Homeostatic Regulation of Neutrophil ROS Production and Lung Injury

吞噬细胞产生大量氧自由基的能力构成了宿主抵抗微生物感染的重要防御机制。然而，相同的氧化剂生成机制也有助于组织损伤，例如由吞噬细胞的不受控制的活化引起的急性肺损伤（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的目的是确定这种负性调节机制，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，集中在我们最近确定的MAP激酶磷酸酶5（MKPS），它抑制多形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的调节机制。在目的1中，我们将确定MKP 5如何调节PMN氧化剂的产生，这是ALI的关键。MKP5的显著负调节作用表明，MKP5的靶点p38 MAPK对PMN NADPH氧化酶的激活至关重要。研究提出了测试的假设，p38 MAPK是必不可少的顺序磷酸化的p47 [phox]由众多的蛋白激酶，导致p47 [phox]的构象变化和NADPH氧化酶的完全激活。我们还将检验p38 MAPK信号通过MK2进一步放大的假设，MK2是一种下游效应子和蛋白激酶，有助于p47 [phox]磷酸化。将使用MK2-/-和MKP5-/-小鼠研究MK2在ALI中的体内功能及其通过MKP5的调节。在目标2中，我们将确定MKP 5通过调节PMN和内皮激活在预防ALI中的中心作用。使用小鼠肺炎症模型，我们将检验MKP 5是减少促炎性细胞因子和趋化因子表达、限制PMN浸润、抑制PMN氧化剂产生和氧化剂介导的肺损伤的重要调节剂的假设。我们还将质疑内皮MKP 5在限制ICAM-1表达中的重要性，从而防止PMN粘附以及氧化剂介导的损伤。这些研究旨在阐明LPS启动PMN氧化产物产生的潜在机制，并探索其在控制ALI中的治疗潜力的新的负性调节机制。

The ability of phagocytes to produce large amounts of oxygen radicals constitutes an important host-defense mechanism against microbial infection. However, the same oxidant-generafing 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 potenfial for dampening lung inflammatory response. Studies are proposed in two substanfial 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 producfion, which is crifical 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 infiammation, we will test the hypothesis that MKP5 is an essential regulator that reduces proinfiammatory cytokine and chemokine expression, limits PMN infiltrafion, and dampens PMN oxidant production and oxidant-mediated lung injury. We will also query the possibility that endothelial MKP5 is important in limifing 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 producfion, and explore a novel negative regulatory mechanism for its therapeutic potential in controlling ALI.