Behavioral dynamics of Langerhans cells in skin

朗格汉斯细胞(LCs)是未成熟的树突状细胞(DC)亚群，执行两个关键的监视任务 (抗原采样和危险信号检测)在环境界面。我们的目标是定义 LC在其自然栖息地(即皮肤)完成这些任务的行为机制。为了这个 目的：我们开发了新的成像系统来获取活体小鼠的LCS的4D图像。在标准中 LCS的成熟模型表现为dSEARCH运动(特征为伸展和收缩 树枝状)、阿米巴状侧向迁移、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。在L-AP-EGFP基因敲除小鼠(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 LCS Express EGFP)。我们将每隔2分钟记录一次EGFP*LCS的3D图像，持续4小时，使用多路延时技术- 用光子激光扫描显微镜测量麻醉小鼠的时空动力学 绿色荧光蛋白LCS的运动活性。我们将通过获取EGFP LCS的图像来研究稳态LC周转 不同时间点的相同微观场(“间歇性”成像)。通过比较细胞 在L-AP-EGFP敲入小鼠和Langerin-EGFP敲入小鼠中记录的运动，我们将评估 皮肤中其他DC子集的行为。2)明确LC对病理刺激的行为反应。我们 将研究局部应用DNFB、UVB辐射后EGFP LCS的周转和运动活性，以及 皮肤感染A组链球菌(GAS)。潜在的机制将在当地进行评估 注射选定的细胞因子、趋化因子和黏附分子的拮抗剂，并通过回交 目的基因缺失突变小鼠的EGFP基因敲除小鼠。3)发展活体内“多色”成像 系统。我们将监测EGFP*LC内化改造的气体菌株的动态过程 表达红色荧光蛋白(DsRed)。绿色荧光蛋白基因敲除小鼠将与：a)IL-1p杂交 启动子驱动的dsRed转基因小鼠研究LC成熟(红色信号)和LC运动(绿色信号) 独立和b)K14启动子驱动的dsRed转基因小鼠研究LCS的动态相互作用 (绿色)与邻近的角质形成细胞(红色)。我们的研究应该为我们的行为提供重要的见解 LCS的生物学和LCS在其中起致病或保护作用的皮肤病的病理生理学。

Langerhans cells (LCs) are immature dendritic cell (DC) subsets that perform two key surveillancetasks (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 after topical DNFB application, UVB radiation, and skin infection with Group A streptococci (GAS).The underlying mechanisms will then be assessed by locally injecting antagonists of selected cytokines, chemokines, and adhesion molecules and by back-crossing the EGFP knock-in mice with mutant mice deficient in target genes. 3) To develop intravital "multi-color" imaging systems. We will monitor dynamic processes in which EGFP* LCs internalize GAS strains engineered to expressing a red 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.