Cellular Selenium Status and PGJ2 Metabolism

描述（由申请人提供）：巨噬细胞在免疫中起核心作用，其中炎症从起始到消退的转变对于稳健和有益的免疫应答至关重要。然而，这种转换的不平衡最终导致在许多疾病状态中观察到的长期炎症。这些细胞的氧化还原状态的不平衡可能是延迟和/或不成功解决的潜在关键决定因素之一。含硒蛋白是细胞抗氧化机制中的一员，它能有效地调节氧化还原的变化，从而为免疫功能带来营养成分。硒缺乏是吸烟者和患有AIDS和乳腺癌的个体的特征，其中巨噬细胞活化伴随着促炎细胞因子和前列腺素（PG）E2（环氧合酶（考克斯）-2途径的氧化花生四烯酸产物）的增加。促炎基因表达受氧化还原敏感性转录因子NF-：B和过氧化物酶体增殖物激活受体（PPAR）-3的差异调节的影响。有趣的是，硒补充巨噬细胞导致优先生产的15 d-PGJ 2共价修饰的两个转录因子。待检验的具体假设是，Se补充通过考克斯途径选择性地调节与花生四烯酸代谢相关的几种酶，从而影响抗炎15 d-PGJ 2的优先形成，其通过调节NF-：B和PPAR-3减弱促炎基因的表达。15 d-PGJ 2的合成发生在细胞内膜-细胞质界面处，容易失活，这可能会削弱其生物活性。待检验的另一个补充假设是，微粒体谷胱甘肽S-转移酶（MGSTA 1）的表达在硒缺乏期间上调，这是15 d-PGJ 2代谢失活的部分原因。将在维持硒缺乏的C57 BL/6小鼠中检验假设。（0.01 ppm），硒充足（0.08 ppm），和硒补充（0.4 ppm）饲料以及从这些小鼠中分离的巨噬细胞中的细胞毒性，具体目的如下：1）检测细胞硒状态对通过考克斯-1和考克斯-2依赖性途径调节PG产生的影响，2）研究硒缺乏时微粒体PGE 2合酶和造血PGD 2合酶在炎症通路中的作用和调节; 3）阐明MGSTA 1在硒缺乏时15 d-PGJ 2代谢中的作用。我们的研究提供了对细胞硒状态和抗炎轴之间以前未被认识的联系的深入了解，通过抑制促炎性PGE 2产生增加的15 d-PGJ 2。拟议的研究将开辟新的研究途径，其中抵消NF-：B依赖性促炎基因表达的饮食干预可用于患有HIV-AIDS、癌症和动脉粥样硬化的个体的长期益处和健康。 项目叙述：专职吞噬细胞（如巨噬细胞）是细胞因子、趋化因子、酶和脂质介质的良好来源，因此不仅在炎症的发作中，而且在消退过程中发挥关键作用。然而，目前还不清楚是什么重新编程巨噬细胞从促炎基因表达网络切换到抗炎程序。基于初步的数据，我们认为，细胞的抗氧化状态可能在这种重编程事件中发挥重要作用。本项目的主要目的是研究硒（一种微量营养素和抗氧化剂）在原代巨噬细胞中抗炎作用的潜在分子机制。拟议的研究将描绘花生四烯酸代谢中的硒依赖性开关，从促炎性前列腺素（PG）E2到抗炎性15-脱氧-12，14-PGJ 2（15 d-PGJ 2），影响炎症的消退过程。本文提出的研究还将阐明硒的抗炎作用机制，特别是通过涉及15 d-PGJ 2的独特机制抑制IKK 2/NF-：B依赖性促炎基因表达失活。

DESCRIPTION (provided by applicant): Macrophages play a central role in immunity, where a switch from initiation to resolution of inflammation is crucial for a robust and beneficial immune response. However, an imbalance in this switch culminates in prolonged inflammation that is seen in many disease states. Imbalances in the redox status of these cells could be one of the underlying crucial determinants of delayed and/or unsuccessful resolution. Selenoproteins, which contain selenium (Se), are a member of the cellular antioxidant machinery that effectively regulate the redox changes, and thus, brings in a nutritional component to immune function. Selenium-deficiency is characteristic of cigarette smokers and individuals with AIDS and breast cancer, where macrophage activation is accompanied by increased pro-inflammatory cytokines and prostaglandin (PG) E2, an oxidized arachidonic acid product of the cyclooxygenase (COX)-2 pathway. The pro- inflammatory gene expression is effected by differential modulation of the redox- sensitive transcription factor, NF-:B, and peroxisome proliferator activated receptor (PPAR)-3. Interestingly, Se-supplementation of macrophages leads to the preferential production of 15d-PGJ2 that covalently modifies the two transcription factors. The specific hypothesis to be tested is that Se-supplementation selectively modulates several enzymes associated with the arachidonic acid metabolism via the COX pathway, thereby influencing the preferential formation of anti-inflammatory 15d- PGJ2, which attenuates the expression of pro-inflammatory genes through the modulation of the NF-:B and PPAR-3. Synthesis of 15d-PGJ2, which occurs at the intracellular membrane-cytoplasmic interface, is susceptible to inactivation that potentially blunts its biologic activity. An additional complementary hypothesis to be tested is that the expression of a microsomal glutathione S-transferase (MGSTA1) is up- regulated during Se-deficiency, which is partially responsible for the metabolic inactivation of 15d-PGJ2. The hypotheses will be tested in C57BL/6 mice maintained on Se-deficient (0.01 ppm), Se-adequate (0.08 ppm), and Se-supplemented (0.4 ppm) diets as well as in the macrophages isolated from these mice in the following Specific Aims: 1) to examine the effect of cellular Se status on the regulation of PG production via the COX-1 and COX-2- dependent pathways, 2) to examine the role and regulation of microsomal PGE2 synthase and hematopoietic PGD2 synthase involved in inflammatory pathways during Se-deficiency, and 3) to elucidate the role of MGSTA1 in 15d-PGJ2 metabolism during Se deficiency. Our studies provide insight into a previously unappreciated link between cellular Se status and anti-inflammatory axis, producing increased 15d-PGJ2, by suppressing pro-inflammatory PGE2. The proposed studies will open new avenues of research where dietary interventions to counteracting NF-:B-dependent pro- inflammatory gene expression may be applied for long-term benefits and well-being of individuals suffering from HIV-AIDS, cancer, and atherosclerosis. Project Narrative: Professional phagocytes, such as macrophages, are good sources of cytokines, chemokines, enzymes, and lipid mediators and, thus, play a pivotal role not only in the onset of inflammation, but also in the resolution process. However, it is unclear as to what reprograms the macrophage to switch from pro-inflammatory gene expression networks to anti-inflammatory programs. Based on the preliminary data, we believe that cellular antioxidant status may play an important role in such reprogramming events. The primary objective of this project is to investigate the underlying molecular mechanism of the anti-inflammatory effects of selenium, a micronutrient and antioxidant, in primary macrophages. The proposed studies will delineate a selenium-dependent switch in arachidonic acid metabolism from pro-inflammatory prostaglandin (PG) E2 to an anti-inflammatory 15-deoxy-12,14-PGJ2 (15d-PGJ2) that effects the resolution process of inflammation. Studies proposed here will also unravel the anti-inflammatory mechanism of action of selenium, particularly on the suppression of IKK2/NF-:B- dependent inactivation of pro-inflammatory gene expression via a unique mechanism involving 15d-PGJ2.