PROJECT SUMMARY (Revised July 15, 2021) – NO CHANGES FROM PARENT GRANT Aqueous-deficient dry eye is among the most common and debilitating clinical manifestations of systemic autoimmune diseases such as Sjögren's, lupus and rheumatoid arthritis. While it is well established that chronic inflammation of the ocular surface and exocrine glands (e.g., lacrimal gland) represents the predominant driving force in dry eye disease, pathological alterations in the cornea are among the most common and debilitating clinical manifestations of autoimmune-mediated dry eye. With the cornea providing approximately two-thirds of the eye's focusing power and protecting the anterior eye from environmental, inflammatory and microbial insult, disease-mediated corruption of tissue function and homeostasis has a vast array of pathological outcomes. Indeed, a multitude of studies in both human patients and mouse models of aqueous-deficient dry eye illustrate late-stage disease encompasses epithelial barrier disruption, epithelial hyperplasia, reduced corneal innervation and inflammatory cell infiltration. Thus, dry eye disease induces pathological changes along multiple levels. However, due to the limited understanding of the cellular makeup of the cornea, including the lineage relationships of specific cell types, the impact of dry eye on cell heterogeneity and lineage outcomes is unknown. To help solve this significant problem, we have begun to define the cellular diversity and differentiation trajectories of corneal cells and how these are perturbed by dry eye disease using single cell nuclei RNAsequencing in combination with the autoimmune regulatory (Aire)- deficient mouse model. Using this model, we have also begun to identify novel changes in corneal cell composition at late disease stages that may underlie disease progression. Based on these data we hypothesize that disease progression is promoted by an alteration in corneal cell fate that compromises the repair and cellular replenishment of the tissue. We propose to explore this hypothesis by first defining epithelial and stromal cell heterogeneity, differentiation trajectories and stem cells in the homeostatic cornea. We will then seek to elucidate the impact of dry eye on the identity of the corneal cell lineages at late stage disease. Outcomes from these studies will aid in the development of therapeutics to halt disease progression, as well as the generation of diagnostic targets for early intervention.