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

项目摘要 维生素A或视黄醇是其中最好的特点食品来源的营养素与不同的免疫- 调节作用饮食中维生素A的缺乏不仅与免疫功能障碍有关， 肠道，但也有几个系统性免疫疾病。为了维持足够的维生素A水平，身体依赖于 对肠上皮细胞或IEC从肠腔摄取视黄醇的影响。经独立选举委员会采纳后， 视黄醇可以被加工用于储存或被进一步代谢成视黄酸（RA）。尽管IEC 是维生素A代谢的中心，在控制膳食维生素A的命运方面起着主导作用， 我们对IEC内在维生素A代谢机制是如何调节的知识是非常肤浅的。通过 通过比较无菌（GF）和常规（CV）小鼠，我们证明了肠道细菌在 调节视黄酸（RA）水平和肠道中维生素A代谢机制的表达 上皮具体来说，我们发现细菌差异调节视黄醇脱氢酶7（RDH 7）的表达， 在肠上皮中参与视黄醇转化为RA的关键基因。通过使用基因小鼠 在IEC中的rdh 7中含有缺失的模型，我们确定IEC内在RA产生调节水平 白细胞介素IL-22，一种控制肠道细菌屏障反应的关键细胞因子。我们的数据表明， 这是第一次肠道细菌依赖性RA合成对于调节局部免疫反应至关重要。 目前的建议旨在了解肠道细菌调节RA合成的分子机制 和维生素A代谢基因rdh 7（Aim 1），建立rdh 7在IEC中表达的调节作用， 肠道中的RA信号传导（目的2），并确定IEC-内源性维生素A代谢对宿主微生物的作用 通过调节肠道中的IL-22水平来维持体内平衡（目的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rdh 7，特别是在IEC。 此外，我们建议利用新的方法，如单相关和无菌小鼠模型， 描绘细菌的线索，调节RA的产生在肠上皮细胞。我们的实验 方法学结合了稳健的分析方法，如串联质谱法（LC-MS/MS）， 使我们能够准确地确定维生素A代谢途径中的通量，以响应离散的细菌 线索这项工作将是重要的，因为它将建立肠道细菌和 肠上皮内源性维生素A代谢途径。此外，它将测试操纵的可行性 通过细菌调节IEC内在维生素A代谢的粘膜免疫用于治疗目的。

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.