to promote chromatin accessibility and neurogenesis. Three weeks later, retinas were harvested for immunofluorescence. The number of MG-derived neurons (GFP+/Otx2+) was compared between CCR2 sufficient and knockout mice. FACS-purified MG and immune cells were also analyzed using scRNA-seq and the Seurat and CellChatDB software.
Results :
We found that monocytes invade into the retina after NMDA injury, and blocking this invasion significantly increased MG-derived neurogenesis. scRNA-seq analysis using CellChatDB produced a ranked list of ligand-receptor interactions between the Monocytes and MG, with the Secreted Phosphoprotein 1 (Spp1) pathway amongst the strongest. The addition of recombinant Spp1 in monocyte-deficient retinas reversed the neurogenic increase seen with monocyte depletion. This suggests that Spp1 is a key monocyte-derived signal that inhibits MG neurogenesis.
Conclusions :
This work implicates peripheral immune invasion as a significant factor in MG reprogramming strategies. We find that monocytes release signals that are detrimental to MG neurogenesis. A more thorough understanding of the interactions between glia and the immune system may guide the development of novel therapeutic approaches.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.