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、肝和骨髓中的巨噬细胞，但这种接触是否会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，将缺乏 CD47 的基因敲除小鼠的红细胞注射到对照小鼠中，发现红细胞被脾巨噬细胞迅速清除 - 即使基因敲除小鼠没有表现出红细胞缺陷或贫血 [Oldenborg Science 2000]。受这一悖论和研究结果的刺激，我们决定关注 CD47“自身”信号与人类的相关性，并首先描述人类与小鼠 CD47 在红细胞上的差异 [Dahl Blood 2003, 2004; Dahl Blood 2003, 2004;萨勃拉曼尼亚血2006]。尽管存在许多结构差异，但我们对人红细胞吞噬作用的体外研究表明，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，防止抗体驱动的进食发生 [Tsai & Discher J Cell Biol 2008]。现在，我们已将人 CD47 还原为 10-20 个氨基酸的“自身”肽，该肽与 hSIRPa 结合，抑制吞噬作用，甚至阻碍纳米颗粒从表达人类相容性 mSIRPa 的 NOD/SCID (NSG) 小鼠的脾脏清除[Rodriguez Science 2013]。延迟清除还增强了纳米颗粒向肿瘤异种移植物的染料和药物的递送。然而，“自我”信号传递受到激烈争论[Willingham PNAS 2012，Burger Blood 2012； Wang Mol Ther 2013]，颗粒来源（生物的和合成的）、尺寸、特性的影响在很大程度上尚不清楚。在我们的 Aim-1 中，将检查“自我”展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”以及与衰老相关的刚性红细胞形状。不同巨噬细胞的吞噬途径将在分子细节上进行比较，部分基于基于质谱的蛋白质组学研究，旨在阐明体内和体外自我信号传导的差异。 “自身”信号传导可能因人类 CD47 对天然 SIRPa 变体的亲和力而变得复杂，在我们的初步研究中，该变体跨越了约 50 倍的范围。新的突变体、多态性和肽将在 Aim-2 中进行研究，以阐明这种广泛变异的机制和含义。根据脾脏和肝脏解剖结构，将通过与循环相关的颗粒和细胞经过固定巨噬细胞的微流，在体外探测“自我”识别的动力学和纳米级力量。将与通过蛋白质化 AFM 尖端获得的单分子力以及通过粘附到与植入物相关的“自身”纳米膜获得的单分子力进行比较。 Aim-3 将重点关注 NSG 小鼠巨噬细胞对人类细胞的“自我”识别与抗体诱导的巨噬细胞拮抗之间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.