Alveolar macrophage protein interactions in response to SP-A and infection

 描述(申请人提供)：肺炎是一种主要的公共卫生负担。肺炎克雷伯菌是引起医院获得性肺炎和社区获得性肺炎的主要原因，尤其是在免疫功能低下或虚弱的患者中，耐药菌株已成为一个日益严重的公共卫生问题。肺泡巨噬细胞(AM)是肺部先天免疫的哨兵细胞，是对感染的第一反应细胞。肺表面活性蛋白A(SP-A)是一种天然免疫蛋白，由肺上皮分泌到AM所在的肺泡内。SP-A调节AM的许多功能，对AM蛋白质组有重要影响，包括与细胞骨架相关的蛋白质的变化。蛋白质，如细胞表面蛋白和我们的蛋白质组学研究确定的那些蛋白质，如何相互作用带来特定的AM功能尚不清楚。我们将使用Toponome成像系统(TIS)来解决这一知识鸿沟，这是第一个具有高功能分辨率(~40 nm)的显微系统，可以在单个细胞中定位多达100个蛋白质。TIS的研究表明，蛋白质是如何组织成簇或组合分子表型(CMP)的，这些组合分子表型是蛋白质信号网络的功能单位，以及CMP组的功能如何依赖于少数几种蛋白质，被称为“铅蛋白”。我们假设SP-A调节AM功能的+/-Kpn感染依赖于特定的CMPS，其完整性依赖于铅蛋白，并且在不同的条件下，CMPS和铅蛋白是不同的。提出了两个特定的目标：1)研究和比较野生型Kpn感染的AM和SP-A-/-小鼠AM中的CMPS，鉴定每个研究组特有的CMPS，并确定CMP组中的领先蛋白(S)；以及2)抑制选定的CMPS的领先蛋白(S)(来自目标1)，并研究这种抑制对细胞骨架变化、细胞大小和CMP完整性的影响。被探测的蛋白质将包括AM细胞表面分子(一些已知与SP-A相互作用)、SP-A调节的蛋白质(重点是细胞骨架蛋白质)和区分巨噬细胞亚群的蛋白质。抑制将使用封闭抗体，或基于铅分子的性质(S)和试剂可用性的其他方法。数据分析将确定特定的CMP基序 并在研究条件之间进行区分。对于抑制研究，将优先考虑在包括最多单个CMP的CMP基序中识别的先导蛋白。所获得的知识将有助于我们理解细胞表面和细胞内蛋白质如何在细胞骨架的调节中相互作用，并识别在这些相互作用中起作用的分子。这项研究的成功完成将有助于深入了解肺部对感染的反应，以及先天免疫蛋白SP-A在这一过程中的作用。重要的是，在这项“原则证明”研究中，识别与AM形态和细胞骨架变化相关的CMP和Lead蛋白，将为未来的研究提供基础 研究中，可以确定导致特定的、更复杂的结果的铅蛋白，如吞噬作用，并将其作为治疗干预的目标，以增强或抑制AM功能。

 DESCRIPTION (provided by applicant): Pneumonia is a major public health burden. Klebsiella pneumoniae (K. pn) is a major cause of nosocomial and community-acquired pneumonia, particularly in immunocompromised or debilitated patients, and antibiotic- resistant strains have become an increasing public health concern. The alveolar macrophage (AM), the sentinel cell of lung innate immunity, is the first responder to infection. Surfactant protein A (SP-A), an innate immune protein, is secreted by the lung epithelium into alveoli where the AM reside. SP-A regulates many AM functions and has a major effect on the AM proteome, including changes in proteins related to the cytoskeleton. How proteins, such as cell surface proteins and those identified by our proteomics studies, interact to bring about specific AM functions is not known. We will address this knowledge gap using the Toponome Imaging System (TIS), the first microscopic system with high functional resolution (~40nm) that can localize up to 100 proteins in a single cell. Studies with TIS have shown how proteins are organized into clusters or combinatorial molecular phenotypes (CMP), which are functional units of protein signaling networks, and how the function of groups of CMPs depend on a few proteins, termed "lead proteins". We hypothesize that SP-A-regulated AM functions +/- K. pn infection depend on specific CMPs, the integrity of which depend on lead proteins, and that both the CMPs and lead proteins differ under different conditions. Two Specific Aims are proposed: 1) To study and compare CMPs in AM from K. pn-infected wild type and SP-A -/- mice, identify CMPs unique to each study group, and identify the lead protein(s) in CMP groups, and 2) to inhibit the lead protein(s) of selected CMPs (from Aim 1) and investigate the impact of this inhibition on cytoskeletal changes, cell size, and disruption of CMP integrity. The proteins probed will include AM cell surface molecules (some known to interact with SP-A), SP-A-regulated proteins (with emphasis on cytoskeletal proteins), and proteins that distinguish macrophage subsets. Inhibition will be accomplished using blocking antibodies, or other approaches based on the nature of lead molecule(s) and reagent availability. Data analysis will identify CMP motifs that are specific for and distinguish among the study conditions. For the inhibition studies priority will be given t lead proteins identified in CMP motifs that include the largest number of individual CMPs. The knowledge gained will contribute to our understanding of how cell surface and intracellular proteins interact in the regulation of the cytoskeleton and identify molecules instrumental in these interactions. The successful completion of this will provide insight into lung pathophysiology in response to infection, and the role of the innate immune protein SP-A in this process. Importantly, identification of CMP and lead proteins associated with AM morphology and cytoskeletal changes in this "proof of principle" study, will provide the foundation for future studies where lead proteins responsible for a specific, more complex outcome, such as phagocytosis, could be identified and targeted for therapeutic intervention to enhance or inhibit AM function.