A novel anti-inflammatory role for the neutrophil NADPH oxidase

描述(由申请人提供)：局部和全身性炎症事件在宿主体内引发免疫激活，以提供基于所感知的危险的必要的促炎反应，随后主动消退并回到基线。这一过程的一个常见的临床例子是全身性炎症反应综合征(SIRS)，它可以在一系列侮辱之后被激活，这些侮辱可能包括感染、休克、过敏反应或创伤。免疫反应的促炎阶段与恢复免疫动态平衡的代偿性抗炎反应重叠。循环中的中性粒细胞(PMN)在调节全身炎症的后果中起关键作用，部分是通过产生活性氧(ROS)来实现的。PMN通过激活NADPH氧化酶(NOx)产生ROS，其中NOX2(Gp91Phox)是该细胞类型中的催化亚基。尽管中性粒细胞来源的ROS在微生物防御中的要求是明确的，但人们清楚地认识到，PMN的激活可以导致宿主组织的损伤。这种经过充分研究的PMN的致炎活性与最近支持NOX2抗炎作用的证据相平衡。目前的建议集中在确定ROS信号在静息PMN中的细胞作用和分子靶点，以及NOX2在抗炎中的抗炎作用。这些目标是基于两组强有力的初步数据：1)在缺乏NADPH氧化酶功能的情况下，显示静息中中性粒细胞中特定的促炎表型变化，以及2)使用无菌全身炎症的小鼠模型，NOX2缺乏的小鼠的炎症增强和持续性炎症以及死亡率增加。这一建议的总体假设是，PMN中的NOX2在调节宿主炎症状态方面具有重要的抗炎作用。此外，我们假设NOX2衍生的ROS既是维持细胞静息状态所必需的，也是炎症性损伤后恢复动态平衡所必需的。这些假说将通过以下目的来验证：1)NOX2在未经刺激的PMN中是活跃的，是维持静息细胞表型所必需的。2)NOX2对p38丝裂原活化蛋白激酶(MAPK)途径介导的PMN具有抗炎作用。实现这些目标的方法将包括对未受刺激的人PMN的亚细胞囊泡/内体进行复杂的分析，以寻找NOX2组装和激活的证据。此外，还将重点探索抗炎ROS信号的细胞靶点，以及该信号的下游后果。原代人体中性粒细胞将用于体外信号通路的分析，并将使用共聚焦显微镜来定位ROS信号。更好地了解宿主炎症的细胞生物学将与许多疾病过程相关。

DESCRIPTION (provided by applicant): Local and systemic inflammatory events elicit immune activation in the host to provide the necessary pro- inflammatory response based on the danger sensed, followed by active resolution and return to baseline. One common clinical example of this process is the Systemic Inflammatory Response Syndrome (SIRS), which can be activated following a range of insults that may include infection, shock, allergic reaction, or trauma. This pro- inflammatory phase of the immune response is overlapped by a compensatory anti-inflammatory response that restores immune homeostasis. Circulating neutrophils (PMN) are critically involved in mediating the consequences of systemic inflammation, in part by the generation of reactive oxygen species (ROS). PMN produce ROS via activation of the NADPH oxidase (Nox), with Nox2 (gp91phox) being the catalytic subunit in this cell type. Despite the unequivocal requirement for neutrophil-derived ROS in microbial defense, it is clearly recognized that PMN activation can lead to host tissue damage. This well-studied proinflammatory activation of PMN is counterbalanced against recent evidence supporting an anti-inflammatory role for Nox2. The current proposal focuses on defining cellular effects and molecular targets of ROS signaling in resting PMNs, and investigation of an anti-inflammatory role of Nox2 in termination of inflammation. These goals are predicated on two subsets of strong preliminary data: 1) demonstration of specific pro-inflammatory phenotypic alterations in resting PMNs in the absence of NADPH oxidase function, and 2) enhanced and persistent inflammation and increased mortality in Nox2-deficient mice using a murine model of sterile generalized inflammation. The overall hypothesis of this proposal is that Nox2 in PMN has an essential anti-inflammatory role in the regulation of the host inflammatory state. Furthermore, we hypothesize that Nox2-derived ROS are required both to maintain resting cellular quiescence and to return to homeostasis following an inflammatory insult. These hypotheses will be tested with the following aims: 1) Nox2 is active in unstimulated PMN and necessary to maintain the resting cell phenotype. 2) Nox2 has anti-inflammatory effects in PMN mediated via the p38 mitogen-activated protein kinase (MAPK) pathway. The approach to these aims will include sophisticated analysis of subcellular vesicles/endosomes of unstimulated human PMN for evidence of assembly and activation of Nox2. In addition, focused exploration of cellular targets of anti-inflammatory ROS signaling, and downstream consequences of this signaling will be undertaken. Primary human PMNs will be used for in vitro analyses of signaling pathways and confocal microscopy will be employed to localize ROS signals. A better understanding of the cell biology of host inflammation will be relevant to numerous disease processes.