Mapping neutrophil heterogeneity across murine models of corneal infection and ocular surface inflammation

Abstract

Purpose : Neutrophils are a critical cell type in ocular surface diseases, including in murine models of microbial keratitis and allergic eye disease (AED) with concomitant meibomian gland dysfunction (MGD). It is now appreciated that neutrophils are transcriptionally heterogeneous, which determines their function in multiple diseases. To characterize neutrophils from distinct ocular pathologies, we integrated single cell RNA sequencing (scRNA-seq) data from naïve mice and from murine models of ocular surface disease.

Methods : In independent experiments, we examined neutrophil transcriptional profiles using the AED mouse model of MGD, and mouse models of bacterial keratitis (Pseudomonas aeruginosa) and fungal keratitis (Aspergillus fumigatus). Corneas were dissected from microbial keratitis mice, or conjunctiva, blood, and tears were collected from naïve C57Bl/6 and AED mice. Samples were collagenase digested, and CD45+ leukocytes were isolated by fluorescence-activated cell sorting, before being analyzed via scRNA-seq. Datasets were analyzed using Seurat, including independent filtering and subsequent integration with anchoring, dimensionality reduction, and shared nearest neighbor clustering. Differentially expressed genes were then identified for each cluster using the Seurat FindAllMarkers function, and heterogeneity across tissues and disease etiologies was evaluated.

Results : Neutrophils were identified using known markers including Ptprc, S100a8, and Wfdc21. In the current analysis, we observed four distinct neutrophil clusters, with some differences by disease. Specifically, neutrophil profiles from bacterial keratitis mice and AED mice were more similar, whereas neutrophils in fungal corneal infection mice were more distinct.

Conclusions : The presence of distinct neutrophil transcriptomes in multiple ocular surface diseases and tissues demonstrates neutrophil heterogeneity at the transcriptional level in these microenvironments. Further, the presence of shared and unique profiles across distinct ocular pathologies may suggest the presence of neutrophil populations with distinct functions. Future work will focus on characterizing the functional significance of these profiles.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.