Homeostatic Regulation of Neutrophil ROS Production and Lung Injury

吞噬细胞产生大量氧自由基的能力是宿主对抗微生物感染的重要防御机制。然而，同样的氧化剂生成机制也会导致组织损伤，例如由吞噬细胞不受控制的激活引起的急性肺损伤（ALI）。虽然对吞噬细胞NADPH氧化酶的研究已经产生了大量关于其激活机制的信息，但对限制吞噬细胞氧化剂产生的负调控机制知之甚少。项目2的目标是明确这种负调控机制，并探索抑制肺部炎症反应的潜力。研究提出了两个实质性的具体目标，重点关注我们最近发现的MAP激酶磷酸酶5 （MKPS）的调节机制，该机制抑制多态核中性粒细胞的氧化剂产生，从而减少lps诱导的炎症性血管损伤。在Aim 1中，我们将确定MKP5如何调节对ALI至关重要的PMN氧化剂的产生。MKP5深远的负调控作用表明，MKP5的靶点p38 MAPK对PMN NADPH氧化酶的激活至关重要。为了验证p38 MAPK对于多种蛋白激酶对p47[phox]的顺序磷酸化至关重要，从而导致p47[phox]构象改变和NADPH氧化酶的完全激活，提出了研究。我们还将验证p38 MAPK信号通过MK2进一步扩增的假设，MK2是一种下游效应物和蛋白激酶，有助于p47[phox]磷酸化。利用MK2 -/-和MKP5 -/-小鼠研究MK2在ALI中的体内功能及其受MKP5的调控。在Aim 2中，我们将定义MKP5通过调节PMN和内皮细胞活化在预防ALI中的核心作用。通过小鼠肺部炎症模型，我们将验证MKP5是一种必要的调节因子，可以降低炎性细胞因子和趋化因子的表达，限制PMN的浸润，抑制PMN氧化剂的产生和氧化介导的肺损伤。我们还将质疑内皮细胞MKP5是否在限制ICAM-1表达中起重要作用，从而阻止PMN粘附以及氧化介导的损伤。总之，这些研究旨在描述LPS启动PMN氧化剂产生的潜在机制，并探索其控制ALI治疗潜力的新的负调控机制。

The ability of phagocytes to produce large amounts of oxygen radicals constitutes an important host-defense mechanism against microbial infection. However, the same oxidant-generating machinery also contributes to tissue injury such as in Acute Lung Injury (ALI) resulting from uncontrolled activation of phagocytes. While research on phagocyte NADPH oxidase has produced a great deal of information regarding its activation mechanisms, little is known about the negative regulatory mechanisms that limit phagocyte oxidant production. The objective of Project 2 is to define such negative regulatory mechanisms and explore the potential for dampening lung inflammatory response. Studies are proposed in two substantial specific aims that focus on our recently identified regulatory mechanism by MAP kinase phosphatase 5 (MKPS), which restrains oxidant production by polymorphnuclear neutrophils, thereby decreasing LPS-induced inflammatory vascular injury. In Aim 1, we will determine how MKP5 regulates PMN oxidant production, which is critical to ALI. The profound negative regulatory effect by MKP5 suggests that p38 MAPK, the target of MKP5, is critical to PMN NADPH oxidase activation. Studies are proposed to test the hypothesis that p38 MAPK is essential for sequential phosphorylation of p47[phox] by a multitude of protein kinases, leading to p47[phox] conformational change and full activation of NADPH oxidase. We will also test the hypothesis that p38 MAPK signaling is further amplified through MK2, a downstream effector and protein kinase that contributes to p47[phox] phosphorylation. The in vivo function of MK2 in ALI and its regulation by MKP5 will be interrogated with the use of MK2 -/- and MKP5 -/- mice. In Aim 2, we will define the central role of MKP5 in preventing ALI through modulation of PMN and endothelial activation. Using mouse models of lung inflammation, we will test the hypothesis that MKP5 is an essential regulator that reduces proinfiammatory cytokine and chemokine expression, limits PMN infiltration, and dampens PMN oxidant production and oxidant-mediated lung injury. We will also query the possibility that endothelial MKP5 is important in limiting ICAM-1 expression and thus prevents PMN adhesion as well as oxidant-mediated injury. Together, these studies aims to delineate the underlying mechanism for LPS priming of PMN oxidant production, and explore a novel negative regulatory mechanism for its therapeutic potential in controlling ALI.