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

基本信息

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
项目类别：
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

项目摘要

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，其中无血管纤维囊将植入物隔离开来，然而，有助于纤维化的细胞机制。胶囊尚未阐明，因此，合成生物材料仅限于那些尽管有 FBR，但身体仍能耐受并发挥作用，并且不会导致生物材料-宿主整合。这是一个高度探索性的项目，我们根据我们团队最近发表的研究结果提出了一个新颖的假设我们证明了细胞FLICE样抑制蛋白（cFLIP）的抑制，这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，我们检验了以下假设： FBR 中巨噬细胞的持续存在是由细胞内 cFLIP 介导的，抑制 cFLIP 会重新敏化我们开发了两种特定的方法来控制巨噬细胞凋亡信号以防止或解决纤维包膜。旨在检验这一假设，并将这一想法转化为针对巨噬细胞中 cFLIP 的生物材料策略为了防止纤维包裹在具体目标#1中，我们将确定巨噬细胞的动力学。 FBR 中对不同植入物的持久性该目标将使用偶联的 hCD68-rtTA 转基因小鼠。使用 Tet-on Cre 系统和荧光 tdTomato 表达，使用该小鼠模型进行了一系列谱系。将进行结合多参数流式细胞术的追踪实验来识别骨髓亚群，包括招募的和组织驻留的巨噬细胞，区分它们在 FBR 中的时间模式和确定纤维化相关基因表达谱的变化在特定目标#2中，我们将暂时进行。抑制巨噬细胞中的 c-FLIP，促进其程序性细胞死亡并减弱形成和目标 #2 的第一部分将确定 FBR 中纤维囊的维护。使用与目标 1 类似的转基因小鼠模型抑制巨噬细胞中的 cFLIP，但该模型是耦合的使用删除 cFLIP 的 tet-On Cre 系统，该小鼠模型将实现 cFLIP 删除的时间效应。目标 #2 的第二部分将确定纤维胶囊的形成及其溶解。专注于设计一种光触发生物材料来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，当通过严格控制，光触发导致 YM155 缓慢释放，YM155 是 cFLIP 的小分子抑制剂。当 YM155 被释放时，基于生物材料的策略局部 cFLIP 抑制的时间效应将是在该项目结束时，我们将 a) 确定巨噬细胞的时间模式。积累以及当它们开始在 FBR 中持续存在时，b) 阐明了 cFLIP 在介导促生存中的作用巨噬细胞的编程及其对纤维封装的影响，c）确定了消耗的最佳时机 cFLIP，d) 开发了可用于预防或解决 FBR 的新策略。