Abstract
Purpose :
The miR-183/96/182 cluster (miR-183C) regulates both corneal resident immune cells (CRICs) and nerves. This study is to uncover the molecular atlas of mouse cornea at a single-cell (sc) level and the roles of miR-183C in CRICs and corneal cellular landscape.
Methods :
Corneas of naïve, adult, female miR-183C knockout (KO) mice and 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). CD45+ cells were enriched by MACS. scRNA libraries were constructed using the Next GEM Single Cell 3’ Reagent kits v3.1 (10xGENOMICS) and sequenced on an Illumina NovaSeq. Data were analyzed using Cell Ranger, Seurat, SingleR/Celldex and DAVID.
Results :
We obtained a total of 29,379 sc transcriptomes of total corneal cells of 2 months old (mo) and 6 mo, and MACS-enriched CRICs of 6 mo WT and miR-183C KO mice. Major findings include:
1) The composition of major cell types of the cornea stayed stable in WT mice from 2 to 6 mo. However, the composition of subtypes of corneal cells shifted in WT mice from 2 to 6 mo, suggesting age-related adaptation to maintain the homeostasis. Inactivation of miR-183C disrupted the stability of major cell types and age-related shifts of subtype of corneal cells.
2) CRICs of naïve cornea are a heterogenous population of myeloid cells and lymphocytes. We discovered previously unrecognized resident neutrophils and fibrocytes in naïve mouse cornea.
3) The diversity of CRICs was increased or enhanced with age.
4) Corneal resident macrophages (ResMΦ) adopted corneal specific function by producing components of extracellular matrix (ECM) and molecules involved in ECM organization and its interaction with cellular components.
5) Naïve cornea is endowed with partially differentiated, proliferative MΦ, providing a mechanism of quick amplification of functional MΦ.
6) Naïve cornea contained microglia-like MΦ; their number was decreased with age.
7) The resident lymphocytes are comprised mainly by innate lymphoid cells, followed by NKT and a small number of γδT cells. They are the major source of innate IL-17a.
8) miR-183C limits the number and the extent of polarity of ResMΦ and other types of CRICs.
Conclusions :
miR-183C serves as a checkpoint for CRICs and imposes a global regulation of the cellular landscape of the cornea.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.