Immunomodulatory effects of arginine supplementation in colitis and colon cancer

描述（由申请人提供）：炎症性肠病（IBD），由溃疡性结肠炎（UC）和克罗恩病（CD）组成，是美国人群中广泛发病率和进展为结肠癌风险的来源，超过100万美国人受到影响。半必需氨基酸l -精氨酸（L-Arg）被吹捧为具有许多潜在益处的补充剂，包括增强免疫力。为了响应RFA AT-07-004，我们将在小鼠模型中进行机制研究，以确定l -精氨酸作为IBD补充剂的潜在价值，并确定它是否可以降低结肠炎相关癌症的风险。在免疫激活条件下，L-Arg利用的两条主要途径是诱导型一氧化氮（NO）合成酶（iNOS）和精氨酸酶途径。精氨酸酶产生l -鸟氨酸，鸟氨酸脱羧酶（ODC）的底物产生多胺，多胺已被证明可以增强伤口修复，并在免疫反应中作为调节因子。我们在两种结肠炎小鼠模型中的数据表明，补充l -精氨酸或删除iNOS可改善结肠炎，而抑制精氨酸酶或ODC则会加重疾病。l -精氨酸进入细胞主要依赖于阳离子氨基酸转运蛋白（CAT），尤其是巨噬细胞中主要的转运蛋白CAT2和无处不在表达的CAT1。我们将展示初步数据，表明l -精氨酸的可用性在细胞功能中很重要，如全局蛋白翻译、伤口修复和对炎症刺激的反应。我们还将证明，CAT2在结肠炎组织和结肠炎相关肿瘤中的表达上调，并且CAT2缺乏的小鼠似乎会加剧结肠炎。我们的假设是，L-Arg的可用性是IBD粘膜炎症的重要调节因子，因此细胞外环境中L-Arg的数量、L-Arg从细胞外到细胞内的运输以及L-Arg利用的竞争代谢途径的平衡在免疫调节中都很重要。我们的具体目标是确定补充l -精氨酸的生物学效应及其在我们将要研究的模型中摄取和代谢的作用：体外：a)上皮完整性；b)巨噬细胞功能；2)。体内结肠炎：a)临床参数；b)组织iNOS和多胺水平，血清NO和l -精氨酸水平；c)结肠上皮细胞和巨噬细胞的生物学效应；d)结肠淋巴细胞的反应；3)。体内结肠炎相关癌症：a)临床参数；b)肿瘤CAT1、CAT2、iNOS、细胞因子和多胺水平，以及血清NO和l -精氨酸水平。在这些研究中，我们试图建立关于l -精氨酸在粘膜免疫学中的作用的新见解，并为补充l -精氨酸在IBD和IBD相关癌症中的潜在有益作用提供证据，同时确定l -精氨酸摄取在这一过程中的重要性。这项工作有望为患者的转化研究提供基础，从而可能导致安全、有效、低成本的新辅助疗法，并改善IBD的长期预后。

DESCRIPTION (provided by applicant): Inflammatory bowel disease (IBD), consisting of ulcerative colitis (UC) and Crohn's disease (CD), is a source of extensive morbidity and risk for progression to colon cancer in the US population, with more than one million Americans affected. The semi-essential amino acid L-arginine (L-Arg) has been touted as a supplement with many potential benefits including enhancement of immunity. In response to RFA AT-07-004 we will conduct mechanistic studies in mouse models to address the potential value of L-Arg as a supplement in IBD and determine if it can reduce risk for colitis-associated cancer. During conditions of immune activation, the two major routes for L-Arg utilization are the inducible nitric oxide (NO) synthase (iNOS) and arginase pathways. Arginase generates L-ornithine, the substrate for the enzyme ornithine decarboxylase (ODC) that produces polyamines, which have been shown to enhance wound repair and have been implicated as regulatory factors in immune responses. Our data in two murine models of colitis indicate that supplementation of mice with L-Arg or deletion of iNOS improves colitis, while inhibition of arginase or ODC exacerbates disease. L-Arg uptake into cells is primarily dependent on the cationic amino acid transporter (CAT) proteins, particularly CAT2, which is the predominant transporter in macrophages and CAT1, which is ubiquitously expressed. We will show preliminary data that L-Arg availability is important in cellular functions such as global protein translation, wound repair, and responses to inflammatory stimuli. We will also show that CAT2 expression is upregulated in colitis tissues and colitis-associated tumors, and that mice deficient in CAT2 appear to have exacerbation of colitis. Our hypothesis is that L-Arg availability is an important regulator of mucosal inflammation in IBD such that the amount of L-Arg in the extracellular environment, the transport of L- Arg from outside to inside cells, and the balance of competing metabolic pathways for the utilization of L-Arg are all important in immunomodulation. Our specific aims are to determine the biological effects of L-Arg supplementation and the role of its uptake and metabolism in models in which we will study: 1.) In vitro: A.) epithelial integrity; and B.) macrophage function; 2.) In vivo colitis: A.) clinical parameters; B.) tissue iNOS and polyamine levels, and serum NO and L-Arg levels; C.) biological effects on colonic epithelial cells and macrophages, D.) responses in colonic lymphocytes; 3.) In vivo colitis-associated cancer: A.) clinical parameters; and B.) tumor CAT1, CAT2, iNOS, cytokine, and polyamine levels, and serum NO and L-Arg levels. In these studies, we seek to establish new insights into the role of L-Arg in mucosal immunology and to provide evidence for the potential beneficial effects of L-Arg supplementation in both IBD and IBD-associated cancer while establishing the importance of L-Arg uptake in this process. This work is expected to provide the groundwork for translational studies in patients that could lead to new adjunctive therapies that would be safe, effective, and low in cost and improve the long-term outcome in IBD. PUBLIC HEALTH REVELANCE: Inflammatory bowel disease (IBD) affects more than one million Americans and results in a substantial amount of suffering and the risk for developing cancer of the colon. In this project we will test the hypothesis that supplementation of the amino acid L-arginine (L-Arg) is beneficial in these diseases and that L-Arg uptake into cells has an essential role in the modulation of intestinal inflammation. We will use in vitro and in vivo models of colitis and colitis-associated cancer to conduct mechanistic studies that are designed to generate new insights into the immunobiology of IBD and lay the groundwork for new approaches to attenuate inflammation and associated carcinogenesis.