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（由适用提供）：肺炎是伯宁的主要公共卫生。肺炎（K. PN）是医院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，抗生素抗药性已成为日益增长的公共卫生问题。肺泡巨噬细胞（AM），肺部免疫组织化学的前哨细胞是感染的第一响应者。表面活性剂蛋白A（SP-A）是一种先天免疫蛋白，由肺上皮分泌到AM居住的肺泡中。 SP-A调节许多AM功能，并对AM蛋白具有重大影响，包括与细胞骨架相关的蛋白质变化。尚不清楚蛋白质（例如细胞表面蛋白质和通过我们的蛋白质组学研究所鉴定的蛋白质）相互作用，以实现特定的AM功能。我们将使用Toponeme Imaging System（TIS）解决此知识差距，这是第一个具有高功能分辨率（〜40nm）的微观系统，该系统可以在单个单元格中定位多达100个蛋白质。 TIS的研究表明，如何将蛋白质组织成簇或联合分子表型（CMP），它们是蛋白质信号网络的功能单位，以及CMP组的功能如何取决于几种蛋白质，称为“铅蛋白”。我们假设SP-A调节的AM功能+/- K. Pn感染取决于特定的CMP，其完整性取决于铅蛋白，并且在不同条件下，CMPS和铅蛋白都不同。 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 正直。所探测的蛋白质包括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。将使用阻断抗体或基于铅分子的性质和试剂可用性的其他方法来实现抑制作用。数据分析将确定特定的CMP基序 为了区分研究条件。对于抑制研究，将优先考虑在CMP基序中鉴定的T铅蛋白，其中包括最多的单个CMP。获得的知识将有助于我们理解细胞表面和细胞内蛋白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发挥作用的分子。成功的完成将为感染的响应以及先天免疫蛋白SP-A在此过程中的作用提供对肺病理生理学的见解。重要的是，在此“原则证明”研究中，CMP和与AM形态和细胞骨架变化相关的CMP和铅蛋白的鉴定将为未来提供基础 可以鉴定并针对治疗干预以增强或抑制AM功能的治疗干预措施的铅蛋白质（例如吞噬作用）的铅蛋白质（例如吞噬作用）。