Behavioral dynamics of Langerhans cells in skin

朗格汉斯细胞 (LC) 是未成熟的树突状细胞 (DC) 亚群，执行两项关键的监视任务 （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）中实现这些任务的行为机制。为此 为此，我们开发了新型成像系统来获取活体小鼠 LC 的 4D 图像。标准中 LC 成熟模型中，LC 表现出 dSEARCH 运动（其特征是 树突）、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 l-Ap-EGFP 敲入小鼠中可视化（其中 EGFP 荧光信号由所有 DC 子集表达）和 langerin-EGFP 敲入小鼠（其中仅 LC 表达 EGFP）。我们将通过“延时”多点记录技术，每 2 分钟记录一次 EGFP* LC 的 3D 图像，持续 4 小时。 光子激光扫描显微镜在麻醉小鼠中测量个体的时空动力学 EGFP LC 的运动活性。我们将通过获取 EGFP LC 的图像来研究稳态 LC 周转率 不同时间点的相同微观视野（“间歇”成像）。通过比较蜂窝 l-Ap-EGFP 敲入小鼠与 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) 确定LC对病理刺激的行为反应。我们 将研究 EGFP LC 在局部 DNFB 应用、UVB 辐射和 A 组链球菌 (GAS) 的皮肤感染。然后将由当地评估潜在的机制 注射选定的细胞因子、趋化因子和粘附分子的拮抗剂，并通过回交 EGFP 敲入小鼠与缺乏目标基因的突变小鼠。 3）开发活体“多色”成像 系统。我们将监测 EGFP* LC 内化 GAS 菌株的动态过程，该菌株被设计为 表达红色荧光蛋白（dsRed）。 EGFP 敲入小鼠将与以下物质杂交： a) IL-1p 启动子驱动的 dsRed 转基因小鼠用于研究 LC 成熟（红色信号）和 LC 运动性（绿色信号） b) K14 启动子驱动的 dsRed 转基因小鼠研究 LC 的动态相互作用 （绿色）与邻近的角质形成细胞（红色）。我们的研究应该为行为提供重要的见解 LC 的生物学以及 LC 发挥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.