The role of beta 2 integrins in macrophage fusion

项目总结 巨噬细胞融合导致多核巨细胞(MGCS)的形成 与慢性炎症相关的各种疾病，包括异物反应(FBR) 由植入的生物材料引发。尽管对快堆的研究有很长的历史，但分子和 巨噬细胞融合的细胞机制，这是植入长期失败的核心事件 人造血管移植物和其他医疗设备仍然知之甚少。在我们的预赛中 利用体内植入模型研究，我们发现mGCS和肉芽组织的形成， 它在种植体周围发展，是不良纤维帽的前驱，几乎 在纤维蛋白原缺陷小鼠中完全消失。令人惊讶的是，MGC的数量在 植入Mac-1基因缺陷小鼠的生物材料大于野生型小鼠，其厚度 肉芽组织的体积较大。我们假设巨噬细胞在生物材料上的融合 取决于沉积的纤维蛋白(原)基质和不存在Mac-1，通过改变 巨噬细胞的粘附性，加剧了FBR。具体目标1是检验这一假设。 利用生物材料植入的小鼠模型和基因靶向小鼠，我们将进行系统的 FBR早期和晚期的分析及mGCS M1/M2表型的确定 来自野生型和Mac-1缺乏的巨噬细胞。使用纳米技术方法，我们将 生物材料上沉积的纤维蛋白基质的粘附性和力学性能表征 在野生型和Mac-1缺陷小鼠中。特定目标2将描述以前未被识别的肌动蛋白- 在植入的生物材料上的mGC之间形成的基于拉链的类似结构(ZLS)。我们开发了 一种复制ZLS形成的体外模型并证明了细胞间隙 在ZLS内充满了连接蛋白E-钙粘素和Nectin-2。我们假设MGCS形式 帮助MGC存活的上皮样连接。利用技术创新 包括一个微流体室，它可以精确地解剖ZLS，然后是蛋白质组学 分析、高分辨率显微镜、活细胞成像和髓系细胞特异性KO的E- 钙粘附素和其他连接点的成分，我们将确定ZLS的组成及其在 联邦调查局。具体目标3是确定真正的融合蛋白合胞素在 巨噬细胞融合。根据我们的发现，巨噬细胞融合是由一种基于肌动蛋白的 突起，我们将使用击倒实验，EM和视频显微镜来验证假设 供体巨噬细胞前沿的融合能力强的突起含有合胞素。工作服， 这些研究将定义巨噬细胞融合的新生物学，并表征新的机制 有可能调节快速堆。

PROJECT SUMMARY Macrophage fusion resulting in the formation of multinucleated giant cells (MGCs) accompanies a variety of maladies associated with chronic inflammation, including the foreign body response (FBR) elicited by implanted biomaterials. Despite the long history of research on FBR, the molecular and cellular mechanisms of macrophage fusion, an event central to the long-term failure of implanted prosthetic vascular grafts and other medical devices, remain poorly understood. In our preliminary studies using in vivo implantation model, we found that the formation of MGCs and granulation tissue, which develops around the implant and is a precursor of the undesirable fibrotic cap, was almost completely abolished in fibrinogen-deficient mice. Surprisingly, the number of MGCs formed on biomaterials implanted into Mac-1-deficient mice was greater than in wild-type mice and the thickness of granulation tissue was larger. We hypothesize that macrophage fusion on biomaterials critically depends on the deposited fibrin(ogen) matrix and the absence of Mac-1, through the alteration of adhesive properties of macrophages, exacerbates the FBR. Specific Aim 1 is to test this hypothesis. Using a mouse model of biomaterial implantation and gene-targeted mice, we will perform systematic analyses of the early and late stages of FBR and determine the M1/M2 phenotype of MGCs derived from wild-type and Mac-1-deficient macrophages. Using nanotechnology approaches we will characterize the adhesive and mechanical properties of fibrin(ogen) matrices deposited on biomaterials in wild-type and Mac-1-deficient mice. Specific Aim 2 will characterize previously unrecognized actin- based zipper-like structures (ZLS) that form between MGCs on implanted biomaterials. We developed an in vitro model that reproduces the formation of ZLS and demonstrated that the intercellular space within ZLS is filled with junctional proteins E-cadherin and nectin-2. We hypothesize that MGCs form epithelial-like junctions that aid the MGC survival. Taking advantage of technological innovations including a microfluidic chamber that allows the precise dissection of ZLS followed by proteomics analyses, high-resolution microscopy, live cell imaging and mice with myeloid cell-specific KO of E- cadherin and other components of junctions, we will determine the composition of ZLS and their role in the FBR. Specific Aim 3 is to determine the role of authentic fusogenic proteins syncytins in macrophage fusion. Based on our finding that macrophage fusion is initiated by an actin-based protrusion, we will use knockdown experiments, EM and video microscopy to test the hypothesis that a fusion-competent protrusion at the leading edge of a donor macrophage contains syncytins. Overalls, these studies will define the novel biology of macrophage fusion and characterize new mechanisms that have the potential to modulate the FBR.