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

描述(由申请人提供)： 幽门螺杆菌的感染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非常关注的健康问题。幽门螺杆菌感染了世界上一半的人口，10%的人会导致消化性溃疡，1%的人会患上胃癌，它是全球癌症死亡的第二大原因。美国军人在世界上感染率非常高、与癌症风险高相关的菌株普遍存在的地区暴露于幽门螺杆菌。因此，改善对缺陷免疫反应的理解是至关重要的。我们对为什么对幽门螺杆菌的持续免疫反应未能根除这种有机体有了新的见解。我们的工作集中在L-精氨酸(L-精氨酸)在幽门螺杆菌对巨噬细胞反应中的作用。L-精氨酸是两条不同的酶途径的共同底物：诱导型一氧化氮合酶(INOS)，产生高产量的NO；精氨酸酶，由精氨酸酶I(Arg1)和精氨酸酶II(Arg2)组成，产生L-鸟氨酸，是鸟氨酸脱羧酶合成多胺的底物。我们已经证明，巨噬细胞摄取L-精氨酸的调节剂阳离子氨基酸转运体2(CAT2)是诱导型一氧化氮合酶蛋白表达所必需的，其功能被抑制。 多胺在一个过程中限制NO的生成，并导致持续的细菌定居和炎症。此外，我们还发现Arg2是幽门螺杆菌无效反应的中心。在目前的模式中，M1巨噬细胞表达iNOS并产生致炎介质，参与宿主对细胞外感染的防御；M2细胞表达Arg1，参与宿主对寄生虫感染的反应，并与肿瘤相关的巨噬细胞有关。然而，对幽门螺杆菌的反应，巨噬细胞不是M1型或M2型。它们表达iNOS和Arg2，但不表达Arg1。在抑制或敲除Arg2后，或在Arg2-/-小鼠的细胞中，iNOS蛋白的翻译/表达增强，并没有产生NO，表明H.Pylori刺激的细胞具有次优的M1反应。Arg2-/-小鼠对幽门螺杆菌感染表现出增强的免疫反应，这与减少定植有关，我们将增强的宿主防御与更多的胃巨噬细胞(GMAC)联系在一起，胃巨噬细胞(GMAC)经历更少的凋亡和更多的iNOS/NO产生，以及Th1/Th17反应增强。我们现在发现，在幽门螺杆菌感染的胃中，很大一部分GMAC是调节性巨噬细胞(Mregs)，这可能有助于感染的持久性。我们推测，诱导型一氧化氮合酶和精氨酸在GMCs中对L-精氨酸的竞争导致无效循环和宿主防御的妥协，从而导致幽门螺杆菌的持续存在、炎症和癌症风险，并且是进行治疗干预的场所。我们的具体目的是：1确定Arg2的免疫抑制作用是否源于对iNOS的抑制。我们将产生Arg2-/-iNOS-/-双基因敲除小鼠，并将其与野生型(WT)、Arg2-/-和iNOS-/-小鼠进行比较，并将使用骨髓嵌合体来检测A)幽门螺杆菌定植和胃炎；B)GMAC表型和功能；以及C)胃组织、脾细胞和淋巴细胞的适应性免疫。2.)以确定Arg2的作用是否由下游的多胺介导。我们将建立Arg2-/-ODC+/-小鼠，并对WT和Arg2-/-小鼠应用DFMO(ODC抑制剂)，评估：A)幽门螺杆菌定植和胃炎；B)GMAC表型和功能；C)获得性免疫。3)确定Arg2是否促进幽门螺杆菌诱导的胃癌发生，是否为干预的靶点，以及是否为识别退伍军人胃癌RI的生物标志物。我们将利用：a)发展为异型增生/癌症的小鼠，与Arg2-/-小鼠杂交。B)发展为异型增生/癌症的沙土鼠，用精氨酸酶抑制剂BEC治疗。C)来自两个生物储存库的人类受试者的胃组织和幽门螺杆菌分离物：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。这些研究将对我们理解幽门螺杆菌的免疫发病机制和胃癌的发生产生重大影响，并将为风险评估和治疗提供新的方法。

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.