Abstract
Purpose :
Retinopathy of prematurity (ROP) is a major cause of blindness in children. The incidence of ROP continues to increase and there is an urgent need to understand the molecular mechanism of ROP development. Immune cells are a source of cytokines and growth factors that may interact with endothelial cells and contribute to retinopathy. We reported that myeloid cells regulated choroidal neovascularization via suppressor of cytokine signaling 3 (SOCS3). However, the role of myeloid cells in ROP is still unknown. We aimed to study the role of myeloid cells and SOCS3 in ROP.
Methods :
Socs3 myeloid-specific knockout mice (Socs3 cKO) were generated by breeding Socs3 flox/flox (Socs3 f/f) mice with LysM-Cre mice. Socs3 myeloid-specific overexpression mice (Socs3 cOE) were generated by breeding Socs3 OE flox/flox (Socs3 OE) mice with LysM-Cre mice. Oxygen induced retinopathy (OIR) mouse model was generated by exposing newborn mice to 75% oxygen from postnatal day (P)7 to P12 and to room air from P12 to P17. CD45 positive cells were isolated from retinas with OIR (age-matched normal retina as control) using flow cytometry for single cell RNA sequencing (10x genomics). Retinas were stained with endothelial cell marker Isolectin B4 (Invitrogen) and flat mounted for phenotypical analysis. Results were presented as mean ± SEM and compared using the unpaired nonparametric Mann-Whitney test. Statistical analyses were performed with GraphPad Prism.
Results :
Using single cell RNA sequencing data, we applied t-Distributed Stochastic Neighbor Embedding (t-SNE) method to analyze the immune cell profile at steady state and during OIR. All clusters were assigned based on cell markers, which mainly included microglia, macrophages, neutrophils, B cells, T cells, and CD45-positive endothelial cells. A few clusters were identified that only appeared in OIR retinas including subtypes of microglia and macrophages as well as potential myeloid lineage derived endothelial cells. In addition, metabolism, inflammation, and cell proliferation related pathways were ranked on the top based on the pathway analysis using differential expressed genes (DEGs) in clusters that are different from OIR retinas compared with normal retinas.
Conclusions :
SOCS3 regulated retinal angiogenesis via modulating myeloid lineage cell recruitment. Manipulating SOCS3 expression in myeloid cells may provide a new way to cure neovascularization in retinas.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.