Lineage tracing reveals the dynamics of monocyte and microglia migration to the subretinal space following retinal detachment

Abstract

Purpose : Retinal detachment (RD) occurs when photoreceptors (PR) are physically separated from the retinal pigment epithelium, compromising PR cell survival. RD triggers an innate immune response, with the attraction of circulating monocytes and resident retinal microglia to the outer nuclear layer (ONL) and to the photoreceptor outer segments (POS) in the subretinal space. Because immune cell markers are not specific, previous studies were unable to differentiate between monocytes and microglia.

Methods : Tamoxifen (Tam)-inducible Cre-driver mice (CX3CR1-CreERT2) were crossed with Cre reporter mice expressing red-fluorescent protein (RFP) in a Cre-dependent fashion (ROSA26-FloxedStop-tdTomato) to specifically lineage trace microglia. A 4-week washout period after Tam treatment was used to eliminate RFP-expressing non-classical monocytes. In like fashion, lineage tracing of circulating monocytes was performed with a novel Cre driver mouse strain with CreERT2 knocked into the RNA binding with multiple splicing gene locus (RBPMS-CreERT2). Blood cell types were identified and examined for RFP expression by flow cytometry. The mice received subretinal injections of 1% hylaluronic acid to cause RD. Retinal sections were obtained at 3, 7 and 14 days post-retinal detachment (dprd). Untreated fellow eyes were used as controls. Retinal and subretinal cells were examined by immunofluorescence for expression of RFP and the myeloid leukocyte marker CD11b.

Results : Lineage tracing with CX3CR1-CreERT2 resulted in RFP-labeling of 92-94% of retinal microglia, with less than 1% labeling of circulating monocytes. With RBPMS-CreERT2, no microglia were labeled, while 91-94% of classical monocytes and 80-86% of non-classical monocytes in circulation were labeled. Linage traced microglia accounted for 50±14% (mean ± SE) of the subretinal cells at 3 dprd; this fraction significantly increased over time to 65±4% at 7 dprd and 86±2% 14 dprd. The RBPMS-Cre mediated lineage tracing showed that 28±5% of subretinal cells were lineage-traced monocytes at 7 dprd.

Conclusions : The initial innate immune response to RD involves an equivalent attraction of resident microglia and circulating monocytes to orphaned POS in the subretinal space. Over time monocytes are depleted and the subretinal cell population becomes dominated by microglia.

This is a 2020 ARVO Annual Meeting abstract.