Functional Immunoimaging in the GI Tract

对于胃肠道中的自身免疫性和炎症性疾病， 炎症性肠病。现有的治疗方法可能有毒副作用， 开发更好的替代品的障碍是粘膜免疫仍然不完全 明白在疾病期间，身体的自然耐受机制会崩溃， 让炎症不受阻碍地发展越来越多的证据表明 调节性T细胞（TCFs）抑制这些有害的免疫反应，但这些细胞不是 完全理解，细胞动力学尚未探索。在本申请中，我们建议 使用功能性免疫成像来研究TdR，因为它们调节肠道炎症， 探索生物素和钙在这一过程中的作用。有几种额外的方法可以定义 所研究的组织和细胞的表型和功能状态，这将补充成像 调查结果。值得注意的是，没有发表的关于在结肠炎期间以这种方式研究TdR的报告。的 已经设计了使用两种结肠炎小鼠模型的实验，一种是过继转移模型， 自发模型，生物素缺乏。这些实验也是在 这样便于翻译到诊所。我们将使用双光子显微镜， 用于实时成像活组织中相互作用的淋巴细胞，以确定细胞免疫调节 这些事件解释了TdR如何抑制结肠炎。在该提案的第一个目标中，我们的研究 集中在肠系膜淋巴结，以观察免疫反应的诱导和进展 假设TCLs从树突状细胞物理置换致病性T细胞。我们还将 使用S1P1激动剂进行实验，S1P1激动剂是一种被认为可以预防 效应T细胞从离开淋巴结，这可能与协同工作，以防止 阻止致病性T细胞到达结肠一种类似的药剂已经在 溃疡性结肠炎的临床试验在该提案的第二个目标中，我们的研究集中在肠道 炎症和组织破坏引起疾病症状的壁。同样，我们将专注于 在这一过程中的作用。我们已经看到，生物素缺乏的小鼠发生自发性疾病， 这完全解决了生物素替代，直接连接生物素肠道炎症。 如果生物素补充剂可以被证明是有效的，那么它将代表一种低成本的治疗， 副作用最后，第三个目标集中在Tcl4如何调节效应T细胞中的钙信号 肠系膜淋巴结和结肠都有钙信号是T细胞发育所必需的 与树突状细胞的免疫突触，用于激活和发病。我们认为， 劫持这一过程以诱导效应T细胞从树突状细胞释放，从而预防疾病。 此外，我们将研究一种名为Orai1通道阻滞剂的新型治疗剂的作用。 Orai1已被确定为该系统中的钙通道，我们相信其阻断也将是一个重要的机制。 有效调节胃肠道中的免疫应答。此外，Orai 1封锁似乎 没有Treg功能的影响，我们将检查它是否可以用于增强Treg介导的抑制， 肠系膜淋巴结和结肠的炎症。我们预计这三个独立的， 但相互关联的目标将增强我们对TdR机制及其如何影响的理解， 胃肠道炎症。此外，我们希望对药物制剂的研究 提到的将推进他们的临床应用，并使我们所服务的退伍军人受益。

There are limited effective treatments for autoimmune and inflammatory diseases in the GI tract like Inflammatory Bowel Disease. The treatments that are available can have toxic side-effects and part of the barrier to developing better alternatives is that mucosal immunity remains incompletely understood. During disease, there is a breakdown in the body's natural tolerance mechanisms and that allows inflammation to progress unopposed. There is a growing body of evidence that supports a role for regulatory T cells (Tregs) in suppressing these deleterious immune responses but these cells are not fully understood and the cellular dynamics have not been explored. In this application, we propose to use functional immunoimaging to study Tregs as they modulate inflammation in the intestine and explore the role of biotin and calcium in this process. There are several additional approaches to define phenotype and functional status of the tissue and cells being studied which will supplement the imaging findings. Notably, there are no published reports on studying Tregs in this manner during colitis. The experiments have been designed to use two mouse models for colitis, an adoptive-transfer model and a spontaneous model that develops with biotin deficiency. The experiments have also been developed in such a way as to facilitate translation to the clinic. We will use two-photon microscopy which allows for real-time imaging of lymphocytes interacting in live tissue to define the cellular immunoregulatory events that explain how Tregs function to suppress colitis. In the first aim of the proposal, our studies are focused on the mesenteric lymph node to look at induction and progression of immune responses hypothesizing that Tregs physically displace pathogenic T cells from Dendritic Cells. We will also perform experiments using an S1P1 agonist which is a pharmacological agent believed to prevents effector T cells from leaving the lymph node and which may work synergistically with Tregs to prevent pathogenic T cells from reaching the colon. A similar agent has already shown promising results in clinical trials for Ulcerative Colitis. In the second aim of the proposal, our studies focus on the intestinal wall where inflammation and tissue destruction create the disease symptoms. Again, we will be focused on the role of Tregs in this process. We have seen that biotin deficient mice develop spontaneous disease that completely resolves with biotin replacement which directly links biotin to intestinal inflammation. If biotin supplementation can be shown to be effective, it would represent a low cost treatment without side-effects. Finally, the third aim focuses on how Tregs modulate calcium signaling in effector T cells in both the mesenteric lymph node and the colon. Calcium signaling is necessary for T cells to develop an immunological synapse with Dendritic Cells for activation and pathogenesis. We believe that Tregs hijack this process to induce the release of effector T cells from Dendritic Cells preventing disease. Additionally, we will study the effects of a novel therapeutic agent called an Orai1 channel blocker. Orai1 has been identified as the calcium channel in this system and we believe its blockade will also be effective in modulating immune responses in the GI tract. Furthermore, Orai1 blockade seems to have no effect of Treg function and we will examine if it can be used to augment Treg-mediated suppression of inflammation in the mesenteric lymph node and the colon. We anticipate that these three independent, but interrelated aims will enhance our understanding of the mechanism of Tregs and how they affect inflammation in the GI tract. Furthermore, we hope that the studies on the pharmacologic agents mentioned will advance their implementation for clinical use and benefit the Veterans we serve.