The miR-183/96/182 cluster is a checkpoint for resident immune cells and shapes the cellular landscape of the cornea

Abstract

Purpose: The conserved miR-183/96/182 cluster (miR-183C) regulates both corneal sensory innervation and corneal resident immune cells (CRICs). This study is to uncover its role in CRICs and in shaping the corneal cellular landscape at a single-cell (sc) level.

Methods: Corneas of naïve, young adult [2 and 6 months old (mo)], female miR-183C knockout (KO) mice and wild-type (WT) littermates were harvested and dissociated into single cells. Dead cells were removed using a Dead Cell Removal kit. CD45⁺ CRICs were enriched by Magnetic Activated Cell Sorting (MACS). scRNA libraries were constructed and sequenced followed by comprehensive bioinformatic analyses.

Results: The composition of major cell types of the cornea stays relatively stable in WT mice from 2 to 6 mo, however the compositions of subtypes of corneal cells shift with age. Inactivation of miR-183C disrupts the stability of the major cell-type composition and age-related transcriptomic shifts of subtypes of corneal cells. The diversity of CRICs is enhanced with age. Naïve mouse cornea contains previously-unrecognized resident fibrocytes and neutrophils. Resident macrophages (ResMφ) adopt cornea-specific function by expressing abundant extracellular matrix (ECM) and ECM organization-related genes. Naïve cornea is endowed with partially-differentiated proliferative ResMφ and contains microglia-like Mφ. Resident lymphocytes, including innate lymphoid cells (ILCs), NKT and γδT cells, are the major source of innate IL-17a. miR-183C limits the diversity and polarity of ResMφ.

Conclusion: miR-183C serves as a checkpoint for CRICs and imposes a global regulation of the cellular landscape of the cornea.

Keywords: Corneal resident fibrocytes (CRFs); Corneal resident immune cells (CRICs); Corneal resident lymphocytes (CRL); Corneal resident myeloid cells (CRMCs); Corneal resident neutrophils (CRNs); Resident macrophages (ResMφ); Single cell RNA sequencing (scRNA seq); miR-183/96/182 cluster (miR-183C); microRNAs (miRNAs).