Project Summary Non-infectious uveitis is comprised of a heterogeneous group of clinically-defined diseases for which empiric therapy fails many patients. Molecular characterization of uveitis, on the other hand, would result in more precise classification of disease subtypes, while simultaneously providing pathophysiologic insight with therapeutic potential. This could spare patients unnecessary toxic side effects or loss of vision due to therapeutic inefficacy. Consequently, there is a critical need to better classify uveitis subtypes in order to discover more effective strategies for targeted immune suppression. The long-term goal is to develop a platform for molecular characterization of uveitis that will eventually facilitate precision medicine treatment strategies. The approach is to utilize validated experimental techniques to explore novel observations obtained from single cell RNA-Sequencing (scRNA-Seq), a high-definition gene expression analysis of ocular immune cells. Specifically, the central hypothesis for this proposal, that type 1 conventional dendritic cells (DC1s) promote the resolution of ocular inflammation, is based on primary observations of human ocular DC1s obtained via scRNA-Seq. The overall objectives of this proposal are to a) ascertain the role of DC1s in ocular inflammation, and b) to determine how patient-specific alterations in DC1s contribute to the chronicity of uveitis. Toward these ends, the following specific aims will be pursued: 1) Test the hypothesis that the ocular microenvironment promotes pro-resolution DC1s. 2) Test the hypothesis that DC1s promote the resolution of ocular inflammation. 3) Test the hypothesis that patient-specific DC1 gene expression signatures predict uveitis chronicity. In aim 1, the effect of the ocular microenvironment on DC1s will be tested by a) analyzing aqueous-exposed human DC1s in vitro and b) analyzing murine DC1s in 2 distinct models of ocular inflammation in vivo. In aim 2, the specific effects of DC1s on disease chronicity during ocular inflammation will be explored using mice deficient in DC1s. Finally, in aim 3, the transcriptional signatures that best predict chronic and acute uveitis will be validated and used to a) identify peripheral blood biomarkers and b) identify potential therapeutic targets. This contribution will be significant because it will improve our ability to define uveitis entities based on pathophysiologic mechanisms that can then be appropriately targeted by precision application of immune therapies. This proposal is innovative in the use of scRNA-Seq to generate testable hypotheses from in-depth analysis of patient samples. Ultimately, improved molecular characterization of uveitis based on identification and validation of differential gene expression provides a scaffold for future analyses that will facilitate a precision medicine approach to immune therapy.

Project Narrative Non-infectious uveitis causes 10% of blindness in the United States and disproportionately affects working- aged people, making it a significant public health issue. Targeted corticosteroid-sparing immune therapies are only variably effective because the underlying pathogenic mechanisms in ocular inflammation are not well understood. If successful, this research will address this knowledge gap and advance the NEI’s goals by: 1) developing advanced molecular diagnostic tools, 2) better defining heterogeneity within uveitis and 3) identifying the roles of specific immune mediators in individual inflammatory syndromes.