The role of indole-induced immune dysregulation in collagen-inducedarthritis

项目总结/摘要 临床前类风湿性关节炎（RA）是一个时期，其中全身性自身免疫性发展在前几年， 出现临床上明显的疾病。在这里，我们的目标是定义导致临床前自身免疫的机制， 这可以为阻断这些通路和预防RA的治疗设计提供信息。我们的团队是不可或缺的 在确定和调查RA的“粘膜起源”假说，利用胶原诱导的关节炎， (CIA)模型来证明疾病发展对微生物组的要求。我们最近发现 细菌衍生的色氨酸代谢物吲哚作为该模型中自身免疫的关键调节剂。枯竭 无论是微生物组（使用广谱抗生素）或饮食色氨酸是对CIA的保护，和 在任一种情况下补充吲哚可逆转这种保护作用。这种吲哚-CIA模型提供了，对于第一个 时间，一个模型，其中一个单一的细菌代谢产物是足够的疾病发展。基于我们 初步发现，Th 17细胞和炎性细胞因子（特别是IL-6）的产生在这种情况下升高， 吲哚-CIA模型中，我假设吲哚是一种生物活性代谢产物，通过倾斜Th 17细胞促进CIA 分化和刺激抗原呈递细胞（APC）以产生IL-6。本文中概述的实验 建议的目的是确定吲哚对CIA的影响。目的1将确定吲哚对CD 4 在吲哚-CIA模型和抗原特异性T细胞应答模型中的效应T细胞应答 利用OT-II转基因小鼠。目的2将确定吲哚介导的IL-6的来源和需求 生产我将利用组合的单细胞RNA/ATACseq来鉴定细胞亚群和信号通路。 其中吲哚诱导IL-6产生。最后，我将测试对树突状细胞（DC）介导的IL-6的需求 吲哚-CIA的生产。成功完成这些目标将（1）确定细胞亚群和功能 在吲哚-CIA模型中受吲哚影响，这将允许未来的机制研究更好地定义和 调节这一过程，（2）确定DC衍生的IL-6在CIA中的作用，这从未被证明，和（3） 产生假设，以确定受吲哚刺激影响的信号通路，这将是 这是理解吲哚引发自身免疫的机制所必需的。总之，这些发现将 填补了关于微生物组在炎症性关节炎发展中的作用的关键知识空白。 此外，通过这些研究，我将成为粘膜免疫学专家，学会设计和测试 严格的假设，并通过分析生物信息学的尖端技术， Aim 2中生成的RNA/ATACseq数据。通过这项建议获得的培训将使我能够很好地- 定位在流变学研究奖学金和应用所需的技能，在未来成功的K 08。

Project Summary/Abstract Pre-clinical Rheumatoid Arthritis (RA) is a period in which systemic autoimmunity develops in the years prior to onset of clinically apparent disease. Here, we aim to define the mechanisms leading to pre-clinical autoimmunity, which could inform the design of therapies to block these pathways and prevent RA. Our group has been integral in identifying and investigating the “mucosal origins” hypothesis of RA, utilizing the collagen-induced arthritis (CIA) model to demonstrate the requirement of the microbiome for disease development. We recently identified a bacterial-derived tryptophan metabolite, indole, as a key regulator of autoimmunity in this model. Depletion of either the microbiome (using broad-spectrum antibiotics) or dietary tryptophan is protective against CIA, and supplementation with indole in either setting reverses this protection. This indole-CIA model provides, for the first time, a model in which a single bacterial metabolite is sufficient for disease development. Based on our preliminary findings that Th17 cells and inflammatory cytokine production (especially IL-6) are elevated in this indole-CIA model, I hypothesize that indole is a bioactive metabolite that promotes CIA by skewing Th17 cell differentiation and stimulating antigen presenting cells (APCs) to produce IL-6. The experiments outlined in this proposal are designed to define the effects of indole on CIA. Aim 1 will determine the effect of indole on CD4 effector T cell responses, both in the indole-CIA model and in a model of antigen-specific T cell responses utilizing OT-II transgenic mice. Aim 2 will then identify the source of and requirement for indole-mediated IL-6 production. I will utilize combined single cell RNA/ATACseq to identify the cell subsets and signaling pathways in which indole induces IL-6 production. Finally, I will test the requirement for dendritic cell (DC)-mediated IL-6 production in indole-CIA. Successful completion of these aims will (1) define the cell subsets and functions affected by indole in the indole-CIA model, which will allow for future mechanistic studies to better define and modulate this process, (2) define the role of DC-derived IL-6 in CIA, which has never been shown, and (3) generate hypotheses to later identify the signaling pathways affected by indole stimulation, which will be necessary to understand the mechanisms by which indole incites autoimmunity. Altogether, these findings will fill a critical knowledge gap regarding the role of the microbiome in the development of inflammatory arthritis. Furthermore, through these studies I will become an expert in mucosal immunology, learn to design and test rigorous hypotheses, and become proficient in cutting-edge bioinformatic techniques through analysis of the RNA/ATACseq data generated in Aim 2. The training gained through this proposal will enable me to be well- positioned for a research fellowship in rheumatology and apply skills needed for a successful K08 in the future.