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

肺泡巨噬细胞蛋白对 SP-A 和感染的相互作用

• 批准号: 8890420
• 负责人: JOANNA FLOROS
• 金额: $ 24.05万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8890420
• 关键词: A Mouse; Actins; Address; Affect; Alveolar Macrophages; Alveolus; Antibiotic Resistance; Architecture; Bacterial Infections; Blocking Antibodies; Cell Nucleus; Cell Size; Cell Surface Proteins; Cell Surface Receptors; Cell physiology; Cell surface; Cells; Cellular Structures; Characteristics; Communities; Complex; Confocal Microscopy; Cytoskeletal Proteins; Cytoskeleton; Data; Data Analyses; Data Set; Development; Epithelium; F-Actin; Foundations; Functional disorder; Future; Image; Immune; Immunocompromised Host; In Situ; Inclusion Bodies; Individual; Infection; Intermediate Filaments; Klebsiella pneumonia bacterium; Knowledge; Lead; Ligands; Link; Lung; Mediating; Microfilaments; Microscopic; Microtubules; Morphology; Names; Natural Immunity; Nature; Organelles; Outcome; Patients; Phagocytosis; Play; Pneumonia; Process; Protein Inhibition; Proteins; Proteome; Proteomics; Public Health; Pulmonary Surfactant-Associated Protein A; Reading; Reagent; Regulation; Research Personnel; Resolution; Role; Sentinel; Signal Transduction; Signaling Protein; Subcellular structure; System; Technology; Testing; Therapeutic Intervention; Tissues; Vesicle; analytical tool; base; combinatorial; emergency service responder; functional outcomes; imaging system; indexing; insight; macrophage; molecular phenotype; new technology; prevent; protein distribution; protein function; public health relevance; research study; resistant strain; response; trafficking

 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.

 描述（由申请人提供）：肺炎是一个主要的公共卫生负担。肺炎克雷伯菌（K. PN）是医院获得性肺炎和社区获得性肺炎的主要原因，特别是在免疫功能低下或虚弱的患者中，并且抗生素抗性菌株已经成为日益增加的公共卫生问题。肺泡巨噬细胞（alveolar macrophage，AM）是肺天然免疫的前哨细胞，是感染后的第一反应者。表面活性蛋白A（SP-A）是一种先天性免疫蛋白，由肺上皮分泌到AM所在的肺泡中。SP-A调节许多AM功能，并对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，如细胞表面蛋白质和我们的蛋白质组学研究所确定的蛋白质，如何相互作用，使特定的AM功能尚不清楚。我们将使用Toponome成像系统（TIS）来解决这一知识缺口，TIS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种蛋白质。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，这是蛋白质信号网络的功能单元，以及CMP组的功能如何取决于一些蛋白质，称为“前导蛋白”。我们假设SP-A调节的AM功能为+/- K。pn感染依赖于特定的CMP，其完整性依赖于前导蛋白，并且CMP和前导蛋白在不同条件下不同。本研究有两个具体目的：1）研究和比较K. pn感染的野生型和SP-A -/-小鼠，鉴定每个研究组特有的CMP，并鉴定CMP组中的前导蛋白，和2）抑制所选CMP的前导蛋白（来自目的1），并研究这种抑制对细胞骨架变化、细胞大小和CMP完整性破坏的影响。所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调节蛋白（重点是细胞骨架蛋白）和区分巨噬细胞亚群的蛋白质。将使用封闭抗体或基于先导分子性质和试剂可用性的其他方法来实现抑制。数据分析将确定特定的CMP基序 并区分研究条件。对于抑制研究，将优先考虑在CMP基序中鉴定的先导蛋白，其包括最大数量的单个CMP。所获得的知识将有助于我们了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，并确定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病理生理反应，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“原理证明”研究中，CMP和铅蛋白与AM形态和细胞骨架变化相关的鉴定将为未来的研究提供基础。 研究中，负责特定的，更复杂的结果，如吞噬作用的先导蛋白可以被识别和靶向治疗干预，以增强或抑制AM功能。