Arginine Availability and Metabolism in the Immune Evasion of H. pylori

描述（由申请人提供）： 幽门螺杆菌的获得及其消化性溃疡疾病和胃癌的临床后遗症仍然是我们退伍军人的重大健康问题。H.幽门螺杆菌感染了世界上一半的人口，10%的感染者会患上消化性溃疡，1%的感染者会患上胃癌，它是世界范围内癌症死亡的第二大原因。美国军人/妇女暴露于H。在世界上感染率非常高并且与高癌症风险相关的菌株普遍存在的地区，因此，提高对缺陷性免疫应答的理解至关重要。我们对为什么对H. pylori不能根除有机体。我们的工作集中在L-精氨酸（L-Arg）在巨噬细胞对H.幽门。L-Arg是两种不同酶途径的共同底物：诱导型一氧化氮（NO）合酶（iNOS），其产生高输出NO;和精氨酸酶，由两种形式组成，精氨酸酶I（Arg 1）和精氨酸酶II（Arg 2），其产生L-鸟氨酸，L-鸟氨酸是鸟氨酸脱羧酶（ODC）合成多胺的底物。我们已经表明，巨噬细胞中L-Arg摄取的调节剂阳离子氨基酸转运蛋白2（CAT 2）是iNOS蛋白表达所必需的，并且其功能受到抑制 通过多胺在限制NO生成的过程中，导致持续的细菌定植和炎症。此外，我们发现Arg 2是对H的无效反应的中心。幽门。在目前的范例中，M1巨噬细胞表达iNOS并产生参与宿主防御细胞外感染的促炎介质，而M2细胞表达Arg 1，参与宿主对寄生虫感染的反应，并被认为是肿瘤相关的巨噬细胞。然而，在回应H. pylori感染时，巨噬细胞不是M1或M2型。它们表达iNOS和Arg 2，但不表达Arg 1。在抑制或敲低Arg 2后，或在来自Arg 2-/-小鼠的细胞中，存在增强的iNOS蛋白翻译/表达和NO产生，表明H.幽门刺激的细胞具有次优的M1应答。Arg 2-/-小鼠对H.幽门螺杆菌感染与减少的定植相关，我们已经将增强的宿主防御与更多的胃巨噬细胞（GMacs）联系起来，GMacs经历更少的凋亡，具有更多的iNOS/NO产生，以及增强的Th 1/Th 17应答。我们现在表明，在H。幽门螺杆菌感染的胃是调节性巨噬细胞（MMPs），这可能有助于感染的持续性。我们推测，在GMacs中，iNOS和Arg 2对L-Arg的竞争导致了一个无效的循环和宿主防御的妥协，从而导致H。幽门螺杆菌的持久性，炎症和癌症的风险，是一个网站的治疗干预。我们的具体目的是：1确定Arg 2的免疫抑制作用是否是由于iNOS的抑制。我们将产生Arg 2-/-iNOS-/-双敲除小鼠，将其与野生型（WT）、Arg 2-/-和iNOS-/-小鼠进行比较，并将使用骨髓嵌合体来检查A）H。幽门螺杆菌定植和胃炎; B）GMac表型和功能;和C）胃组织、脾细胞和淋巴细胞中的适应性免疫。2.）确定Arg 2的作用是否由下游多胺介导。我们将产生Arg 2-/-ODC+/-小鼠，并向WT和Arg 2-/-小鼠施用DFMO（ODC抑制剂），并评估：A）H。幽门螺杆菌定植和胃炎; B）GMac表型和功能;和C）适应性免疫。3)为了确定Arg 2是否促进H. pylori诱导的胃癌发生，是干预的靶点，也是识别退伍军人胃癌风险的生物标志物。我们将利用：A）发展发育异常/癌症的小鼠，与Arg 2-/-小鼠杂交。B）用BEC酶抑制剂治疗的发展发育异常/癌症的沙鼠。C）胃组织和H.来自两个生物储存库中的人类受试者的幽门螺杆菌分离物：来自哥伦比亚的低和高胃癌风险地区，以及来自纳什维尔VAMC的退伍军人。这些研究将对我们对H. pylori免疫发病机制和胃癌发生机制的研究，将为风险评估和治疗带来新的方法。

DESCRIPTION (provided by applicant): The acquisition of Helicobacter pylori and its clinical sequelae of peptic ulcer disease and gastric cancer remain substantial health concerns for our Veterans. H. pylori infects half of the world's population, causes peptic ulcers in 10% and gastric cancer in 1% of those infected, and it is the second leading cause of cancer death worldwide. American Servicemen/women are exposed to H. pylori in regions of the world where infection rates are very high and strains associated with high cancer risk are prevalent. Thus, improved understanding of the defective immune response is crtical. We have gained new insights into why the sustained immune response to H. pylori fails to eradicate the organism. Our work has focused on the role of L-arginine (L-Arg) in the macrophage response to H. pylori. L-Arg is the common substrate for two divergent enzymatic pathways: inducible nitric oxide (NO) synthase (iNOS), that generates high output NO; and arginase, consisting of two forms, arginase I (Arg1) and arginase II (Arg2) that generate L-ornithine, which is the substrate for polyamine synthesis by ornithine decarboxylase (ODC). We have shown that the regulator of L-Arg uptake in macrophages, cationic amino acid transporter-2 (CAT2), is required for protein expression of iNOS and that its function is inhibited by polyamines in a process that restricts