Nanoscience of 'Self' - reductionist approaches to hCD47 inhibition of phagocytes

“自我”的纳米科学 - 吞噬细胞 hCD47 抑制的还原论方法

• 批准号: 8764516
• 负责人: Dennis E. Discher
• 金额: $ 38.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8764516
• 关键词: Address; Adhesions; Adhesives; Affinity; Amino Acids; Anatomy; Anemia; Antibodies; Binding; Biological; Biological Assay; Blood; Blood Cells; Blood Circulation; Blood Platelets; Blood flow; CD47 gene; Cell Shape; Cell Survival; Cell Therapy; Cells; Colloids; Color; Complement; Cytoskeleton; Data; Defect; Development; Disorder by Site; Dyes; Eating; Engineering; Equilibrium; Erythrocytes; Excretory function; Film; Gel; Gene Transduction Agent; Genetic Polymorphism; Goals; Gold; Hematopoietic; Human; Image; Implant; In Vitro; Injury; Kinetics; Knockout Mice; Left; Liposomes; Liquid substance; Liver; Lung; Magnetism; Maps; Marrow; Measures; Mediating; Membrane Glycoproteins; Membrane Proteins; Methods; Molecular; Mononuclear; Mouse Strains; Mus; Myosin ATPase; Nanotechnology; Occupations; Particle Size; Pathway interactions; Peptides; Phagocytes; Phagocytosis; Pharmaceutical Preparations; Polystyrenes; Property; Proteins; Proteomics; Recombinants; Reporting; Research; Research Design; Role; Science; Self Perception; Serum; Shapes; Signal Transduction; Site; Source; Spleen; Spottings; Structure; Subfamily lentivirinae; Surface; System; Testing; Time; Validation; Variant; Virus; Work; Xenograft procedure; base; biomaterial compatibility; comparative; design; in vivo; inhibitor/antagonist; intravenous injection; killings; macrophage; man; mutant; nano; nanoparticle; nanoscale; nanoscience; nanotool; overexpression; particle; public health relevance; receptor; senescence; single molecule; tumor; tumor xenograft; uptake

DESCRIPTION (provided by applicant): Blood cells often contact Macrophages in the spleen, liver, and marrow, but whether such contacts activate the macrophage and promote blood cell clearance is a basic question of broad importance not only to cell survival but also to biocompatibility and nanotechnologies. Some years ago, RBCs from knockout mice lacking CD47 were injected into control mice, and the RBCs were found to be cleared rapidly by splenic macrophages - even though the knockout mice showed no RBC defects or anemia [Oldenborg Science 2000]. Stimulated by this paradox and the findings, we decided to focus on the relevance of CD47 'Self' signaling to humans, and we began by characterizing differences between human vs mouse CD47 on RBCs [Dahl Blood 2003, 2004; Subramanian Blood 2006]. Despite many structural differences, our studies of human RBC phagocytosis in vitro showed that human-CD47 can indeed inhibit eating, with signaling to the cytoskeleton against antibody- driven eating occurring through SIRPa on a human macrophage [Tsai & Discher J Cell Biol 2008]. We have now reduced human-CD47 to a 10-20 amino acid 'Self' peptide that binds hSIRPa, inhibits phagocytosis, and even impedes splenic clearance of nanoparticles from the circulation of NOD/SCID (NSG) mice expressing a human-compatible mSIRPa [Rodriguez Science 2013]. Delayed clearance also enhances nanoparticle delivery of dyes and drugs to tumor xenografts. However, 'Self' signaling is hotly debated [Willingham PNAS 2012, Burger Blood 2012; Wang Mol Ther 2013], and effects of particle source (biological and synthetic), size, properties are all largely unclear. In our Aim-1, pathways will be examined for 'Self'-displaying particles or cells that range from Exosomes or Platelet-like Particles and Lentivirus to Gold or Magnetic Nanoparticles, monomeric 'Self Colloids', and also Rigidified RBC shapes relevant to senescence. Phagocytosis pathways in diverse Macrophages will be compared in molecular detail, based in part on Mass Spec-based proteomics studies designed to elucidate differences in Self signaling in vivo as well as in vitro. 'Self' signaling is perhaps complicated by affinitis of human-CD47 for natural SIRPa variants that span a ~50-fold range in our initial studies. New mutants, polymorphisms, and peptides will be studied in Aim-2 to clarify mechanisms and implications of such wide variation. Kinetics and nano-scale forces of 'Self' recognition will be probed in vitro with circulation-relevant microflows of particles and cells past stationary macrophages, per spleen and liver anatomy. Comparisons will be made to single molecule forces obtained with proteinated AFM tips and also via adhesion to Nano-films of 'Self' relevant to implants. Aim-3 will focus on the in vivo balance in nanoscale signaling between 'Self' recognition of human cells by NSG mouse Macrophages and Ab-induced Antagonism of the Macrophage. Our ultimate goal is to clarify mechanisms of 'Self' recognition from a perspective of blood and through an array of nanotechnology developments.

