Abstract
Purpose :
Meibomian gland dysfunction (MGD) is a leading cause of evaporative dry eye disease, yet the mechanism driving pathogenesis remains poorly understood. Recent studies in the Allergic Eye Disease (AED) mouse model have demonstrated the role of a Th17 and neutrophil-driven pathway in MGD pathogenesis. However, the manner by which neutrophils cause MGD in these mice is unclear. We addressed this knowledge gap by performing single cell RNA sequencing on cells, primarily leukocytes, from blood, tears, and conjunctiva collected from the AED mouse model, and compared results against tissues collected from naïve mice.
Methods :
AED was induced in 9-week-old female C57Bl/6 mice by immunizing against ovalbumin (OVA) in the presence of pertussis toxin and, following a two-week incubation, OVA was applied to the eye, daily, for one week before collecting tissues, as previously described (Reyes NJ, 2018). Blood, conjunctiva, and tear samples from AED (n=9) and naïve (n=6) mice were harvested and prepared as single cell suspensions. FACS sorting was used to isolate cells from tears (live singlets), neutrophils from blood (live singlets, CD45+, Ly6G+), and leukocytes from conjunctiva (live singlets, CD45+). Sorted cells were pooled by treatment group and by tissue before sequencing. Data was analyzed using Seurat 4 with R version 4.1.
Results :
The major immune cell (Ptprc) types identified included neutrophils (S100a8, Cd33), eosinophils (Ccr3, Siglecf), macrophages (Aif1), mast cells (Cd200r3), Th2 αβ T cells (Trac, Gata3), Th17 γδ T cells (Trdc, Rorc), dendritic cells (Flt3, Ccr7), and B cells (Ms4a1). We observed that AED mouse ocular tissues had increased neutrophil abundance and unique neutrophil cluster prevalence relative to naïve ocular tissues. Most notably, of the four identified neutrophil clusters, only a single cluster was associated with disease. This unique population was nearly absent in both the conjunctiva of naïve mice and the blood of either AED or naïve mice.
Conclusions :
The notable presence of this disease-associated neutrophil cluster in the conjunctiva and tears of AED mice, but not naïve tissues or AED mouse blood, indicates that this phenotype arises as a result of the local ocular inflammatory environment. Further, it suggests that these neutrophils may be involved in MGD pathogenesis in the AED model.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.