NO generation and leads to sustained bacterial colonization and inflammation. In addition, we found that Arg2 is at the center of the ineffective response to H. pylori. In the current paradigm, M1 macrophages express iNOS and produce pro-inflammatory mediators involved in host defense against extracellular infections, while M2 cells express Arg1, are involved in host response to parasitic infection, and are implicated as tumor-associated macrophages. However, in response to H. pylori, macrophages are not M1 or M2 type. They express iNOS and Arg2, but not Arg1. Upon inhibition or knockdown of Arg2, or in cells from Arg2-/- mice, there is enhanced iNOS protein translation/expression and NO generation, indicating that H. pylori-stimulated cells have sub-optimal M1 response. Arg2-/- mice exhibit increased immune responses to H. pylori infection that are correlated with decreased colonization, and we have linked enhanced host defense to more gastric macrophages (GMacs), which undergo less apoptosis and have more iNOS/NO production, and an enhanced Th1/Th17 response. We now show that a substantial portion of GMacs in the H. pylori-infected stomach are regulatory macrophages (Mregs), which may contribute to persistence of the infection. We hypothesize that the competition between iNOS and Arg2 for L-Arg in GMacs leads to a futile cycle and a compromise of host defense that results in H. pylori persistence, inflammation, and cancer risk, and is a site for therapeutic intervention. Our specific aims are: 1 To determine if immunosuppressive effects of Arg2 are due to inhibition of iNOS. We will generate Arg2-/-iNOS-/- double knockout mice that will be compared to wild-type (WT), Arg2-/-, and iNOS-/- mice, and bone marrow chimeras will be used, to examine A) H. pylori colonization and gastritis; B) GMac phenotype and function; and C) Adaptive immunity in gastric tissues, splenocytes, and lymphocytes. 2.) To determine if effects of Arg2 are mediated by downstream polyamines. We will generate Arg2-/-ODC+/- mice, and administer DFMO (ODC inhibitor) to WT and Arg2-/- mice, and assess: A) H. pylori colonization and gastritis; B) GMac phenotype and function; and C) Adaptive immunity. 3) To determine if Arg2 facilitates H. pylori-induced gastric carcinogenesis, is a target for intervention, and is a biomarker for identifying gastric cancer ris in Veterans. We will utilize: A) Mice that develop dysplasia/cancer, crossed with Arg2-/- mice. B) Gerbils that develop dysplasia/cancer, treated with an arginase inhibitor, BEC. C) Gastric tissues and H. pylori isolates from human subjects in two biorepositories: from areas of low and high gastric cancer risk in Colombia, and from Veterans at the Nashville VAMC. These studies will have a major impact on our understanding of H. pylori immunopathogenesis and gastric carcinogenesis, and will lead to new approaches for risk assessment and treatment.