Microscale models of inflammation and its resolution

项目摘要 中性粒细胞是先天免疫系统的主要细胞，是宿主防御所必需的， 然而，持续的嗜中性粒细胞炎症导致组织损伤和慢性炎症， 包括心血管疾病、自身免疫性疾病和癌症在内的广泛疾病。先前认为 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 吞噬作用;然而，我们最近发现中性粒细胞也会留下组织炎症部位 通过称为中性粒细胞反向迁移的过程。在这里，我们应用微尺度 器官型模型，通过1）分析迁移， 和通过原代中性粒细胞或诱导多能干细胞的3D共培养的信号传导 （iPSC）衍生的嗜中性粒细胞，2）在诱导分化过程中时空分离和回收嗜中性粒细胞， 正向和反向迁移和3）模拟中性粒细胞迁移和反向迁移， 生理相关的3D微环境，以及。我们的目标是开发一种生理上相关的 体外模型，以复制中性粒细胞募集到和清除部位的关键步骤， 炎症该应用的一个关键优势是使用器官型微尺度模型， 诱导和分解中性粒细胞的基本几何形状和细胞相互作用的复制 炎症拟议研究的一个广泛目标是提供一个合作的，多学科的 （工程，生物学家，临床医生）的方法，以有关人类健康的基本问题-即， 炎症及其在组织损伤部位的消退。我们致力于开发实用的工具， 这些方法将改变我们识别修饰中性粒细胞前向和后向信号通路的能力， 逆转迁移并具有治疗潜力。特别关注的是旁分泌信号 由巨噬细胞、内皮细胞和中性粒细胞在共培养和三培养中产生， 诱导性多能干细胞衍生的中性粒细胞作为原代人的模型的表征 中性粒细胞（Aim 1）。此外，分层开放式微流体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，我们将研究人类中性粒细胞的贩运及其 与巨噬细胞和内皮细胞的相互作用，以响应不同的炎症刺激， 生理学相关的器官型体外模型，以检查中性粒细胞的反向迁移， 改变疾病（目标3）。我们希望我们的发现能够解决理解细胞如何 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.