Abstract
Purpose :
To compare immune cell phenotype, gene expression profiles, and cornea wound healing response between RXRα mutant Pinkie mouse strain and wild-type C57BL/6 (B6) using single-cell RNA sequencing (scRNA-seq) and alkali burn animal model.
Methods :
Cd45+ immune cells were sorted from the cornea of Pinkie and wild-type B6 mice using flow cytometry and scRNA-seq was performed using 10x Genomics. The data analysis was performed in R using the Seurat package. A standard 2 mm central cornea wound was created using 1N NaOH. Cornea thickness was measured with Optical Coherence Tomography (OCT).
Results :
scRNA-seq analysis revealed that resident CD45+ cells in the cornea of B6 and Pinkie mice belonging to fourteen distinct cell clusters, which include 58% myeloid (two clusters of neutrophils (Neut): Neut, Ccl3hi-Neut, two clusters of macrophages (MP): MP, Vegfhi-MP, cDC2, and two distinct clusters of monocytes (Mo): Acehi Mo, Ly6c2hi Mo) and 42% lymphoid (gdT, CD8, CD4, NK, B, ILC2, and Pre-T/B cells) cells. Neutrophils (38.4% vs 7.2%), Il17-producing gdT (19.2% vs 4.1%), Il1β and Vegfa-producing inflammatory MP (Vegfhi-MP) (3.3% vs 0.0%), and Ly6c2hi Mo (3.3% vs 5.0%) were the predominantly increased cell populations in Pinkie corneas as compared to B6. Il10, Apoe, and C1qa/c expressing regulatory MP cell population decreased in Pinkie (4.3%) as compared to wild-type B6 (30.3%). CCR2K0-Pinkie strain showed a significant decrease in the expression of cytokines/chemokines such as Il1α/β, S100a8, S100a9, Il17a/f, Il23a, and Cxcr6. Opacity score and corneal thickness measured by OCT initially increased, but significantly improved in wild-type B6 mice by day 10. By day 21, 100% of alkali-burned Pinkie corneas developed corneal opacification, neovascularization, and ulceration.
Conclusions :
These findings indicate that RXRα suppresses the activation and recruitment of cornea disease-promoting cells such as gdT cells, inflammatory Mo, and neutrophils and that CCR2 was identified as a potential therapeutic target in corneal disease.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.