In vivo behavior of monocytes in resting and inflammatory conditions

描述(由申请人提供)：对受伤或感染组织的保护涉及到迁移白细胞的协调输送。因此，目前的研究努力旨在确定控制这些细胞进出特定组织的机制。在这里，我们将研究单核细胞，因为这些细胞在组织炎症的协调和消退中发挥着核心作用，因此是治疗中有吸引力的靶点。被招募到组织中的单核细胞被认为完全来自血液和骨髓。然而，最近的研究表明，脾中含有真正的、未分化的单核细胞，其数量超过了它们的血液等价物。此外，在适当的刺激下，脾单核细胞可以被大量动员到炎症部位。这些发现构成了我们假设的基础，即脾是单核细胞的储存和快速部署的场所，脾单核细胞是人体用来控制炎症的重要来源。目前，有必要阐明控制单核细胞稳态和功能的与脾相关的机制。在第一个目标中，我们将测量定义静止脾中单核细胞生物学的参数(细胞周转、教育与维护、位置和组织以及体内行为)。这些研究对于揭示单核细胞行为的基本方面以及脾作为单核细胞储存器官的作用是必要的。在第二个目标中，我们将定义动员脾单核细胞的机制。我们将研究AT-1受体-血管紧张素II(Ang II)信号，因为它是一个新发现的控制SPM动员的途径。我们将定义SPM对Ang II信号的反应，并研究是否可以利用Ang II信号的调制来控制单核细胞的反应。我们的数据表明，Ang II信号的抑制取消了SPM的部署，从而抑制了远端单核细胞的反应。因此，这些研究具有重要的治疗潜力。我们将使用实验室中提供的最先进的动物模型和读数；其中包括用于询问体内单核细胞行为的延时活体细胞成像，以及用于在不同组织中进行全面分析的“最小接触”细胞的轮廓。在过去的三年里，我们已经开发和/或验证了这项技术，现在将把它应用于回答与单核细胞相关的生物学问题。我们的实验将比较两个单核细胞亚群(Ly-6chi和Ly-6Clo)，因为它们似乎在免疫中扮演不同的角色，但它们的相对贡献需要澄清。体内成像将使用新生成的Cx3cr1gfp/+Ccr2rfp/+小鼠，因为这些小鼠中的单核细胞亚群可以单独跟踪。该项目将与当地(MGH的Weissleder、Luster和Xille；HMS的von Andrian)和外部(加州大学旧金山分校的Charo)免疫学、成像和系统生物学小组密切互动。

DESCRIPTION (provided by applicant): Protection of injured or infected tissue involves the coordinated delivery of migratory leukocytes. Thus, current research efforts aim to define the mechanisms that control the movement of these cells into and out of specific tissues. Here, we will study monocytes because these cells play a central role in the orchestration and resolution of tissue inflammation, and thus represent attractive targets in therapy. Monocytes recruited to tissue are thought to originate exclusively from blood and bone marrow. However, recent work indicates that the spleen contains bona fide, undifferentiated monocytes that outnumber their blood equivalents. Also, splenic monocytes can be mobilized massively to inflammatory sites upon appropriate stimulation. These findings form the basis of our hypothesis that the spleen is a site for storage and rapid deployment of monocytes, and that splenic monocytes represent a significant resource that the body exploits to control inflammation. At present, there is a need to clarify the spleen-associated mechanisms that control monocyte homeostasis and function. In a first aim, we will measure parameters that define the biology of monocytes in the resting spleen (cell turnover, education vs maintenance, location and organization, and in vivo behavior). These studies are necessary to uncover fundamental aspects of monocyte behavior and the role of the spleen as a monocyte storage organ. In a second aim, we will define mechanisms that are employed to mobilize splenic monocytes. We will study AT-1 receptor-Angiotensin II (Ang II) signaling because it is a newly identified pathway that controls mobilization of spM. We will define the spM response to Ang II signals, and study whether modulation of Ang II signaling can be used to control the monocyte response. Our data indicate that suppression of Ang II signaling abolishes the deployment of spM and consequently suppresses the monocyte response at distant sites. Thus, these studies have important therapeutic potential. We will use state-of-the-art animal models and readouts that are available in the laboratory; these include time-lapse intravital cellular imaging to interrogate the behavior of monocytes in vivo, and profiling of 'minimally- touched' cells for comprehensive analysis in distinct tissues. We have developed and/or validated the technology over the past three years, and will now apply it to answer the biological questions relevant to monocytes. Our experiments will compare two monocyte subsets (Ly-6Chi and Ly-6Clo) because they appear to play different roles in immunity, yet their relative contributions need clarification. In vivo imaging will use newly- generated Cx3cr1gfp/+ Ccr2rfp/+ mice because monocyte subsets in these mice can be followed separately. The project will interact closely with local (Weissleder, Luster and Xavier at MGH; von Andrian at HMS) and outside (Charo at UCSF) immunology, imaging and systems biology groups.