Abstract
Purpose :
Previously we identified that the miR-183/96/182 cluster (miR-183C) regulates corneal resident immune cells (CRICs) and nerves. This study is to uncover its roles in shaping the cellular landscape of the cornea, with a focus on CRICs.
Methods :
Corneas anterior to the limbus of naïve, adult, female miR-183C knockout mice (ko) and their wild-type littermates (wt) were dissociated into single cells. Dead cells were removed using a Dead Cell Removal kit and Magnetic Activated Cell Sorting (MACS). To enrich CRIC, CD45+ cells were enriched by MACS. scRNA libraries of total corneal cells or enriched CRICs were prepared using the Next GEM Single Cell 3’ Reagent kits v3.1 (10xGENOMICS) before sequencing on an Illumina NovaSeq, followed by alignment and tabulation with Cell Ranger. Analysis were performed in R using Seurat and SingleR packages.
Results :
We obtained sc transcriptomes of 8,163 and 7316 total corneal cells of 2-3 months old (mo), and 8,783 and 6,211 corneal cells of 6-8 mo wt and ko mice, respectively; 1,981 (wt) and 2915 (ko) MACS-enriched CRICs of 6-8 mo mice.
In total corneal cells, 11 populations (clusters) were identified, including 4 stromal-keratocyte and/or endothelial, 6 epithelial clusters and 1 CRIC cluster. Mononuclear phagocyte system (MPS), including monocytes, macrophages (Mf) and dendritic cells, were increased in ko vs wt mice, consistent with our previous observation by immunostaining and flow cytometry. Multiple populations changed their representations at both ages and/or at the younger vs the older ages in ko vs wt mice.
In CRICs, 13 clusters were uncovered, 6 of which were CRICs for further analysis. Preliminary data suggest that the number of resident MPS cells and the expression of both classical M1 and M2 signature genes in resident Mf are simultaneously increased of ko vs wt mice. Small subpopulations of CRICs, positive to microglial or T-cell or IL-17 or proliferative cell markers, were also increased in the ko mice.
Further analyses on differentially expressed genes and functional annotation in various cell types between the ko vs wt mice are ongoing to uncover cell type-specific targets and molecular pathways regulated by miR-183C.
Conclusions :
scRNA seq provides unprecedented molecular insights into the roles of miR-183C regulating the cellular composition and properties of the cornea.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.