Characterization of Recruited Macrophages Following Corneal Injury

Abstract

Purpose : Macrophages are tissue-resident or monocyte-derived immune cells critical for corneal innate immunity and wound repair. Recent studies have shown that macrophages are plastic cells whose phenotype and function can vary depending on the time after injury. The purpose of this study is to better understand the phenotype of recruited, monocyte-derived macrophages during corneal wound healing using an epithelial/stromal injury model. Furthermore, the effect of MMP12, a macrophage secreted factor, on regulating macrophage phenotype following corneal injury is assessed.

Methods : Chemical injuries were performed on corneas of wild-type (WT) and MMP12^−/− mice by topical application of 0.1N NaOH. Unwounded and wounded corneas were collected 1, 3, and 6 days after injury and processed for flow cytometry assays using pre-conjugated antibodies (CD45, CD11B, F4/80, Ly-6C, CD64, CD11C). Flow cytometric data analyses was performed using FCS Express 7 software.

Results : Two subpopulations of macrophages (CD11b+/F4/80hi and CD11b+/F4/80int) were observed and their expression varied depending on the time point after injury. CD11b+/F4/80hi resident macrophages were predominant in unwounded WT and MMP12^−/− corneas. CD11b+/F4/80int recruited macrophages became abundant 1 day post-injury in both WT and MMP12^−/− corneas and were further subdivided into 3 subpopulations with Ly6C staining. Ly6Chi (inflammatory) cells were most abundant 1 day post-injury in both WT and MMP12^−/− corneas. Ly6Cint cells were highly expressed 3 days post-injury and Ly6Clo (patrolling) cells became the predominant cell type 6 days post-injury. The CD11b+/F4/80int recruited macrophages further subdivided into 2 subpopulations with CD64 and CD11C staining. CD64-CD11C- monocytes were significantly higher 1 day post-injury in both WT and MMP12^−/− corneas. CD64-CD11C+ dendritic cells were highly expressed in MMP12^−/− corneas 6 days post-injury.

Conclusions : These findings show the phenotypic plasticity of recruited, monocyte-derived macrophages in the acute corneal repair response and provide insight into the functional roles of macrophages following corneal injury. Further analyses of these distinct macrophage populations using lineage tracing systems and single-cell transcriptomics are needed and may guide future development of macrophage-targeted therapies.

This is a 2020 ARVO Annual Meeting abstract.