Regulation and Function of Mkp-1 During Sepsis.

项目摘要 败血症是常见的，具有高死亡率，并且在美国是非常资源密集型的医疗保健问题。 脓毒症与细胞因子风暴有关，细胞因子风暴是一种加剧的免疫反应，导致过度的免疫反应。 血管舒张和严重的血管渗漏导致循环休克和多器官衰竭。如败血症 如果持续存在，患者往往会出现免疫麻痹的特征。大多数与休克有关的死亡发生在 这种继发性免疫功能低下状态，可能是由于未能清除初始感染和/或继发性免疫缺陷所致。 抗生素治疗后仍发生院内感染。我们发现，缺乏丝裂原激活的 蛋白激酶（MAPK）磷酸酶（Mkp）-1，p38和JNK MAPK的可诱导负调节因子， 在细菌感染后表现出恶化的炎症。这些小鼠经历了一个实质性的 增强的细胞因子风暴，并显示与血管渗漏相关的死亡率显著增加， 循环衰竭考虑到炎症反应的增强，矛盾的是，Mkp-1-/-小鼠表现出 细菌清除缺陷。这张图片显示炎症反应增强，但无法清除 细菌，模仿什么是经常看到的脓毒症患者，继续发展休克和多器官衰竭。 然而，导致Mkp-1-/-小鼠杀菌活性受损的机制尚不清楚。 此外，使用骨髓重建的小鼠，我们发现非骨髓来源的细胞中的Mkp-1， (such作为内皮细胞）在维持血管屏障功能中起关键作用。我们的核心假设 充分增强Mkp-1表达可以抑制炎症，增强抗细菌能力， 免疫防御，并改善屏障功能，最终导致患者更好的结果， 败血症尽管Mkp-1在先天免疫应答中至关重要，但其调节仍然很差 明白在初步研究中，我们发现Mkp-1的C-末端的两个丝氨酸残基的突变， 其模拟ERK的磷酸化，使Mkp-1的稳定性增加20倍而不影响Mkp-1的泛素化。 基于计算机的分析表明，Mkp-1的C-末端，邻近这两个丝氨酸残基， 含有一个内在无序的结构域，这两个丝氨酸残基的磷酸化降低， 这个领域的混乱。这表明ERK途径通过增加Mkp-1的表达顺序来稳定Mkp-1。 一个内在的无序域。因此，我们建议阐明Mkp-1在吞噬细胞中的功能， 内皮细胞，并确定ERK稳定Mkp-1蛋白的机制。我们还将 确定增强ERK活性是否可以是维持Mkp-1表达的有效策略，从而 阻止p38活化的第二阶段，最终抑制细胞因子的产生。的目的 应用是了解Mkp-1在吞噬细胞和内皮细胞中的功能和调节， 细菌性败血症，并确定选择性ERK激活是否可以是减轻炎症的有效手段。

PROJECT SUMMARY Sepsis is common, has a high mortality rate, and is a very resource-intensive healthcare problem in the U.S.A. Sepsis is associated with cytokine storm, an exacerbated immune response, resulting in excessive vasodilation and severe vascular leakage leading to circulatory shock and multiple organ failure. As sepsis persists, patients often develop features of immunoparalysis. The majority of shock-related deaths occur during this secondary hypo-immune state, likely due to the failure to clear the initial infection and/or secondary nosocomial infection despite antibiotic treatment. We have found that mice deficient in mitogen-activated protein kinase (MAPK) phosphatase (Mkp)-1, an inducible negative regulator of the p38 and JNK MAPKs, exhibit exacerbated inflammation following bacterial infections. These mice experienced a substantially enhanced cytokine storm and showed markedly increased mortality associated with vascular leak and circulatory collapse. Given the enhanced inflammatory response, paradoxically, Mkp-1-/- mice exhibited defective bacterial clearance. This picture of an augmented inflammatory response, and yet an inability to clear bacteria, mimics what is often seen in septic patients that go on to develop shock and multi-organ failure. However, the mechanisms leading to the impaired bactericidal activity in Mkp-1-/- mice are not known. Additionally, using bone marrow reconstituted mice, we found that Mkp-1 in cells of non-bone marrow origin (such as endothelial cells) plays a pivotal role in maintaining vascular barrier function. Our central hypothesis is that adequately enhancing Mkp-1 expression may dampen inflammation, enhance anti-bacterial immune defense, and improve barrier function, ultimately leading to better outcomes for patients with sepsis. Although Mkp-1 is critically important in the innate immune response, its regulation remains poorly understood. In preliminary studies we found that mutation of two serine residues at the C-terminus of Mkp-1, which mimics phosphorylation by ERK, increased Mkp-1 stability 20-fold without affecting Mkp-1 ubiquitination. Computer-based analysis suggests that the C-terminus of Mkp-1, adjacent to these two serine residues, contains an intrinsically disordered domain and that phosphorylation of these two serine residues decreases the disorder of this domain. This suggests that the ERK pathway stabilizes Mkp-1 via increasing the order of an intrinsic disorderd domain. Therefore, we propose to elucidate the function of Mkp-1 in phagocytes and endothelial cells and to define the mechanism by which ERK stabilizes the Mkp-1 protein. We will also determine if enhancing ERK activity can be an effective strategy for sustaining Mkp-1 expression, thereby preventing the second phase of p38 activation, ultimately inhibiting cytokine production. The objective of this application is to understand the function and regulation of Mkp-1 in phagocytes and endothelial cells during bacterial sepsis and determine if selective ERK activation can be an effective means to attenuate inflammation.