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

描述（由申请人提供）：血细胞经常与脾脏、肝脏和骨髓中的巨噬细胞接触，但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问题，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的重要性。几年前，将缺乏CD47的基因敲除小鼠的红细胞注射到对照小鼠体内，发现这些红细胞被脾巨噬细胞迅速清除——尽管基因敲除小鼠没有红细胞缺陷或贫血[Oldenborg Science 2000]。受到这一悖论和研究结果的刺激，我们决定将重点放在CD47“自我”信号与人类的相关性上，我们首先描述了人类和小鼠红细胞上CD47的差异[Dahl Blood 2003, 2004；Subramanian Blood 2006]。尽管存在许多结构差异，我们在体外对人红细胞吞噬的研究表明，人cd47确实可以抑制进食，通过人巨噬细胞上的SIRPa向细胞骨架发出信号，对抗抗体驱动的进食[Tsai & Discher J Cell Biol 2008]。我们现在已经将人cd47还原为一个10-20个氨基酸的“自我”肽，它可以结合hSIRPa，抑制吞噬，甚至阻碍脾脏清除NOD/SCID （NSG）小鼠循环中的纳米颗粒，这些小鼠表达了与人兼容的mSIRPa [Rodriguez Science 2013]。延迟清除也增强了纳米颗粒对肿瘤移植的染料和药物递送。然而，“自我”信号引发了激烈的争论[Willingham PNAS 2012； Burger Blood 2012；Wang Mol Ther 2013]，而颗粒来源（生物和合成）、大小、性质的影响在很大程度上都不清楚。在我们的Aim-1中，将检查“自我”显示颗粒或细胞的途径，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”，以及与衰老相关的硬化红细胞形状。不同巨噬细胞的吞噬途径将在分子细节上进行比较，部分基于基于质量特异性的蛋白质组学研究，旨在阐明体内和体外自我信号传导的差异。在我们最初的研究中，人类cd47对天然SIRPa变异的亲和性可能使“自我”信号传导变得复杂，这些变异的范围约为50倍。Aim-2将研究新的突变体、多态性和多肽，以阐明这种广泛变异的机制和含义。动力学和“自我”识别的纳米级力将在体外通过固定巨噬细胞的颗粒和细胞的循环相关微流进行探测，根据脾脏和肝脏解剖。将与用受保护的AFM尖端获得的单分子力进行比较，也将通过与植入物相关的“自我”纳米膜的粘附进行比较。Aim-3将重点关注NSG小鼠巨噬细胞对人类细胞的“自我”识别和抗体诱导的巨噬细胞拮抗之间的纳米级信号在体内的平衡。我们的最终目标是通过一系列纳米技术的发展，从血液的角度阐明“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.