Effects of altered meibum quality on meibomian gland population dynamics in the AWAT2 KO mouse.

Abstract

Purpose : Recently, several transgenic mouse models of meibomian gland dysfunction have been developed based on deletion of critical genes encoding enzymes required for the synthesis of meibum that lead to altered meibum quality, gland plugging, ductal dilation and a dry eye phenotype. The purpose of this study was to identify changes in cell populations in the wax ester deficient, AWAT2 KO mouse using single cell and spatial transcriptomics.

Methods : The tarsal plate from male AWAT2 KO and wildtype C57Bl/6 mice were excised and single cells isolated for analysis using the 10× Genomics Chromium system followed by Illumina NovaSeq 6000 sequencing. The data were subject to UMAP visualization, as well as Seurat, Monocle2, MetaNeighbor, and SCENIC analysis. For spatial transcriptomics, frozen tissue sections were probed with 100 gene transcripts targeted for probing meibomian and interstitial cells using the Resolve Biosciences Molecular Cartography platform and analyzed using Seurat.

Results : The cell populations identified in the WT tarsal plate were also identified in the AWAT2 KO mouse tarsal plate but in significantly different proportions. While meibocytes and keratinized ductal epithelial cells were unchanged or decreased, there was a marked increase in the non-keratinized ductal epithelial population indicating that the increased gland size in the KO mouse was largely due to ductal hyperplasia. Inflammatory signaling by the meibomian gland was also increased with upregulation of Il23a by keratinized ductal cells and increased Lcn2 by both meibocytes and duct. The most dramatic changes were in the immune cell populations which showed the emergence of a KO-exclusive neutrophil population that expressed S100a8, S100a9 and Mmmp9, along with an increase in the proportion of T cells expressing IL17a. Inflammatory signaling was also increased in the conjunctiva with increased expression of S100a9.

Conclusions : The detection of enhanced expression of IL23a/Lcn2 by the meibomian gland coupled with increased T cell Il17a and recruitment of neutrophils expressing S100a8/S100a9 suggest a psoriasis-like pathology caused by altered meibum quality in the AWAT2 KO mouse. We hypothesize that ductal dilation and stretching of the ductal epithelium may be a complicating factor leading to an autoimmune response in MGD and dry eye disease.

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