Plasma Gelsolin and Host Defense After Lung Injury

描述（由申请人提供）：细菌性肺炎是急性肺损伤的常见并发症。典型的例子是继发性肺炎，通常发生在流感之后，这是季节性流感和流行性流感死亡的主要原因。损伤后易感性的一个关键原因是众所周知的：肺的“第一反应”细胞，肺泡巨噬细胞（AM），在流感或其他肺损伤后大大降低了抗菌功能。缺少的是新的，基于机制的治疗方法，以增强AM宿主在这种高风险时期的防御功能。我们的建议集中在正常的人类蛋白质，血浆凝溶胶蛋白（pGSN），其潜力是一种新型的免疫调节剂，可以降低肺炎的风险。常见病原体链球菌的初步研究。pGSN在体外快速提高巨噬细胞细菌杀伤，并且雾化的pGSN在体内提高细菌清除。该机制的其他线索涉及肺巨噬细胞一氧化氮合酶3（N 0 S3）的活化，当加入N 0 S3-/-巨噬细胞时，pGSN的有益作用丧失突出显示。该项目的两个主要目标是：1）评估pGSN在流感引起的典型肺损伤后改善肺宿主防御的潜力; 2）表征pGSN增强AM抗菌功能的机制。目的1探讨血浆凝溶胶蛋白在机体防御肺炎中的作用。这些研究将使用一种非致死性流感模型，该模型在缓解期对继发性肺炎球菌肺炎的易感性增强。我们将描述急性肺损伤后肺中pGSN水平和功能的变化，并将测试pGSN预防性治疗对细菌清除和继发性肺炎攻毒存活率的影响。目的2：研究血浆凝溶胶蛋白增强AM抗菌功能的机制。将使用pGSN增强细菌杀伤的体外测定、NOS抑制剂和来自NOS 3-/-小鼠的AM以及通过Akt磷酸化级联激活NOS 3的信号传导途径的评价来测试AM NOS 3的假定作用。我们还将研究血浆凝溶胶蛋白是否通过递送存在于肺液中的信号磷脂来发挥功能，并且已知通过结合和竞争测定以及使用S1 P受体敲除小鼠和细胞来激活NOS 3，例如1-磷酸鞘氨醇至其受体（S1 PR 1）。该提案解决了流感和其他急性肺损伤后发病率和死亡率的主要原因，并将评估一种具有巨大潜力的内源性免疫调节剂转化为治疗益处。

DESCRIPTION (provided by applicant): Bacterial pneumonia is a frequent complication of acute lung injury. The prototypical example is the secondary pneumonia that often follows influenza, and which is a major cause of deaths from both seasonal and epidemic flu. A critical cause of post-injury susceptibility is well known: the lung's "1st responder" cell, the alveolar macrophage (AM), has profoundly diminished antibacterial function after influenza or other lung injuries. What is missing are novel, mechanism-based therapeutics to enhance AM host defense function during such high-risk periods. Our proposal focuses on the normal human protein, plasma gelsolin (pGSN), and its potential to be a novel immunomodulator that can reduce risk of pneumonia. Pilot studies with the common pathogen Strep. pneumoniae indicate that pGSN rapidly improves macrophage bacterial killing in vitro, and that aerosolized pGSN improves bacterial clearance in vivo. Additional clues to the mechanism implicate activation of lung macrophage nitric oxide synthase 3 (NOS3), highlighted by loss of beneficial effects of pGSN when added to NOS3 -/- macrophages. The two main goals of this project are: 1) to assess the potential of pGSN to improve lung host defense after the prototypical lung injury caused by influenza; 2) to characterize the mechanisms by which pGSN enhances AM antibacterial function. Aim 1 will determine the role of plasma gelsolin in host defense against pneumonia in vivo. These studies will use a model of non-lethal influenza with enhanced susceptibility to secondary pneumococcal pneumonia during the resolution phase. We will characterize changes in pGSN levels and function in the lung that follow acute lung injury, and we will test the effect of prophylactic treatment with pGSN on bacterial clearance and survival of the secondary pneumonia challenge. Aim 2 will characterize mechanisms by which plasma gelsolin enhances AM anti-bacterial function. The postulated role for AM NOS3 will be tested using in vitro assays of pGSN enhancement of bacterial killing, NOS inhibitors and AMs from NOS3 -/- mice, and evaluation of signaling pathways for activation of NOS3 through Akt phosphorylation cascades. We will also investigate whether plasma gelsolin functions by delivering signaling phospholipids present in lung fluids and known to activate NOS3, e.g. sphingosine-1-phosphate to its receptor (S1PR1), through binding and competition assays, and use of S1P receptor knockout mice and cells. The proposal addresses a major cause of morbidity and mortality after influenza and other acute lung injuries, and will evaluate an endogenous immunomodulator with great potential for translation into therapeutic benefit.