Behavioral dynamics of Langerhans cells in skin

描述（由申请人提供）：朗格汉斯细胞（LC）是未成熟的树突状细胞（DC）亚群，在环境界面执行两项关键监测任务（抗原采样和危险信号检测）。我们的目标是定义LC在其自然栖息地（即，皮肤）。为此，我们开发了新的成像系统，以获取活体小鼠LC的4D图像。在LC成熟的标准模型中，LC表现出树突状运动（其特征在于树突的延伸和收缩）、阿米巴样横向迁移、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。LC将在I-Ap-EGFP敲入小鼠（其中EGFP荧光信号由所有DC亚群表达）和langerin-EGFP敲入小鼠（其中仅LC表达EGFP）中可视化。我们将在麻醉小鼠中通过“延时”多光子激光扫描显微镜每2分钟记录EGFP* LC的3D图像，持续4小时，以测量EGFP+ LC的个体运动活性的时空动力学。我们将通过在不同时间点在相同显微镜视野中获取EGFP+ LC的图像（“间歇”成像）来研究稳态LC周转。通过比较l-Ap-EGFP敲入与langerin-EGFP敲入小鼠中记录的细胞运动，我们将评估皮肤中其他DC亚群的行为。2)识别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敲入小鼠将与以下小鼠杂交：a）IL-1 β启动子驱动的dsRed转基因小鼠，以独立地研究LC成熟（红色信号）和LC运动性（绿色信号），和B）K14启动子驱动的dsRed转基因小鼠，以研究LC（绿色）与相邻角质形成细胞（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发挥致病或保护作用的皮肤病的病理生理学提供重要的见解。

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.