Immunomodulatory effects of arginine supplementation in colitis and colon cancer

描述（由申请方提供）：炎症性肠病（IBD），包括溃疡性结肠炎（UC）和克罗恩病（CD），是美国人群中广泛发病率和进展为结肠癌风险的来源，有超过100万美国人受影响。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。针对RFA AT-07-004，我们将在小鼠模型中进行机制研究，以探讨L-Arg作为IBD补充剂的潜在价值，并确定其是否可以降低结肠炎相关癌症的风险。在免疫激活的条件下，L-Arg利用的两个主要途径是诱导型一氧化氮（NO）合酶（iNOS）和谷胱甘肽转移酶途径。精氨酸酶产生L-鸟氨酸，L-鸟氨酸是产生多胺的酶鸟氨酸脱羧酶（ODC）的底物，多胺已被证明可增强伤口修复，并被认为是免疫应答中的调节因子。我们在两种小鼠结肠炎模型中的数据表明，补充L-Arg或缺失iNOS的小鼠可改善结肠炎，而抑制L-Arg或ODC可加重疾病。细胞摄取L-Arg主要依赖于阳离子氨基酸转运蛋白（CAT），特别是CAT 2，它是巨噬细胞中的主要转运蛋白，CAT 1广泛表达。我们将显示的初步数据，L-Arg的可用性是重要的细胞功能，如全球蛋白质翻译，伤口修复，并对炎症刺激的反应。我们还将证明CAT 2表达在结肠炎组织和结肠炎相关肿瘤中上调，并且CAT 2缺陷的小鼠似乎具有结肠炎恶化。我们的假设是，L-Arg可用性是IBD中粘膜炎症的重要调节剂，使得细胞外环境中L-Arg的量、L-Arg从细胞外到细胞内的转运以及利用L-Arg的竞争代谢途径的平衡在免疫调节中都是重要的。我们的具体目标是确定补充L-Arg的生物学效应及其在模型中的吸收和代谢作用，我们将研究：1。体外：A.）上皮完整性;和B.）巨噬细胞功能; 2.）体内结肠炎：A.）临床参数; B.）组织iNOS和多胺水平以及血清NO和L-Arg水平; C.）对结肠上皮细胞和巨噬细胞的生物学作用，D.）结肠淋巴细胞中的反应; 3.）体内结肠炎相关癌症：A.）临床参数;和B.）肿瘤CAT 1、CAT 2、iNOS、细胞因子和多胺水平，以及血清NO和L-Arg水平。在这些研究中，我们试图建立新的见解，L-Arg在粘膜免疫学中的作用，并提供证据，为IBD和IBD相关癌症中补充L-Arg的潜在有益作用，同时建立L-Arg摄取在这一过程中的重要性。这项工作预计将为患者的转化研究提供基础，这些研究可能导致安全，有效，成本低的新的连续疗法，并改善IBD的长期结局。 公共卫生部门：炎症性肠病（IBD）影响超过一百万美国人，并导致大量的痛苦和发展结肠癌的风险。在这个项目中，我们将测试的假设，补充氨基酸L-精氨酸（L-Arg）是有益的，在这些疾病和L-Arg摄取到细胞中有一个重要的作用，在肠道炎症的调制。我们将使用结肠炎和结肠炎相关癌症的体外和体内模型进行机制研究，旨在对IBD的免疫生物学产生新的见解，并为减轻炎症和相关致癌作用的新方法奠定基础。

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.