Resolution of ocular inflammation: the role of type 1 conventional dendritic cells

项目摘要 非感染性葡萄膜炎由一组不同的临床定义的疾病组成，这些疾病的经验 许多病人的治疗失败了。另一方面，葡萄膜炎的分子特征将导致更多 疾病亚型的精确分类，同时提供病理生理洞察 治疗潜力。这可以避免患者因以下原因而产生不必要的毒副作用或视力丧失 治疗无效。因此，迫切需要更好地对葡萄膜炎亚型进行分类，以便 发现更有效的靶向免疫抑制策略。我们的长期目标是开发一个 葡萄膜炎的分子表征平台，最终将促进精准药物治疗 战略。这种方法是利用经过验证的实验技术来探索获得的新观察结果 来自单细胞RNA测序(scRNA-Seq)的眼免疫高清基因表达分析 细胞。具体地说，这一提议的中心假设是，1型常规树突状细胞(DC1s) 促进眼部炎症的消退，是基于对人类眼部DC 1的初步观察 通过scRNA-Seq获得。这项建议的总体目标是：a)确定Dc1在眼部的作用 炎症，以及b)确定DC 1中患者特有的改变如何导致慢性 葡萄膜炎。为了达到这些目的，将追求以下具体目标：1)检验眼睛 微环境促进DC1s的前分辨率。2)检验DC1s促进DC1s分辨率的假设 眼部发炎。3)检验患者特定的DC1基因表达特征可预测的假设 葡萄膜炎是慢性的。在目标1中，将通过a)分析来测试眼微环境对DCI的影响 体外房水暴露的人DC1s和b)在两种不同的眼部模型中分析小鼠DC1s 体内的炎症。在目标2中，DC1s在眼部炎症过程中对疾病慢性化的特定作用将 使用缺乏DC1s的小鼠进行探索。最后，在目标3中，最好的预测转录签名 将验证慢性和急性葡萄膜炎，并将其用于a)识别外周血生物标志物和b)识别 潜在的治疗靶点。这一贡献将是重大的，因为它将提高我们定义 基于病理生理机制的葡萄膜炎实体，然后可以通过精确地进行适当的靶向 免疫疗法的应用。该方案在使用scRNA-Seq生成可测试序列方面具有创新性 对患者样本进行深入分析后得出的假设。最终，改进了分子特征 基于差异基因表达识别和验证的葡萄膜炎为未来提供了一个支架 这些分析将促进以精确医学方法进行免疫治疗。

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.