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

描述(申请人提供)：血细胞经常接触脾、肝和骨髓中的巨噬细胞，但这种接触是否激活巨噬细胞并促进血细胞清除是一个不仅对细胞生存而且对生物兼容性和纳米技术具有广泛重要性的基本问题。几年前，缺乏CD47的基因敲除小鼠的红细胞被注射到对照组小鼠中，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]。在这一悖论和研究结果的刺激下，我们决定将重点放在CD47‘自我’信号与人类的相关性上，并从表征人和小鼠的CD47在红细胞上的差异开始[Dahl布拉德2003,2004；苏布拉马尼亚血液2006]。尽管有许多结构上的差异，我们在体外对人类红细胞吞噬功能的研究表明，人-CD47确实可以抑制进食，并通过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-CD47减少到一个10-20个氨基酸的“自身”肽，它结合hSIRPA，抑制吞噬，甚至阻止纳米颗粒从表达与人类相容的mSIRPA的NOD/SCID(NSG)小鼠的循环中清除[Rodriguez Science 2013]。延迟清除也会增加纳米颗粒对肿瘤移植瘤的染料和药物的输送。然而，“自我”信号的争论非常激烈[Willingham PNAS 2012，Burger布拉德2012；Wang Mol Ther 2013]，颗粒源(生物和合成)、大小、特性的影响都很不清楚。在我们的Aim-1中，将检查途径中的“自我”--显示颗粒或细胞的范围从外体或血小板样颗粒和慢病毒到金黄色或磁性纳米颗粒、单体“自胶体”，以及与衰老相关的僵硬的红细胞形状。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MS-Spec的蛋白质组学研究，旨在阐明体内和体外自我信号的差异。在我们最初的研究中，自然SIRPA变体的人-CD47的亲和炎可能会使‘自我’信号变得复杂，范围是原来的50倍。新的突变体、多态和多肽将在AIM-2中进行研究，以阐明这种广泛变异的机制和意义。“自我”识别的动力学和纳米尺度的力量将在体外用循环相关的微粒和细胞流过固定巨噬细胞的微流来探索，根据脾和肝脏的解剖结构。我们将与通过蛋白质AFM针尖获得的单分子作用力以及通过附着到与植入物相关的“自身”纳米膜上的单分子作用力进行比较。AIM-3将专注于体内纳米级信号在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平衡。我们的最终目标是从血液的角度和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.