Plasma Gelsolin and Host Defense After Lung Injury

描述（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，它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抗菌功能显著减弱。在这样的高风险时期，缺乏新的、基于机制的治疗方法来增强AM宿主的防御功能。我们的建议侧重于正常人类蛋白，血浆凝胶（pGSN），及其作为一种新型免疫调节剂的潜力，可以降低肺炎的风险。对常见病原体链球菌进行初步研究。研究表明，pGSN在体外可迅速提高巨噬细胞的细菌杀伤能力，雾化pGSN可提高体内细菌清除能力。机制的其他线索涉及肺巨噬细胞一氧化氮合酶3 （NOS3）的激活，当添加到NOS3 -/-巨噬细胞中时，pGSN的有益作用丧失。本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)研究pGSN增强AM抗菌功能的机制。目的1将确定血浆凝胶在宿主体内防御肺炎中的作用。这些研究将使用非致死性流感模型，在解决阶段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，我们将测试pGSN预防性治疗对继发性肺炎挑战的细菌清除和生存的影响。目的2将描述血浆凝胶增强AM抗菌功能的机制。AM NOS3的假设作用将通过体外实验进行验证，包括pGSN增强细菌杀灭、NOS抑制剂和来自NOS3 -/-小鼠的AMs，以及评估通过Akt磷酸化级联激活NOS3的信号通路。我们还将通过结合和竞争试验，以及使用S1P受体敲除小鼠和细胞，研究血浆凝胶蛋白是否通过将存在于肺液中的已知可激活NOS3的信号磷脂（例如鞘氨醇-1-磷酸）传递到其受体（S1PR1）来发挥作用。该提案解决了流感和其他急性肺损伤后发病和死亡的主要原因，并将评估一种内源性免疫调节剂，该调节剂具有转化为治疗益处的巨大潜力。

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.