Arginase, Nitric oxide, and the Defective Immune Response to Helicobacter pylori

描述（由申请人提供）：本提案的总体目标是提供一个培训工具，使申请人能够为成功的职业生涯做好准备，成为生物医学研究领域的独立研究者。这项研究的重点是细菌病原体幽门螺杆菌，尽管有强烈的先天和适应性粘膜免疫反应，但幽门螺杆菌在宿主的胃中持续存在一生。长期目标是阐明精氨酸酶在诱导型一氧化氮（NO）合酶（iNOS）依赖性宿主防御幽门螺杆菌中的作用。该提案的假设是，精氨酸酶II的l -精氨酸（L-Arg）代谢通过限制inos依赖性宿主防御和通过多胺的产生促进炎症，在幽门螺杆菌感染中起致病性。具体目的是：目的1，体外：证明精氨酸酶II代谢L-Arg在巨噬细胞对幽门螺杆菌的反应中具有重要的生物学作用。我们认为精氨酸酶II有效地与iNOS竞争L-Arg，并通过降低细胞内L-Arg的浓度来抑制iNOS的翻译和NO的合成。使用精氨酸酶抑制剂和精氨酸酶ii特异性siRNA/shRNA处理的RAW 264.7小鼠巨噬细胞，以及从精氨酸酶all -/-和野生型小鼠中分离的胃巨噬细胞，我们将评估：a) i. iNOS表达、蛋白质翻译和活性；和二世。杀灭幽门螺杆菌；b)细胞凋亡和多胺水平。目的2，在体内：确定精氨酸酶II代谢l -精氨酸在幽门螺杆菌感染中的生物学重要性。我们将使用两种幽门螺杆菌感染模型，一种是慢性模型，小鼠被幽门螺杆菌感染4个月，另一种是急性模型，小鼠被感染48小时。使用这两种系统，野生型小鼠将接受精氨酸酶抑制剂治疗，并将精氨酸酶全/全和野生型小鼠进行比较。在这些时间点，将评估以下内容：a)胃组织幽门螺杆菌定植、组织学和iNOS表达；b)离体胃巨噬细胞iNOS表达、NO生成和凋亡。期望这些研究将为幽门螺杆菌感染的免疫发病机制提供新的见解。与公共卫生相关：幽门螺杆菌感染了大约50%的世界人口和20-40%的美国人。它在所有个体中引起慢性胃炎，是世界上胃腺癌的主要原因，也是非药物性消化性溃疡疾病的主要原因。由于所有感染者中有10-20%会出现临床表现，因此疾病负担是巨大的。并非所有患者都能用抗生素成功治疗，而且没有适当的筛查策略；因此，正如本研究所追求的那样，对宿主免疫反应失败的原因有更深入的了解是非常必要的。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to provide a training vehicle that will allow the applicant to be prepared for a successful career as an independent investigator in biomedical research. The research is focused on the bacterial pathogen Helicobacter pylori, which persists in the human stomach for the life of the host despite a vigorous innate and adaptive mucosal immune response. The long-term goal is to elucidate the role of the enzyme arginase in inducible nitric oxide (NO) synthase (iNOS)-dependent host defense against H. pylori. The hypothesis of this proposal is that L-arginine (L-Arg) metabolism by arginase II is pathogenic in H. pylori infection by restricting iNOS-dependent host defense and by contributing to the inflammation by generation of polyamines. The specific aims are: Aim 1, in vitro: To demonstrate that L-Arg metabolism by arginase II has important biological effects in the macrophage response to H. pylori. We propose that arginase II effectively competes with iNOS for L-Arg and inhibits iNOS translation and NO synthesis by decreasing the intracellular concentration of L-Arg. Using RAW 264.7 murine macrophages treated with arginase inhibitors and arginase II-specific siRNA/shRNA, and gastric macrophages isolated from arginase ll-/- and wild-type mice, we will assess: A.) i. iNOS expression, protein translation, and activity; and ii. killing of H. pylori; and B.) apoptosis and polyamine levels. Aim 2, in vivo: To determine the biological importance of L-Arg metabolism by arginase II in H. pylori infection. We will use two models of H. pylori infection, a chronic model in which mice are challenged with H. pylori for four months, and an acute model in which mice are challenged for 48 hours. Using both systems, wild-type mice will be treated with arginase inhibitors, and arginase ll-/- and wild-type mice will be compared. At these time points, the following will be assessed: A.) H. pylori colonization, histology, and iNOS expression in gastric tissues; B.) iNOS expression, NO generation, and apoptosis in isolated gastric macrophages. It is expected that these studies will provide new insights into the immunopathogenesis of H. pylori infection. Relevance to public health: H. pylori infects approximately 50% of the world's population and 20-40% of Americans. It causes chronic gastritis in all individuals and is the major cause of gastric adenocarcinoma in the world and the primary cause of peptic ulcer disease that is not drug-induced. Because 10-20% of all of those infected will present with clinical manifestations, the disease burden is enormous. Not all patients can be successfully treated with antibiotics, and there is no screening strategy in place; therefore, greater understanding of the reasons for the failed host immune response, as is being pursued in this study, are greatly needed.