Mouse Corneal Immune Cell Heterogeneity Revealed by Single-Cell RNA Sequencing

Authors

Ebru Yaman
Nicole Heyer
Cintia S de Paiva
Mary Ann Stepp
Stephen C Pflugfelder
Jehan Alam

Publication Date

10-1-2024

Journal

Investigative Ophthalmology & Visual Science

DOI

10.1167/iovs.65.12.29

PMID

39432400

PMCID

PMC11500044

PubMedCentral® Posted Date

10-21-2024

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Animals, Mice, Mice, Inbred C57BL, Female, Single-Cell Analysis, Cornea, Male, Sequence Analysis, RNA, Dendritic Cells, Macrophages, cornea, heterogeneity, single-cell RNA seq, sexually dimorphic, macrophage

Abstract

PURPOSE: This study aimed to define the heterogeneity, spatial localization, and functional roles of immune cells in the mouse cornea using single-cell RNA sequencing (scRNA-seq) and immunofluorescent staining.

METHODS: Enriched mouse corneal immune cells (C57BL/6 strain, age 16-20 weeks) underwent single-cell RNA sequencing library preparation, sequencing, and analysis with Seurat, Monocle 3, and CellChat packages in R. Pathway analysis used Qiagen Ingenuity Pathway Analysis software. Immunostaining confirmed cell distribution.

RESULTS: We identified 14 distinct immune cell clusters (56% myeloid and 44% lymphoid). Myeloid populations included resident macrophages, conventional dendritic cells (cDC2s), Langerhans cells, neutrophils, monocytes, and mast cells. Additionally, lymphocyte subsets (B, CD8, CD4, γδT, natural killer, natural killer T, and group 2 innate lymphoid cells) were found. We also found three new subtypes of resident macrophages in the cornea. Trajectory analysis suggested a differentiation pathway from monocytes to conventional dendritic cells, resident macrophages, and LCs. Intercellular communication network analysis using cord diagram identified amyloid precursor protein, chemokine (C-C motif) ligands (2, 3, 4, 6, 7, 9, and 12), Cxcl2, Mif, Tnf, Tgfb1, Igf1, and Il10 as prominent ligands involved in these interactions. Sexually dimorphic gene expression patterns were observed, with male myeloid cells expressing genes linked to immune regulation (Egr1, Foxp1, Mrc1, and Il1rn) and females showing higher expression of antigen presentation genes (Clic1, Psmb8, and Psmb9). Finally, immunostaining confirmed the spatial distribution of these cell populations within the cornea.

CONCLUSIONS: This study unveils a diverse immune landscape in the mouse cornea, with evidence for cell differentiation and sex-based differences. Immunostaining validates the spatial distribution of these populations, furthering our knowledge of corneal immune function.