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

描述（由申请人提供）：血细胞经常接触脾脏、肝脏和骨髓中的巨噬细胞，但这种接触是否激活巨噬细胞并促进血细胞清除是一个不仅对细胞存活而且对生物相容性和纳米技术都具有广泛重要性的基本问题。几年前，将来自缺乏CD 47的基因敲除小鼠的RBC注射到对照小鼠中，发现RBC被脾巨噬细胞迅速清除-即使基因敲除小鼠未显示RBC缺陷或贫血[Oldenborg Science 2000]。受这一悖论和研究结果的刺激，我们决定专注于CD 47“自身”信号传导与人类的相关性，并从表征人与小鼠RBC上CD 47之间的差异开始[Dahl Blood 2003，2004; Subramanian Blood 2006]。尽管存在许多结构差异，但我们对体外人RBC吞噬作用的研究表明，人-CD 47确实可以抑制进食，其中通过人巨噬细胞上的SIRPa向细胞骨架发出信号以对抗抗体驱动的进食[Tsai & Discher J Cell Biol 2008]。我们现在已经将人-CD 47还原为10-20个氨基酸的“自身”肽，其结合hSIRPa，抑制吞噬作用，甚至阻碍纳米颗粒从表达人相容性mSIRPa的NOD/SCID（NSG）小鼠的循环中的脾清除[Rodriguez Science 2013]。延迟清除还增强染料和药物向肿瘤异种移植物的纳米颗粒递送。然而，“自我”信号传导是激烈争论的[威灵厄姆PNAS 2012，Burger Blood 2012; Wang Mol Ther 2013]，并且颗粒源（生物和合成）、大小、性质的影响在很大程度上都是不清楚的。在我们的Aim-1中，将检查“自我”展示颗粒或细胞的途径，这些颗粒或细胞的范围从外泌体或血小板样颗粒和慢病毒到金或磁性纳米颗粒，单体“自我胶体”，以及与衰老相关的刚性红细胞形状。在不同的巨噬细胞的吞噬途径将进行比较的分子细节，部分基于质谱为基础的蛋白质组学研究，旨在阐明在体内以及在体外的自我信号的差异。 “自身”信号传导可能因人-CD 47对天然SIRPa变体的亲和力而复杂化，在我们的初始研究中，天然SIRPa变体跨越约50倍的范围。新的突变体，多态性和肽将在目标2研究，以澄清机制和这种广泛的变化的影响。根据脾脏和肝脏解剖结构，将在体外用经过静止巨噬细胞的颗粒和细胞的循环相关微流探测“自我”识别的动力学和纳米级力。将进行比较，以获得单分子力与蛋白化的AFM提示，也通过粘附到“自我”相关的植入物的纳米膜。目的-3将集中于NSG小鼠巨噬细胞对人细胞的“自我”识别和Ab诱导的巨噬细胞拮抗作用之间的纳米级信号传导的体内平衡。我们的最终目标是从血液的角度并通过一系列纳米技术的发展来阐明“自我”识别的机制。

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.