Behavioral dynamics of Langerhans cells in skin

描述（由申请人提供）：朗格汉斯细胞（LCs）是未成熟的树突状细胞（DC）亚群，在环境界面执行两个关键的监测任务（抗原采样和危险信号检测）。我们的目标是定义LCs在其自然栖息地（即皮肤）中完成这些任务的行为机制。为此，我们开发了新的成像系统来获取活体小鼠LCs的4D图像。在LC成熟的标准模型中，LC表现出dSEARCH运动（以树突的伸展和收缩为特征）、变形虫样的横向迁移、垂直迁移和LC-LC接触形成。我们的目标是：J_)定义稳态LC行为。LCs将在l-Ap-EGFP敲入小鼠（所有DC亚群都表达EGFP荧光信号）和langerin-EGFP敲入小鼠（只有LCs表达EGFP）中可见。采用延时多光子激光扫描显微镜对麻醉小鼠EGFP* LCs每2 min拍摄一次3D图像，持续4 h，测量EGFP+ LCs个体运动活性的时空动力学。我们将通过在不同时间点获取相同显微镜场中EGFP+ LC的图像（“间歇性”成像）来研究稳态LC转换。通过比较l-Ap-EGFP敲入小鼠和兰根素- egfp敲入小鼠记录的细胞运动，我们将评估皮肤中其他DC亚群的行为。2)确定LC对病理刺激的行为反应。我们将研究EGFP+ LCs荧光蛋白（dsRed）的周转和运动活性。EGFP敲入小鼠将与：a) IL-1p启动子驱动的dsred转基因小鼠杂交，独立研究LC成熟（红色信号）和LC运动（绿色信号）；b) K14启动子驱动的dsred转基因小鼠，研究LC（绿色）与邻近角质形成细胞（红色）的动态相互作用。我们的研究将为LCs的行为生物学和LCs在其中发挥致病或保护作用的皮肤病病理生理学提供重要见解。

DESCRIPTION (provided by applicant): Langerhans cells (LCs) are immature dendritic cell (DC) subsets that perform two key surveillance tasks (antigen sampling and detection of danger signals) at the environmental interface. Our objective is to define behavioral mechanisms by which LCs achieve these tasks in their natural habitat (i.e., skin). For this purpose, we have developed novel imaging systems to acquire 4D images of LCs in living mice. In standard models of LC maturation, LCs exhibited dSEARCH motion (characterized by extension and retraction of dendrites), amoeba-like lateral migration, vertical migration, and LC-LC contact formation. Our aims are: J_) To define steady-state LC behaviors. LCs will be visualized in the l-Ap-EGFP knock-in mice (in which EGFP fluorescent signals are expressed by all DC subsets) and in the langerin-EGFP knock-in mice (in which only LCs express EGFP). We will record 3D images of EGFP* LCs every 2 min for 4 h by "time-lapse" multi- photon laser scanning microscopy in anesthetized mice to measure spaciotemporal kinetics of individual motile activities of EGFP+ LCs. We will study steady-state LC turnover by acquiring images of EGFP+ LCs in the same microscopic fields at different time points ("intermittent" imaging). By comparing the cellular movement recorded in the l-Ap-EGFP knock-in versus langerin-EGFP knock-in mice, we will assess the behaviors of other DC subsets in the skin. 2) To identify LC behavioral responses to pathological stimuli. We will study the turnover and motile activities of EGFP+ LCs fluorescent protein (dsRed). The EGFP knock-in mice will be cross-bred with: a) IL-1p promoter-driven dsRed-transgenic mice to study LC maturation (red signals) and LC motility (green signals) independently, and b) K14 promoter-driven dsRed-transgenic mice to study dynamic interactions of LCs (green) with neighboring keratinocytes (red). Our study should provide important insights into the behavioral biology of LCs and the pathophysiology of skin disease in which LCs play pathogenic or protective roles.