5-Lipoxygenase Activating Protein (FLAP) Dependent Leukotriene Biosynthesis Inhibition (MK591) Attenuates Lipid A Endotoxin-Induced Inflammation

The Lipid A moiety of endotoxin potently activates TLR-4 dependent host innate immune responses. We demonstrate that Lipid-A mediated leukotriene biosynthesis regulates pathogen-associated molecular patterns (PAMP)-dependent macrophage activation. Stimulation of murine macrophages (RAW264.7) with E. coli 0111:B4 endotoxin (LPS) or Kdo2-lipid A (Lipid A) induced inflammation and Lipid A was sufficient to induce TLR-4 mediated macrophage inflammation and rapid ERK activation. The contribution of leukotriene biosynthesis was evaluated with a 5-lipoxygenase activating protein (FLAP) inhibitor, MK591. MK591 pre-treatment not only enhanced but also sustained ERK activation for up to 4 hours after LPS and Lipid A stimulation while inhibiting cell proliferation and enhancing cellular apoptosis. Leukotriene biosynthesis inhibition attenuated inflammation induced by either whole LPS or the Lipid A fraction. These responses were regulated by inhibition of the key biosynthesis enzymes for the proinflammatory eicosanoids, 5-lipoxygenase (5-LO), and cyclooxygenase-2 (COX-2) quantified by immunoblotting. Inhibition of leukotriene biosynthesis differentially regulated TLR-2 and TLR-4 cell surface expression assessed by flow cytometry, suggesting a close mechanistic association between TLR expression and 5-LO associated eicosanoid activity in activated macrophages. Furthermore, MK591 pre-treatment enhanced ERK activation and inhibited cell proliferation after LPS or Lipid A stimulation. These effects were regulated in part by increased apoptosis and modulation of cell surface TLR expression. Together, these data clarify the mechanistic association between 5-lipoxygenase activating protein-mediated leukotriene biosynthesis and 5-LO dependent eicosanoid metabolites in mediating the TLR-dependent inflammatory response after endotoxin exposure typical of bacterial sepsis.内毒素的脂质A（Lipid A）结构域可强效激活依赖Toll样受体4（TLR-4）的宿主固有免疫应答。本研究证实，脂质A介导的白三烯生物合成可调控病原体相关分子模式（PAMP）依赖的巨噬细胞活化。以大肠杆菌0111:B4内毒素（脂多糖，LPS）或Kdo2-脂质A（脂质A）刺激小鼠巨噬细胞RAW264.7，可诱导炎症反应；且脂质A足以介导TLR-4依赖的巨噬细胞炎症反应，并快速激活细胞外调节蛋白激酶（ERK）。本研究通过5-脂氧合酶激活蛋白（FLAP）抑制剂MK591评估白三烯生物合成的作用，结果显示：MK591预处理不仅可增强LPS与脂质A刺激后ERK的激活水平，还将其持续时间延长至4小时；同时该处理可抑制细胞增殖，并促进细胞凋亡。抑制白三烯生物合成可减弱完整LPS或脂质A组分诱导的炎症反应，上述效应通过抑制促炎性类二十烷酸的关键合成酶——5-脂氧合酶（5-LO）与环氧合酶2（COX-2）得以调控，该结论通过免疫印迹法量化验证。通过流式细胞术检测发现，抑制白三烯生物合成可差异性调控TLR-2与TLR-4的细胞表面表达水平，这提示活化巨噬细胞中TLR表达与5-LO相关的类二十烷酸活性之间存在紧密的机制关联。此外，MK591预处理可增强LPS或脂质A刺激后的ERK激活，并抑制细胞增殖，该效应部分通过细胞凋亡增加与细胞表面TLR表达的调控实现。综上，本研究数据阐明了5-脂氧合酶激活蛋白介导的白三烯生物合成与5-LO依赖的类二十烷酸代谢物之间的机制关联，该关联介导了细菌脓毒症典型内毒素暴露后TLR依赖的炎症反应。