Neutrophil Migration in Three Dimensions

描述(由申请人提供)：中性粒细胞是天然宿主防御的关键，因此外周血中中性粒细胞数量的减少表明对威胁生命的感染的易感性增加。除了产生足够数量的中性粒细胞外，宿主防御还取决于中性粒细胞有效发挥作用的能力。白细胞黏附缺陷患者的中性粒细胞数量正常，但整合素的表达存在遗传缺陷。慢性肉芽肿性疾病患者的中性粒细胞不能产生足够水平的杀菌氧化物种。在这两种情况下，这些功能缺陷使患者容易感染机会性病原体、反复感染发作和伤口愈合障碍。或者，过度的中性粒细胞反应可能导致以附带组织损伤为特征的疾病状态，如呼吸窘迫综合征、哮喘、炎症性肠病、缺血/再灌注损伤、脉管炎和类风湿性关节炎。因此，中性粒细胞必须对感染或损伤做出充分但精细调控的反应，以促进体内平衡的恢复。不管不同的身体组织在成分和结构上有很大的差异，中性粒细胞都会渗透到体内任何受损的组织中，以启动炎症反应。像所有的细胞一样，中性粒细胞表达一组特定的细胞表面受体，这些受体引起对微环境中的生物元素的功能性反应。很明显，身体上的 微环境的性质，如其相对刚性，也是功能的重要调节因素。中性粒细胞用来对物理提示做出反应的机制并不像受体-配体诱导的反应那样清楚。我们实验室之前的工作假设，柔软、有弹性的组织，如脑，对中性粒细胞功能的影响可能不同于较硬的组织，如皮肤或肌肉。事实上，中性粒细胞的黏附、牵引力的产生和迁移在纤维连接蛋白涂层的基质上有显著的不同，只有硬度不同。这些硬度都在人体组织硬度的生理范围内。尽管该模型提供了比塑料和玻璃等坚硬材料更相关的生理基础，但它的局限性在于炎性中性粒细胞在三维组织环境中发挥作用。这是一项设计一种可调的3D体外系统的建议，该系统将允许在更接近模拟组织微环境的条件下研究中性粒细胞。此外，我们的系统的新奇之处在于，给定的细胞可以在2维空间中跟踪，然后在3维空间中进行跟踪，从而将维度分离为单个实验变量。实验还将确定与2D系统相比，整合素是否在3D系统中调节牵引力的产生并控制中性粒细胞的迁移。考虑到器官和组织的显著机械特征的3D体外系统将提供更好的手段来预测用于控制炎症的治疗方法的有效性。 与公共卫生相关：当炎症反应不足或过度时，病理结果。旨在优化炎症的新疗法开发缓慢的原因之一是，对中性粒细胞等炎性白细胞的研究通常是在实验室的培养皿和玻片上进行的，这些培养皿和玻片由玻璃和塑料组成，比我们的身体组织坚硬得多。此外，这些平坦的表面只允许进行二维实验。我们之前的工作表明，中性粒细胞在不同硬度的表面上发挥不同的功能。也有新的迹象表明，细胞会 在2D表面上与在3D表面上不同，3D实验更好地反映了活组织中的细胞行为。这是一项关于设计一种系统的建议，在该系统中，细胞功能可以在与身体组织具有相同硬度的表面上实时研究。此外，通过增加顶层矩阵，我们将能够直接比较单个细胞在 2D系统和3D系统。这将揭示在接近现实生活中遇到的条件下在显微镜下研究细胞的重要性。

DESCRIPTION (provided by applicant): Neutrophils are essential to innate host defense such that a decrease in the number of these cells in the peripheral blood presents heightened susceptibility to life threatening infections. In addition to being produced in adequate number, host defense also depends on the ability of neutrophils to function effectively. Patients with Leukocyte Adhesion Deficiency have normal numbers of neutrophils but they are genetically deficient in expression of integrins. Neutrophils in patients with Chronic Granulomatous Disease fail to produce adequate levels of microbiocidal oxidative species. In both cases, these functional deficits predispose patients to infections with opportunistic pathogens, recurrent infectious episodes and impairment of wound healing. Alternatively, an excessive neutrophil response can result in disease states characterized by collateral tissue damage such as respiratory distress syndrome, asthma, inflammatory bowel disease, ischemia/reperfusion injury, vasculitis and rheumatoid arthritis. Therefore, neutrophils must execute a sufficient but finely regulated response to infection or injury to promote a return to homeostasis. Neutrophils infiltrate any compromised tissue in the body in order to initiate an inflammatory response regardless of the fact that different bodily tissues offer substantial variations in composition an structure. Like all cells, neutrophils express a set of specific cell surface receptors that elicita functional response to biological elements within a microenvironment. It is clear that the physical nature of a microenvironment, such as its relative stiffness, is also an important regulator of function. The mechanisms that neutrophils use to respond to physical cues are not as clear as they are for receptor-ligand induced responses. Prior work from our laboratory hypothesized that soft, elastic tissues such as brain, may affect neutrophil function differently than stiffer tissues such as skin or muscle. Indeed, neutrophil adhesion, production of traction forces and migration were significantly different on fibronectin-coated matricies that varied only in stiffnes. The stiffnesses were all within the physiological range of tissue stiffnesses found in the body. Although this model provided a more relevant physiologic substrate than rigid materials such as plastic and glass, it is limited in that inflammatory neutrophils function within a 3-dimensional tissue environment. This is a proposal to engineer a tunable 3D in vitro system that will permit neutrophils to be studied under conditions that more closely model a tissue microenvironment. Moreover, the novelty of our system lies in that a given cell can be tracked in 2-dimensions and then in 3-dimensions thereby isolating dimension as a single experimental variable. Experiments will also determine whether integrins mediate the generation of traction forces and control migration of neutrophils in 3D as compared to 2D systems. A 3D in vitro system that accounts for the salient mechanical features of organs and tissues will provide a better means to predict effectiveness of therapeutics indicated for control of inflammation. PUBLIC HEALTH RELEVANCE: Pathology results when an inflammatory response is either insufficient or excessive. One of the reasons new therapeutics designed to optimize inflammation are slow to develop is that research on inflammatory white blood cells such as neutrophils is usually conducted on laboratory dishes and slides composed of glass and plastic that are much stiffer than our bodily tissues. In addition, these flat surfaces only allow for a tw dimensional experiments to be performed. Our previous work showed that neutrophils function differently on surfaces of different stiffnesses. There are also new indications that cells respond differently on 2D surfaces than on 3D surfaces, and that 3D experiments better reflect cell behavior in living tissues. This is a proposal to engineer a system in which cell function can be studied in real time on surfaces that are the same stiffness as in bodily tissues. Furthermore, by adding a top layer of matrix we will be able to directly compare the function of a single cell on a 2D system and a 3D system. This will reveal the importance of studying cells under a microscope under conditions that are close to that encountered in real life.