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抵抗微生物感染的重要宿主防御机制。然而，同样的氧化剂生成机制也会导致组织损伤，如由于吞噬细胞失控激活而导致的急性肺损伤(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的目标是确定这种负性调节机制，并探索抑制肺炎性反应的潜在作用。我们最近发现了MAP-KP5(MKPS)的调控机制，MKPS可以抑制多形核中性粒细胞产生氧化剂，从而减轻内毒素诱导的炎性血管损伤。在目标1中，我们将确定MKP5如何调节PMN氧化剂的产生，这对ALI至关重要。MKP5的深刻负性调控作用表明，作为MKP5靶点的p38MAPK对PMN NADPH氧化酶的激活起着关键作用。有研究表明，p38MAPK对于一系列蛋白激酶对p47[Phox]的顺序磷酸化是必不可少的，从而导致p47[Phox]构象变化和NADPH氧化酶的完全激活。我们还将测试p38 MAPK信号通过MK2进一步放大的假设，MK2是一种下游效应器和蛋白激酶，有助于p47[Phox]磷酸化。用MK2-/-和MKP5-/-小鼠研究MK2在ALI中的体内功能以及MKP5对MKP5的调节。在目标2中，我们将确定MKP5通过调节PMN和内皮激活在预防ALI中的核心作用。利用小鼠肺部炎症模型，我们将验证MKP5是一种重要的调节因子的假设，它减少炎症细胞因子和趋化因子的表达，限制PMN的侵袭，抑制PMN氧化剂的产生和氧化剂介导的肺损伤。我们还将质疑内皮细胞MKP5在限制ICAM-1表达从而防止PMN黏附和氧化剂介导的损伤中发挥重要作用的可能性。综上所述，这些研究旨在阐明内毒素启动PMN氧化剂产生的潜在机制，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-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.