Microscale models of inflammation and its resolution

项目摘要 中性粒细胞是天然免疫系统的主要细胞，是宿主防御所必需的， 然而，持续的中性粒细胞炎症会导致组织损伤和慢性炎症。 广泛的疾病，包括心血管疾病、自身免疫性疾病和癌症。之前人们认为 中性粒细胞炎症的消退是通过中性粒细胞死亡和巨噬细胞实现的。 吞噬作用；然而，我们最近发现，中性粒细胞也会离开组织炎症部位 通过一个被称为中性粒细胞反向迁移的过程。在这里，我们应用微尺度 通过1)分析迁移来分析炎症发生和消退的器官类型模型 以及通过3D共培养原代中性粒细胞或诱导的多能干细胞来传递信号 (IPSCs)来源的中性粒细胞，2)时空分离和回收中性粒细胞 正向迁移和反向迁移以及3)模拟中性粒细胞迁移和反向迁移 生理上相关的3D微环境，以及。我们的目标是开发一种与生理相关的 复制中性粒细胞募集和清除的关键步骤的体外模型 发炎。这个应用的一个关键优点是使用了器官类型的微型模型，它允许 诱导和分解中性粒细胞的基本几何构型和细胞相互作用的复制 发炎。拟议研究的一个广泛目标是提供一个协作的、多学科的 (工程学、生物学家、临床医生)解决与人类健康有关的基本问题的方法--即， 组织损伤部位的炎症及其消退。我们努力开发实用的工具和 这些方法将改变我们识别信号通路的能力，这些信号通路改变了中性粒细胞的转发和 反向迁移，具有治疗潜力。一个特别的焦点是旁分泌信号。 巨噬细胞、内皮细胞和中性粒细胞在共培养和三重培养中产生的 诱导多能干细胞来源的中性粒细胞作为原代人类模型的特征 中性粒细胞(目标1)。此外，分层开放式微流控技术的使用将使我们能够 在双向迁移的不同阶段分离中性粒细胞以确定时空调节 中性粒细胞行为(目标2)。最后，我们将调查人类中性粒细胞及其 巨噬细胞和内皮细胞对不同炎症刺激的相互作用 检测中性粒细胞反向迁移的生理相关器官表型体外模型及其机制 疾病改变(目标3)。我们希望我们的发现能够弥补在理解细胞如何 在炎症的发生和消退以及iPS的进一步发展过程中进行沟通 衍生的中性粒细胞，可能为人类疾病的治疗干预提供新的途径。

Project Summary Neutrophils are primary cells of the innate immune system that are necessary for host defense, however, persistent neutrophil inflammation contributes to tissue damage and chronic inflammation in broad diseases including cardiovascular disease, autoimmune disease and cancer. It was previously thought that resolution of neutrophil inflammation occurred through neutrophil death and macrophage phagocytosis; however, we recently discovered that neutrophils also leave sites of tissue inflammation through a process referred to as neutrophil reverse migration. Here we apply microscale organotypic models to analyze onset and resolution of inflammation by 1) analyzing migration and signaling through 3D co-culture of primary neutrophils or induced pluripotent stem cells (iPSCs) derived neutrophils, 2) spatiotemporally separating and retrieving neutrophils during forward and reverse migration and 3) modeling neutrophil migration and reverse migration in a physiologically relevant 3D microenvironment, and. It is our goal to develop a physiologically relevant in vitro model to replicate key steps in neutrophil recruitment to and clearance from a site of inflammation. A key strength in this application is the use of organotypic microscale models that allow for the replication of the essential geometries and cellular interactions that induce and resolve neutrophil inflammation. A broad goal for the proposed research is to provide a collaborative, multi-disciplinary (engineering, biologists, clinicians) approach to a fundamental problem relevant to human health – namely, inflammation and its resolution at sites of tissue injury. We strive to develop practical tools and methods that will transform our ability to identify signaling pathways that modify neutrophil forward and reverse migration and have therapeutic potential. A particular focus is the paracrine signals generated by macrophages, endothelial cells, and neutrophils in co- and tri-culture and the characterization of induced pluripotent stem cell derived neutrophils as a model for primary human neutrophils (Aim 1). Furthermore, the use of layered open microfluidics technology will allow us to separate neutrophils at different stages in bidirectional migration to identify spatiotemporal regulation of neutrophil behavior (Aim 2). Finally, we will investigate the trafficking of human neutrophils and their interactions with macrophages and endothelial cells in response to different inflammatory stimuli in a physiologically relevant organotypic in vitro model to examine neutrophil reverse migration and how it is altered in disease (Aim 3). We expect our findings to address a gap in understanding how cells communicate during the onset and resolution of inflammation, and the further development of iPS derived neutrophils that may provide novel avenues for therapeutic interventions for human disease.