Temporo-spatial distribution and transcriptional profile of retinal microglia cells in the oxygen-induced retinopathy mouse model

Abstract

Purpose : Myeloid cells such as resident retinal microglial cells (MG) or infiltrating blood-derived macrophages (Mφ) accumulate at sites of retinal neovascularization (RNV). The temporo-spatial distribution and function of retinal MG and infiltrating Mφ in ischemic retinal disease, however, remain unclarified. The aim of this study is to determine the extent of MG proliferation and Mφ infiltration at sites of retinal neovascularization and to assess the transcriptional profile of MG in the oxygen-induced retinopathy (OIR) mouse model.

Methods : Cx3cr1^CreER/+:Rosa26-Tom^fl/+ reporter mice were treated with intraperitoneal injection of tamoxifen at postnatal day 1 (P1), leading to specific fluorescent tdTomato labeling of myeloid cells. Pups were exposed to 75% oxygen from P7 to P12. Myeloid cells of the retina and peripheral blood were examined by flowcytometry and immunofluorescence at P7, P12 and P17. At P17, tomato-positive retinal MG were isolated by flowcytometry to perform bulk RNA-Seq and Gene Ontology (GO) Cluster Analysis. Furthermore, the cell proliferation marker EdU (5-Ethynyl-2'-Deoxyuridin) was applied daily from P12 to P16.

Results : Tamoxifen-treated Cx3cr1^CreER/+:Rosa26-Tom^fl/+ mice revealed a long-term and specific tdTomato labeling of all retinal MG. In contrast, blood monocytes lost the tdTomato signal within the first two weeks of life. At P17, MG constituted the dominant myeloid cell population at sites of RNV (mean ± SD = 93.8 ± 4.0%) while Mφ appeared rarely (6.2 ± 4.0%). At P17, thirty percent (± 3.6%) of retinal MG were EdU-positive indicating local MG cell expansion. RNA-Seq analysis revealed an upregulation of the GO clusters “cell division” (p = 10^-39) and “interferon beta signaling” (p = 10^-4) in retinal MG of OIR mice compared to untreated controls. Chemokines such as Il1b, Ccl2 and TNF were significantly enriched in MG in the OIR group.

Conclusions : Cx3cr1^CreER/+:Rosa26-Tom^fl/+ mice are a suitable tool to distinguish retinal MG and blood-derived Mφ in the OIR model. Our data demonstrates that retinal MG proliferate and represent the major myeloid cell population at sites of RNV in the OIR model while infiltrating Mφ play a minor role. RNA Seq analysis confirms local MG cell expansion and indicates both pro- and anti-inflammatory effects of MG in ischemic retinal disease.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

MG (* orange) and Mφ (↓ green) shaking hands at a site of RNV (blue)