Lung innate immunity against bacterial infection

描述(申请人提供)：根据美国疾病控制与预防中心的数据，由包括铜绿假单胞菌(PA)在内的各种病原体引起的医院感染在美国每年需要大约450亿美元的医疗费用。尽管有强烈的兴趣，肺泡巨噬细胞(AM)在PA感染中的作用仍然不清楚。巨自噬(以下简称自噬)是一种保守的动态平衡机制，通过这种机制，细胞成分被隔离到自噬小体中，通过泛素化进行降解。自噬机制可能参与先天免疫，提高细菌的清除能力。然而，目前尚不清楚自噬是否会影响PA感染。我们的初步数据显示，PA感染可以诱导自噬，并随后增加细菌的降解。我们还发现，多效性的Src激酶Lyn与Toll样受体(Toll Like Receptor，TLRs)相互作用，促进自噬活性。此外，我们还发现ATG-7在吞噬小体-溶酶体融合中起关键作用。重要的是，饥饿增加了AM对PA的吞噬作用，自噬抑制剂3-甲基腺嘌呤(3MA)阻断了自噬，降低了吞噬作用和随后的细菌清除。因此，我们假设PA诱导的自噬通过促进吞噬和细菌降解来增强宿主防御。我们的长期目标是发现调节宿主抵抗PA入侵所需的关键因素，以开发新的治疗策略。这项建议的目的是研究Lyn/ATG-7调节KO小鼠吞噬作用的生理意义，并利用生化和整体动物成像方法描述潜在的分子机制。目的1：明确ATG-7在假单胞菌肺部感染中的作用。我们的工作假设是，ATG-7是预防PA感染所必需的。目的2：利用小鼠模型和原代小鼠AM评价自噬对肺部细菌感染吞噬功能的影响根据我们的发现，PA感染可以诱导AM的自噬并减少细菌负荷，我们的工作假设是PA诱导的自噬增强了巨噬细胞的吞噬作用。目的3：确定TLR-2、Lyn和ATG-7在宿主抗菌反应中自噬小体形成和吞噬小体融合中的作用。因此，我们的工作假设是TLR-2、LYN和ATG-7是将细菌运送到溶酶体进行降解的关键元件。这些拟议的研究将揭示AM根除细菌的新机制，并提出新的治疗靶点。

DESCRIPTION (provided by applicant): Hospital-acquired infections derived from various pathogens including P. aeruginosa (Pa) require approximately $45 billion in annual medical expenses in the U.S., according to the CDC. Despite intense interest, the function of alveolar macrophages (AM) in Pa infection remains elusive. Macroautophagy (hereafter autophagy) is a conserved homeostasis mechanism by which cellular components are sequestered to autophagosomes for degradation through ubiquitination. The autophagosome machinery may contribute to the innate immunity to enhance bacterial clearance. However, it is unknown whether autophagy impacts Pa infection. Our preliminary data revealed that Pa infection can induce autophagy, and subsequently increasing bacterial degradation. We also showed that the pleiotropic Src kinase, Lyn, interacts with toll like receptors (TLRs) to boost autophagic activity In addition, we found that Atg-7 is critical for phagosome-lysosome fusion. Importantly, starvation increases AM phagocytosis of Pa and blocking autophagy by autophagy inhibitor 3-methyladenine (3MA) decreased phagocytosis and subsequent bacterial clearance. Thus, we hypothesize that Pa-induced autophagy augments host defense by facilitating phagocytosis and bacterial degradation. Our long-term goal is to discover the key factors required for regulating host defense against Pa invasion in order to develop novel therapeutic strategies. The objective of this proposal is to examine the physiological significance of Lyn/Atg-7 modulated phagocytosis with KO mice and to delineate the underlying molecular mechanisms using biochemical and whole animal imaging approaches. Aim 1: Define the functional role of Atg-7 in Pseudomonas pulmonary infection. Our working hypothesis is that atg-7 is required for protection against Pa infection. Aim 2: Evaluate the impact of autophagy on phagocytosis in pulmonary bacterial infection utilizing a mouse model and primary mouse AM. Based on our finding that Pa infection can induce autophagy and reducing bacterial burdens in AM, our working hypothesis is that Pa-induced autophagy augments macrophage phagocytosis. Aim 3: Determine the role of TLR-2, Lyn, and Atg-7 in autophagosome formation and phagolysosome fusion in host response against bacteria. Thus, our working hypothesis is that TLR-2, Lyn, and Atg-7 are key elements for delivery of bacteria to lysosomes for degradation. These proposed studies will reveal a novel mechanism for AM to eradicate bacteria and suggest novel therapeutic targets.