PROJECT SUMMARY The nature and function of ocular surface nerves play a critical role in maintaining ocular surface health while preventing disease. Disruption of ocular surface sensory nerves can lead to blinding keratitis, graft rejection, dry eye disease, and ocular surface pain. The advent of “-omics” studies have provided a platform to understand how ocular surface nerves participate in the broader context of ocular health and disease. Here, we outline a proposal that will create large datasets detailing the nature of ocular surface innervation. We will take a systems-level approach towards analyzing our data using bioinformatics and machine learning platforms, so that we can gain a practical understanding of the ocular surface environment during health and disease. Specifically, this proposal will address how ocular surface nerves, epithelial cells, and immune cells interact. We have recruited a multidisciplinary research team that consists experts in ophthalmology, neuroimmunology, neurology, proteomics, systems immunology, and bioinformatics. With this team, we plan to comprehensively analyze all three levels of research outlined in the RFA, which includes anatomical and morphological characterization, defining cellular and molecular properties of neuronal and non-neuronal cell types, and assessing functional properties of neuronal and non-neuronal cells. Because innervation patterns and functionality changes with disease, we will use multiple ocular surface disease models (viral keratitis and aqueous dry eye disease) to better understand how neurons, epithelial cells, and immune cells affect neuronal functionality and subsequent disease. We will achieve our goal by pursuing four specific aims: 1) We will perform single-cell (sc)-omics on neuonrs and non-neuronal cells of the cornea and ocular surface innervating ganglia. These data will characterize neuronal identities and describe their functionality during disease. Additionally, we will be able to molecularly characterize immune and epithelial cells that influence the functionality of ocular surface afferents during disease. 2) We will use mass spectrometry to analyze the proteomic signatures of the cornea during health and disease. An unbiased shotgun approaches and a targeted approach focusing on neurotrophic factors will be used to identify proteins that influence ocular surface nerve functionality. 3) We will use our imaging techniques and histocytometry to comprehensively analyze the location and morphology of neuronal and non-neuronal cells within the cornea, so that we may be able to make conclusions about how nerves, epithelial, and immune cells interact in close proximity at the ocular surface. 4) We will use DREADD (designer receptor exclusively activated by designer drugs) technology to perturb neuronal sensitivity, so that we can assess how altering neuronal responsiveness to stimuli may affect the ocular surface homeostasis and progress of disease. A systems-level analysis...