Role of epithelial cell intrinsic vitamin A metabolism in regulating immune function in the gut

项目总结 维生素A或视黄醇是最具特点的食源性营养物质之一，具有多种免疫功能- 起到调节作用。饮食中缺乏维生素A不仅与免疫功能障碍有关，还与 肠道，但也有几种全身性免疫紊乱。为了保持足够的维生素A水平，身体依赖于 肠上皮细胞从肠腔摄取视黄醇。在被IECS吸收后， 视黄醇既可以加工储存，也可以进一步代谢成维甲酸(RA)。即使IECS 它们处于维生素A代谢的中心，在控制饮食中维生素A的命运方面起着主导作用， 我们对IEC固有的维生素A代谢机制是如何调控的了解非常肤浅。通过 比较无菌(GF)和常规(CV)小鼠，我们证明肠道细菌在 调节肠道维甲酸水平和维生素A代谢机制的研究 上皮组织。具体地说，我们发现细菌对视黄醇脱氢酶7(Rdh7)的表达有不同的调节。 肠上皮细胞中视黄醇转化为视黄酸的关键基因。通过使用遗传小鼠 在IECS中含有rdh7缺失的模型，我们建立了IEC固有的RA产生调节水平 白介素IL-22，一种控制肠道细菌屏障反应的关键细胞因子。我们的数据演示了 这是第一次肠道细菌依赖的RA合成对调节局部免疫反应至关重要。 目前的提案试图了解肠道细菌调节RA合成的分子机制 和IECS中的维生素A代谢基因rdh7(Aim1)，确定了rdh7在IECS中的表达在调控中的作用 肠道中的RA信号(目标2)和确定IEC固有的维生素A代谢对宿主微生物的作用 通过调节肠道中的IL-22水平实现动态平衡(目标3)。为了实现这些目标，我们开发了 创新的遗传方法使我们能够有条件地删除或过度表达rdh7，特别是在IECS中。 此外，我们建议利用新的方法，如单关联和诺生菌小鼠模型来 描述调节肠道上皮中类风湿因子生成的细菌线索。我们的实验 方法学结合了强大的分析方法，如串联质谱仪(LC-MS/MS)， 使我们能够准确地确定维生素A代谢途径中的流量，以响应离散细菌 暗示。这项工作意义重大，因为它将在肠道细菌和 肠上皮固有的维生素A代谢途径。此外，它还将测试操纵的可行性 用于治疗目的的通过细菌调节IEC固有的维生素A代谢的粘膜免疫。

PROJECT SUMMARY Vitamin A or retinol is amongst the most well characterized food-derived nutrients with diverse immune- modulatory roles. Deficiency in dietary vitamin A has not only been associated with immune dysfunctions in the gut, but also with several systemic immune disorders. To maintain sufficient levels of vitamin A, the body relies on the uptake of retinol from the intestinal lumen by intestinal epithelial cells or IECs. After uptake by IECs, retinol can either be processed for storage or be further metabolized into retinoic acid (RA). Even though IECs are at the center of Vitamin A metabolism and play a dominant role in controlling the fate of dietary vitamin A, our knowledge of how IEC intrinsic vitamin A metabolic machinery is regulated is extremely superficial. By comparing germ-free (GF) and conventional (CV) mice we demonstrate that gut bacteria play a critical role in modulating retinoic acid (RA) levels and expression of vitamin A metabolic machinery in the intestinal epithelium. Specifically, we find bacteria differentially regulate expression of retinol dehydrogenase 7 (rdh7), a key gene involved in conversion of retinol into RA in the intestinal epithelium. By employing genetic mouse models harboring a deletion in rdh7 in IECs, we establish that IEC intrinsic RA production regulates the levels of Interleukin IL-22, a key cytokine that controls barrier responses to gut bacteria. Our data demonstrates for the first time that gut bacteria-dependent RA synthesis is critical for regulating local immune responses. Current proposal seeks to understand the molecular mechanism by which gut bacteria regulate RA synthesis and vitamin A metabolic gene rdh7 in IECs (Aim1), establish the role of rdh7 expression in IECs in regulating RA-signaling in the gut (Aim 2) and determine the role of IEC-intrinsic vitamin A metabolism on host-microbe homeostasis via modulation of IL-22 levels in the gut (Aim 3). To accomplish these goals, we have developed innovative genetic approaches that enable us to conditionally delete or over-express rdh7 specifically in IECs. Further we propose to utilize novel methodologies such as monoassociated and gnotobiotic mouse models to delineate the bacterial cues that regulate RA generation in the intestinal epithelium. Our experimental methodology incorporates robust analytical approaches such as tandem mass-spectrometry (LC-MS/MS) that enables us to accurately determine the flux in vitamin A metabolic pathway in response to discrete bacterial cues. This work will be significant because it will establish mechanistic links between gut bacteria and intestinal epithelium intrinsic vitamin A metabolic pathway. Moreover, it will test the feasibility of manipulating mucosal immunity by bacterial modulation of IEC intrinsic vitamin A metabolism for therapeutic purposes.