Decoding temporal epithelial signaling programs to restore homeostasis in acute lung injury

摘要 急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)是一种常见的 病死率高的综合征。ALI/ARDS源于肺与肺之间的沟通障碍 上皮细胞和免疫细胞，是严重呼吸道感染病例的主要因素， 包括新冠肺炎。ALI/ARDS的治疗目前受到模型不足的限制 信号通路与肺上皮细胞行为之间的复杂关系。一个核心 信号通路网络控制着上皮结构和细胞因子的释放，是一种 对ALI/ARDS的重要的未开发的干预点，通过现有的靶向激酶抑制剂或 代谢调节剂。这个核心网络包括核因子-kB，它是一种主要的转录因子， 细胞因子释放和炎症，以及ERK、AKT、JNK、p38、mTOR和AMPK， 调节细胞因子的产生，以响应细胞的生长和代谢状态。 值得注意的是，这个信号网络在肺癌中也起到了因果作用，并参与了其他 肺上皮疾病，包括纤维化和慢性阻塞性肺病。我们的实验室已经开发出一种 广泛的活细胞技术平台，用于跟踪信号事件的时间程序- 不同的时间、强度和这些途径之间的协调模式--这决定了 上皮细胞命运的决定。我们的总体目标是定量破译细胞因子 分泌是由多个激酶活性程序指定的。我们的假设是， 单细胞水平上信号活动的重合阶段将显著改善 预测细胞因子分泌的整体和局部变异性，并将确定代谢 减少炎症的手法。我们的方法将开发所需的技术 检测和操纵时间信号程序以恢复肺上皮细胞的动态平衡 ALI/ARDS。目标1将开发一个高内容的数据驱动模型，将信令程序关联起来 在多个条件下逐个细胞地释放细胞因子。目标2将开发 描述肺上皮亚区局部炎症环境的能力 通过检查10-100个相邻细胞组中的激酶信号的动态程序。 在此过程中，我们将提供使用固定免疫荧光板的原则证明。 用于捕获动态信号模式的标记。目标3将调查潜在的 用现有的药理学药物操纵时间信号程序，重点是 AMPK激活剂作为调节更大的信令网络的途径正在显现的好处。我们的 多学科团队将跨越肺部护理、细胞生物学和 计算系统建模，以创建一个连接现代单机版的技术平台 肺部疾病中紧迫挑战的细胞信号转导分析。我们预计我们的 该项目将确定ALI/ARDS潜在的新干预途径，并揭示 细胞因子信号通路中的一般概念，与治疗广泛相关 肺部恶性肿瘤和炎症性疾病。

Abstract Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a common pulmonary syndrome with high mortality. ALI/ARDS stems from a communication breakdown between lung epithelial cells and immune cells and is a major factor in severe cases of respiratory infection, including COVID-19. Treatments for ALI/ARDS are currently limited by inadequate models of the complex relationships between signaling pathways and lung epithelial cell behavior. A core network of signaling pathways control epithelial structure and cytokine release and are an important unexploited point of intervention in ALI/ARDS via existing targeted kinase inhibitors or metabolic modulators. This core network includes NF-kB, a primary transcription factor for cytokine release and inflammation, and the kinases ERK, AKT, JNK, p38, mTOR and AMPK, which modulate cytokine production in response to cellular growth and metabolic status. Notably, this signaling network also plays a causal role in lung cancer and is involved in other diseases of the lung epithelium, including fibrosis and COPD. Our lab has developed an extensive live-cell technology platform for tracking temporal programs of signaling events – different patterns of timing, intensity, and coordination between these pathways – that govern epithelial cell fate decisions. Our overall objective is to quantitatively decode how cytokine secretion is specified by multi-kinase activity programs. Our hypothesis is that accounting for coinciding phases of signaling activity at the single cell level will significantly improve the prediction of both overall and local variability in cytokine secretion and will identify metabolic manipulations that reduce inflammation. Our approach will develop the technology needed to detect and manipulate temporal signaling programs to restore lung epithelial homeostasis in ALI/ARDS. Aim 1 will develop a high-content data-driven model correlating signaling programs to cytokine release on a cell-by-cell basis across multiple conditions. Aim 2 will develop the capacity to characterize the localized inflammatory environment in subregions of lung epithelium by examining the dynamic programs of kinase signaling in groups of 10-100 neighboring cells. In the process, we will provide proof of principle for using panels of fixed immunofluorescence markers to capture dynamic signaling patterns. Aim 3 will investigate the potential for manipulating temporal signaling programs with existing pharmacological agents, focusing on the emerging benefits of AMPK activators as a route to modulate the larger signaling network. Our multi-disciplinary team will work across the boundaries of pulmonary care, cell biology, and computational systems modeling to create a technology platform that connects modern single- cell signal transduction analysis to pressing challenges in lung disease. We anticipate that our project will identify potential new routes of intervention for ALI/ARDS, as well as revealing general concepts in cytokine signaling pathways that are broadly relevant for the treatment of lung malignancies and inflammatory disease.