描述（由申请人提供）：血细胞经常接触脾，肝脏和骨髓中的巨噬细胞，但是这种接触是否激活巨噬细胞并促进血细胞清除是一个基本的问题，这不仅是对细胞存活的重要性，而且对生物相容性和纳米技术。几年前，将缺乏CD47的敲除小鼠的RBC注入对照小鼠中，并且发现RBC被脾巨噬细胞迅速清除 - 尽管敲除小鼠没有RBC缺陷或贫血[Oldenborg Science 2000]。受到这种悖论和发现的刺激，我们决定关注CD47“自我”信号与人的相关性，我们首先是在RBC上表征人与小鼠CD47之间的差异[Dahl Blood 2003，2004; Subramanian Blood 2006]。尽管存在许多结构上的差异，但我们对人类RBC吞噬作用的研究表明，人CD47确实可以抑制饮食，并向细胞骨架发出信号，以防止通过人类巨噬细胞在人类巨噬细胞上通过siRPA发出抗体驱动的饮食[Tsai＆Discher J Cell Biol 2008]。现在，我们已将人类CD47降低到结合HSIRPA，抑制吞噬作用的10-20个氨基酸“自我”肽，甚至阻碍了纳米颗粒的脾气间隙，无法从nod/scID（NSG）小鼠的循环中循环，表达人类与人类相关的MSSIRPA [Rodrrpa [Rodrrrpa [Rodrrrpa [Rodrrpa Science 2013]]。延迟清除还可以增强染料和药物向肿瘤异种移植物的递送。然而，“自我”信号引起了人们的争论[Willingham PNAS 2012，Burger Blood 2012; Wang Mol Ther 2013]，以及粒子源（生物学和合成）的影响，大小，性质在很大程度上都不清楚。在我们的AIM-1中，将检查途径的“自我播放颗粒或细胞”，这些颗粒或细胞从外泌体或血小板状颗粒和慢烟病到金或磁性纳米颗粒，单体“自胶体”，以及与衰老相关的僵化的RBC形状。分子细节将比较不同的巨噬细胞中的吞噬作用途径，部分基于基于质量规格的蛋白质组学研究，旨在阐明体内自信号传导的差异以及体外。 对于天然SIRPA变体的人类CD47的炎症，“自我”信号传导可能是复杂的，在我们的初步研究中占50倍。将在AIM-2中研究新的突变体，多态性和肽，以阐明这种广泛变化的机制和含义。每次脾脏和肝脏解剖结构，将在体外探测“自我”识别的动力学和纳米级力。比较将与用蛋白质的AFM尖端获得的单分子力以及与植入物相关的“自我”的纳米膜的粘附。 AIM-3将集中于NSG小鼠巨噬细胞对人类细胞的“自我”识别与AB诱导的巨噬细胞拮抗作用之间的体内平衡。我们的最终目标是从血液的角度和一系列纳米技术发展阐明“自我”认可的机制。