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

C-Flip 在介导异物反应中促生存巨噬细胞中的作用

• 批准号: 10063721
• 负责人: Stephanie J Bryant
• 金额: $ 21.11万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063721
• 关键词: Apoptosis; Apoptotic; Attenuated; Biocompatible Materials; CASP8 and FADD-like apoptosis regulating protein; CASP8 gene; Cell Death; Cells; Cessation of life; Chemistry; Coupled; Data; Doxycycline; Expression Profiling; Fibrosis; Flow Cytometry; Foreign Bodies; Gene Expression; Genes; Hydrogels; Immunologics; Immunology; Impaired wound healing; Implant; Inflammatory; Kinetics; Lead; Light; Maintenance; Mediating; Medical Device; Methods; Mus; Myelogenous; Myeloid Cells; Pattern; Performance; Polymers; Prevention; Protein Inhibition; Proteins; Publishing; Reporting; Research; Role; Series; Signal Transduction; Silicones; System; Testing; Tetanus Helper Peptide; Time; Tissue Engineering; Tissues; Transgenic Mice; Transgenic Organisms; Translating; base; capsule; cellular targeting; design; experimental study; functional improvement; high reward; high risk; innovation; macrophage; monocyte; mouse model; multidisciplinary; novel; novel strategies; polyetheretherketone; prevent; recruit; response; small molecule inhibitor; success; wound healing

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.

巨噬细胞是外国体内反应（FBR）对植入生物材料的关键参与者，其中 植入物上的无血管纤维胶囊壁。但是，有助于纤维的细胞机制 胶囊尚未阐明。结果，基于合成的生物材料仅限于那些 身体可容忍和哪个功能目的地是FBR，并且不会导致生物材料宿主整合。在这个 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颖假设 在组织纤维化中。我们证明了抑制细胞果实样抑制蛋白（CFLIP），这是 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，我们检验了以下假设 FBR中的巨噬细胞持久性是由细胞内CFLIP介导的，并抑制CFLIP脱气 巨噬细胞到凋亡死亡信号，以防止或解决纤维封装。我们开发了两个特定的 旨在检验假设，并将这一想法转化为靶向巨噬细胞CFLIP的生物材料策略 为了防止纤维封装。在特定的目标＃1中，我们将确定巨噬细胞的动力学 在FBR中坚持不懈。此目标将使用耦合的HCD68-RTTA转基因鼠标 带有Tet-On Cre系统和荧光TDTOMATO表达。使用此鼠标模型，一系列谱系 将进行追踪实验，以结合多参数流式细胞术，以识别髓样子集， 包括招募和组织居民巨噬细胞，区分其在FBR中的临时模式 确定与纤维化相关基因的表达谱的变化。在特定的目标＃2中，我们将暂时 抑制巨噬细胞中的C翼，以促进其程序性细胞死亡并减弱形成和 维护FBR中的纤维囊。 AIM＃2的第一部分将确定 使用类似的转基因小鼠模型在巨噬细胞中的CFLIP抑制与AIM 1中的抑制作用，但已耦合 带有删除CFLIP的Tet-On Cre系统。该鼠标模型将使CFLIP缺失的暂时效应 可以在纤维囊的形成及其溶解下确定。 AIM＃2的第二部分将 专注于设计可触发的生物材料，以临时抑制CFLIP，并在巨噬细胞中局部局部。 这将通过嵌入生物材料中的光状微粒来实现，当 由光导致YM155的缓慢释放，YM155是CFLIP的小分子抑制剂。通过严格控制 释放YM155时，基于生物材料的策略局部CFLIP抑制的临时影响将是 决定。在该项目的结论结束时，我们将有a）确定巨噬细胞的临时模式 累积以及当它们开始持续在FBR中时，b）阐明了CFLIP在介导促生物中的作用 巨噬细胞中的编程及其对纤维封装的影响，c）确定了耗尽的最佳时机 CFLIP和D）开发了可用于预防或解决FBR的新型策略。