The Role of C-Flip in Mediating Pro-Survival Macrophages in the Foreign Body Response

巨噬细胞是植入性生物材料异物反应(FBR)的关键分子。 植入物上的无血管纤维囊壁。然而，导致纤维性疾病的细胞机制 胶囊还没有被阐明。因此，基于合成的生物材料仅限于那些 身体耐受，即使有快速反应也能发挥作用，并且不会导致生物材料-宿主的整合。在这 高度探索性的项目，我们提出了一个新的假设，它是基于我们团队最近发表的发现 在组织纤维化方面。我们证明了抑制细胞FLICE样抑制蛋白(CFLIP)是一种 巨噬细胞命运的主要调节者，可以预防组织纤维化。在这个项目中，我们测试了这样的假设 巨噬细胞在FBR中的持久性是由细胞内cFLIP和抑制cFLIP再敏感性所介导的 巨噬细胞向凋亡发出死亡信号，以防止或化解纤维包膜。我们开发了两种特定的 旨在验证这一假设，并将这一想法转化为针对巨噬细胞中cFlIP的生物材料策略 以防止纤维包裹体。在特定的目标#1中，我们将确定巨噬细胞的动力学 在FBR中对不同植入物的持久性。这一目标将使用hCD68-RTTA转基因小鼠 带有Tet-On Cre系统和荧光tdTomato表达。使用这个小鼠模型，一系列的血统 将进行示踪实验，结合多参数流式细胞术来识别髓系亚群， 包括招募的和组织驻留的巨噬细胞，区分它们在FBR和 确定它们在纤维化相关基因表达谱中的变化。在具体目标2中，我们将暂时 抑制巨噬细胞中的c-flip以促进其程序性细胞死亡和减弱形成 快堆内纤维膜的维护。目标#2的第一部分将确定 使用与Aim 1相似的转基因小鼠模型抑制巨噬细胞中的cFlIP，但这是耦合的 使用可删除cFLIP的tet-on CRE系统。此小鼠模型将启用cFLIP删除的时间效应 对纤维包膜的形成和溶解情况进行测定。目标#2的第二部分将 专注于设计一种可光触发的生物材料，以暂时和局部抑制巨噬细胞中的cFLIP。 这将通过嵌入在生物材料中的不稳定光的微粒来实现，当 光触发导致cFLIP小分子抑制物YM155的缓慢释放。通过严格控制 当YM155发布时，基于生物材料的策略对局部cFLIP抑制的时间效应将是 下定决心。在本项目结束时，我们将有a)确定巨噬细胞的时间模式 当它们开始在FBR中持续存在时，b)阐明了cflip在促进生存中的作用。 巨噬细胞中的程序化及其对纤维包膜的影响，c)确定了消耗的最佳时机 和d)开发了可用于预防或解决FBR的新策略。

Macrophages are key players in the foreign body response (FBR) to implanted biomaterials, in which an avascular fibrous capsule walls off the implant. However, the cellular mechanisms that contribute to the fibrous capsule have not yet been elucidated. As a result, synthetic-based biomaterials have been limited to those that the body tolerates and which function despite a FBR and does not lead to biomaterial-host integration. In this highly exploratory project, we put forth a novel hypothesis that is based on our team’s recent published findings in tissue fibrosis. We demonstrated that inhibition of cellular FLICE-like inhibitory protein (cFLIP), which is a major regulator of macrophage cell fate, can prevent tissue fibrosis. In this project, we test the hypothesis that that macrophage persistence in the FBR is mediated by intracellular cFLIP and inhibiting cFLIP resensitizes macrophages to apoptotic death signals to prevent or resolve fibrous encapsulation. We developed two specific aims to test the hypothesis and to translate this idea to a biomaterial strategy that targets cFLIP in macrophages to prevent fibrous encapsulation. In specific Aim #1, we will determine the kinetics of macrophage persistence in the FBR to distinct implants. This aim will use the hCD68-rtTA transgenic mouse that is coupled with a Tet-on Cre system and fluorescent tdTomato expression. Using this mouse model, a series of lineage tracing experiments will be performed that combine multiparameter flow cytometry to identify myeloid subsets, including recruited and tissue-resident macrophages, distinguish their temporal patterns in the FBR and determine changes in their expression profiles for fibrosis-relevant genes. In specific Aim #2, we will temporally inhibit c-FLIP in macrophages to promote their programmed cell death and attenuate formation and maintenance of the fibrous capsule in the FBR. The first part of Aim #2 will determine the temporal effects of cFLIP inhibition in macrophages using a similar transgenic mouse model as in Aim 1, but which is coupled with a tet-On Cre system that deletes cFLIP. This mouse model will enable the temporal effects of cFLIP deletion to be determined on both the formation of fibrous capsule and on its dissolution. The second part of Aim #2 will focus on designing a phototriggerable biomaterial to inhibit cFLIP temporally and locally in macrophages. This will be achieved through photo-labile microparticles that are embedded within a biomaterial, which when triggered by light lead to the slow release of YM155, a small molecule inhibitor of cFLIP. By tightly controlling when YM155 is released, the temporal effects of local cFLIP inhibition by a biomaterial-based strategy will be determined. At the conclusion of this project, we will have a) determined the temporal patterns of macrophage accumulation and when they begin to persist in the FBR, b) elucidated the role of cFLIP in mediating pro-survival programming in macrophages and its effect on fibrous encapsulation, c) identified the optimal timing for depleting cFLIP, and d) developed novel strategies that can be applied for preventing or resolving the FBR.