Particle-Assisted Control over Macrophage-Neutrophil interactions (Pac-Man)

项目摘要 容积性肌肉丧失(VML)是一种由于创伤或疾病对骨骼肌造成的衰弱损伤 导致丧失能力的纤维化和肢体功能丧失。我们和其他人已经确认了延迟的通关 凋亡的中性粒细胞通过对卫星细胞和巨噬细胞的有害作用成为纤维化的主要介导者 行为。因为巨噬细胞是伤口愈合的关键调节细胞，也是清除 中性粒细胞凋亡巨噬细胞疗法是一种很有前途的治疗方法 方法，但受到两个主要挑战的限制：1)巨噬细胞是高度可塑性的细胞，快速转移 对微环境提示作出反应的表型。因此，需要一种策略来控制它们的表型。 在给药后的原位，以防止它们对促炎信号做出反应而改变表型 在受伤的地方。2)高昂的制造成本和监管障碍阻碍了自体(患者- 来源)巨噬细胞，特别是因为需要非常高的数量，但同种异体(供体来源) 巨噬细胞可诱导强烈的T细胞介导的不良免疫反应。我们开发了一种创新的 生物材料介导的巨噬细胞治疗策略，同时应对这两个挑战。 在这个策略中，被称为巨噬细胞-中性粒细胞相互作用的粒子辅助控制(Pac-Man)， 巨噬细胞首先在体外被装载有聚合物可生物降解的微粒，这些微粒会缓慢释放 地塞米松细胞内，从而从内到外控制巨噬细胞表型 对受伤部位进行管理。地塞米松之所以被选为地塞米松是因为它能产生抗炎/促炎作用 巨噬细胞的再生表型，增加其对凋亡细胞的吞噬作用，并抑制其 激活T细胞的能力。因此，地塞米松在给药后的细胞内释放 巨噬细胞到损伤部位有望使它们清除有害的中性粒细胞，化解炎症，并 抑制T细胞活化，防止同种异体细胞排斥。在目标1中，细胞的功能表型 过继转移的Pac-Man巨噬细胞将随着时间的推移在体外和体内使用 流式细胞术，基因表达谱，以及它们与凋亡的中性粒细胞的相互作用的分析。对……的影响 肌肉修复将通过组织学和功能机械测试进行评估。在目标2中，使用 一种异基因细胞来源将在体外和体内使用来自混合捐赠者的原代人类免疫细胞进行测试 以及来自不同品系的小鼠。该项目将推进一种创新的现成、翻译细胞疗法，以 增强对凋亡细胞的清除，这将对治疗纤维化损伤具有变革性，如 VML和其他许多人。

Project Summary Volumetric muscle loss (VML) is a debilitating injury caused by trauma or disease to skeletal muscle that leads to incapacitating fibrosis and loss of limb function. We and others have identified delayed clearance of apoptotic neutrophils as a primary mediator of fibrosis via detrimental effects on satellite cell and macrophage behavior. Because macrophages are critical regulators of wound healing and also the primary cell type that clear apoptotic neutrophils in a process called efferocytosis, macrophage cell therapy is a promising therapeutic approach, but is limited by two main challenges: 1) Macrophages are highly plastic cells that rapidly shift phenotype in response to microenvironmental cues. Therefore, a strategy is needed to control their phenotype in situ following administration, to prevent them from changing phenotype in response to pro-inflammatory cues at the site of injury. 2) High manufacturing costs and regulatory hurdles prevent the use of autologous (patient- derived) macrophages, especially because very high numbers are required, but allogeneic (donor-derived) macrophages elicit a strong T cell-mediated adverse immune response. We developed an innovative biomaterial-mediated macrophage cell therapy strategy that simultaneously addresses both of these challenges. In this strategy, referred to as Particle-Assisted Control over Macrophage-Neutrophil interactions (Pac-Man), the macrophages are first loaded ex vivo with polymeric biodegradable microparticles that slowly release dexamethasone intracellularly, thus controlling macrophage phenotype from the inside out following their administration to sites of injury. Dexamethasone was selected because it causes an anti-inflammatory/pro- regenerative phenotype in macrophages, increases their efferocytosis of apoptotic cells, and suppresses their ability to activate T cells. Thus, the intracellular release of dexamethasone following administration of the macrophages to sites of injury is expected to make them clear detrimental neutrophils, resolve inflammation, and suppress T cell activation to prevent rejection of allogeneic cells. In Aim 1, the functional phenotype of the adoptively transferred Pac-Man macrophages will be rigorously characterized over time in vitro and in vivo using flow cytometry, gene expression profiling, and analysis of their interactions with apoptotic neutrophils. Effects on muscle repair will be assessed using histology and functional mechanical testing. In Aim 2, the potential to use an allogeneic cell source will be tested in vitro and in vivo using primary human immune cells from mixed donors and mice from different strains. This project will advance an innovative off-the-shelf, translational cell therapy to enhance clearance of apoptotic cells, which would be transformative for the treatment of fibrotic injuries such as VML and many others.