Project 2: Genes required for dendritic cell responses to pathogens and T cells

哺乳动物先天免疫系统的树突状细胞(DC)负责检测微生物 遭遇以及炎症和获得性免疫的启动。树突状细胞在宿主生理学中的核心作用 引起了人们的极大关注，这些细胞是积极调查的对象。尽管有人赞赏 尽管DC的重要性，但我们对其机制的了解仍然存在巨大和根本的差距 监管。例如，树突状细胞编码几种检测细菌脂多糖(LPS)的受体，但 只有一个(TLR4)的功能得到了广泛的研究。我们和其他人最近的研究发现 内毒素受体CD14和caspase-11能够诱导新的信号通路 TLR4上游(CD14)或与TLR4平行(caspase-11)。CD14、TLR4和TLR4的集体行动 Caspase-11在DC激活和宿主防御中起重要作用，但其作用机制较差。 已定义。因此，在我们的知识中存在着很大的空白来解释DC与 细菌。我们也缺乏对DC在感染后期如何与T细胞相互作用的了解，但它是 显然，这些相互作用导致两种细胞类型的活动发生了深刻的变化。大部分的研究 在这些相互作用中，重点是DC如何促进T细胞活动的变化。相比之下，我们有一个 对T细胞如何影响树突状细胞活性的了解很少。在此应用程序中，我们建议填充这些 通过在小鼠中对新的调节因子进行正向遗传筛选，我们对DC生物学的了解存在差距。 基于CRISPR的技术在基因组编辑方面的最新进展为这一方法提供了便利。一个 基因发现的管道将通过使用已建立的和新兴的基于FACS的 化验。这些检测方法将在体内和体外用于鉴定缺乏DC调节因子的DC 与微生物或T细胞的相互作用。所有屏幕将取决于 与此U19应用程序相关的信息学、鼠标扰动和CRISPR库核心。后续 候选调节蛋白的功能分析将与其他研究人员合作进行 在这笔赠款上。这些努力的累积结果将是一系列新的基因集，它们应该定义 解释DC生物学的许多方面的途径和过程，因为它们与宿主防御有关。

The dendritic cells (DCs) of the mammalian innate immune system are responsible for detection of microbial encounters and the initiation of inflammation and adaptive immunity. This central role of DCs in host physiology has attracted much attention, and these cells are the subject of active investigation. Despite the appreciation of the importance of DCs, there remain large and fundamental gaps in our knowledge of their mechanisms of regulation. For instance, DCs encode several receptors that detect bacterial lipopolysaccharides (LPS), yet the functions of only one (TLR4) have been examined extensively. Recent work by us and others have identified the LPS receptors CD14 and caspase-11 as being capable of inducing novel signaling pathways that proceed either upstream of TLR4 (CD14) or in parallel to TLR4 (caspase-11). The collective actions of CD14, TLR4 and caspase-11 are important for DC activation and host defense, yet their mechanisms of action are poorly defined. Thus, significant gaps in our knowledge exist to explain the earliest stages of DC interactions with bacteria. We also lack an understanding of how DCs interact with T cells at later stages of infection, yet it is clear that these interactions lead to profound changes in the activities of both cell types. Much of the research into these interactions has focused on how DCs promote changes in T cell activities. In contrast, we have a minimal understanding of how T cells influence the activity of DCs. In this application, we propose to fill these gaps in our knowledge of DC biology through forward genetic screening for novel regulatory factors in mice. This approach is facilitated by recent advances in genome editing provided by CRISPR-based technologies. A pipeline of gene discovery will be generated through the use of established and emerging FACS-based assays. These assays will be used in vivo and ex vivo to identify DCs that are deficient for regulators of DC interactions with microbes or T cells. All screens will depend on significant interactions between the informatics, mouse perturbation and CRISPR library Cores associated with this U19 application. Subsequent functional analysis of candidate regulatory proteins will be performed in collaboration with other investigators on this grant. The cumulative result of these efforts will be a series of novel gene sets that should define pathways and processes that explain numerous aspects of DC biology as they relate to host defense.