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

巨噬细胞是植入生物材料的异物反应（FBR）的关键参与者，其中 无血管纤维囊壁脱离植入物。然而，有助于纤维化的细胞机制 胶囊尚未被阐明。因此，基于合成的生物材料仅限于那些 机体耐受，尽管FBR仍起作用，不导致生物材料-宿主整合。在这 作为一个高度探索性的项目，我们提出了一个基于我们团队最近发表的发现的新假设 组织纤维化我们证明，细胞FLICE样抑制蛋白（cFLIP）的抑制，这是一个 巨噬细胞命运的主要调节剂，可以防止组织纤维化。在这个项目中，我们测试的假设， 巨噬细胞在FBR中的持续存在是由细胞内cFLIP介导的，抑制cFLIP可使FBR再敏感。 巨噬细胞凋亡死亡信号，以防止或解决纤维包囊。我们开发了两个具体的 旨在验证这一假设，并将这一想法转化为靶向巨噬细胞中cFLIP的生物材料策略 以防止纤维封装。在具体目标#1中，我们将确定巨噬细胞的动力学 FBR中的持久性与不同的植入物有关。该目的将使用偶联的hCD 68-rtTA转基因小鼠， 用Tet-on Cre系统和荧光tdTomato表达。使用这种小鼠模型， 将进行结合联合收割机多参数流式细胞术的示踪实验以鉴定骨髓亚群， 包括募集的和组织驻留的巨噬细胞，区分它们在FBR中的时间模式， 确定纤维化相关基因表达谱的变化。在具体目标#2中，我们将暂时 抑制巨噬细胞中c-FLIP以促进其程序性细胞死亡并减弱形成， 维持FBR中的纤维囊。目标#2的第一部分将确定 使用与Aim 1中类似的转基因小鼠模型，在巨噬细胞中抑制cFLIP，但其偶联 用一个删除cFLIP的tet-On Cre系统。该小鼠模型将使cFLIP缺失的时间效应成为可能。 根据纤维囊的形成及其溶解来确定。目标#2的第二部分将 重点在于设计一种可光致降解的生物材料，以暂时和局部抑制巨噬细胞中的cFLIP。 这将通过嵌入生物材料中的光不稳定微粒来实现， 由光引发的光致发光导致cFLIP的小分子抑制剂YM 155的缓慢释放。通过严格控制 当释放YM 155时，通过基于生物材料的策略进行的局部cFLIP抑制的时间效应将是 测定在这个项目的结论，我们将有一个）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在介导促存活中的作用 c）确定了消耗巨噬细胞的最佳时机， cFLIP，以及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.