Monocytes undergo transcriptional and biochemical changes, allowing expression of microglia markers P2ry12, TMEM119, FCRLS and Iba1 upon engraftment into the retina.

Abstract

Purpose : Our previous studies have shown that peripheral monocytes engraft permanently into the neuroretina upon injury, differentiate to microglia-like cells, and express microglia “specific” markers P2ry12, TMEM119, FCRLS and Iba1. Here we explore the role of chromatin regulation in monocyte expression of P2ry12, TMEM119, FCRLS and AIF1 gene (IBA1) upon engraftment into the retina.

Methods : A bone marrow chimera model was employed to differentiate microglia from engrafted monocytes. Flow sorting was used to isolate retinal microglia, engrafted monocytes, and circulating blood monocytes for further analysis with ATAC-seq. Changes in gene accessibility for microglia markers were evaluated and correlated to transcriptional and protein data.

Results : Monocyte exhibit significant changes in chromatin accessibility upon engraftment into the retina, with more than 3000 chromatin accessibility peaks becoming different after infiltration into the retina. Chromatin changes perdure for the long-term (45 days), allowing engrafted monocytes to acquire similar open chromatin signatures to yolk sac-derived microglia. We identified three open chromatin peaks for P2ry12 gene that were conserved in circulating, engrafted and resident monocytes/microglia and one peak that was missing only from the circulating monocytes. FCRLS peaks were similar among the groups, however, engrafted monocytes and microglia had stronger peaks compare to blood monocyte. Likewise, open chromatin peaks of AIF1 gene (IBA1) were similar between the groups, and remained stable for the entire study (45 days). In contrast, TMEM119 chromatin was inaccessible in circulating monocytes, but upon infiltration, it became accessible and similar to that of microglia. Single cell RNAseq and protein studies showed similar trends in terms of mRNA and protein expression of the above mentioned genes, before and after engraftment into the retina.

Conclusions : Peripheral monocytes are epigenetically plastic and undergo changes in open chromatin that allow them to express P2ry12, FCRLS, Aif1 (IBA1) and TMEM119 microglia “specific” genes. This process appears to be adaptational and likely controlled by the retinal microenvironment. Further work is required to understand the implication of these findings in the epigenetic reprograming of engrafted monocytes.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.