Functions of TBK1 in neutrophils during bacterial pneumonia

项目摘要 尽管有抗生素，细菌性肺炎在世界范围内仍会导致严重的发病率和死亡率。 免疫系统必须平衡清除病原体的相互竞争的要求，同时避免局部 器官受损。对于限制伤害或促进治疗的宿主导向疗法的需求仍然没有得到满足 在不影响病原体控制的情况下实现分辨率。中性粒细胞构成对细菌性肺炎的初始反应 并通过直接杀灭细菌和产生包括趋化因子在内的介质来控制感染， 细胞因子、蛋白酶和活性氧(ROS)。本R03应用程序旨在定义新的功能 肺炎期间中性粒细胞中的天然免疫激酶坦克结合蛋白1(TBK1)。TBK1促销 通过磷酸化干扰素调节转录因子表达1型干扰素 在病毒感染过程中，其家族(IRF)发挥作用，但其在细菌性肺炎和中性粒细胞中的作用尚不清楚。 我的K08奖研究了在流感感染期间，TBK1如何在单核细胞和巨噬细胞中发挥作用。 来自该项目和我们之前发表的数据显示，在肺炎链球菌肺炎期间，肺 中性粒细胞上调编码TBK1及其附属蛋白的基因，但不表达常见的 下游产物，1型干扰素，表明中性粒细胞以一种独特的方式使用TBK1信号模块。 我们发现，在肺炎链球菌肺炎中，中性粒细胞功能的多个方面都需要TBK1， 包括多种细胞因子的阐述、活性氧(ROS)的产生和细菌 通行证。我们假设中性粒细胞TBK1调节IRF依赖和IRF非依赖 转录程序以及其他细胞程序，如新陈代谢。建议的目标 专注于确定中性粒细胞中TBK1信号的细胞和分子细节，以响应感染 肺炎链球菌。在目标1中，我们将确定TBK1在肺中性粒细胞中驱动的转录程序， 通过评估转录因子靶标的表达来具体表征1)转录因子的激活 基因和2)调节代谢过程的基因的表达。在目标2中，我们将确定如何 Tbk1促进中性粒细胞ROS的产生，主要集中在NADPH氧化酶组装和中性粒细胞 新陈代谢。我们将评估肺中性粒细胞的葡萄糖摄取、自噬和线粒体功能。 WT和TBK1 KO小鼠肺炎模型的建立。建议的研究将探讨 该药的最终目标是改善这种常见形式的肺损伤患者的预后。这些 与我的K08奖不同的是，研究的重点是中性粒细胞和细菌性肺炎及其需求 对于我不熟悉的技术。他们将增加我参与研究计划的广度和深度 既有髓系细胞也有病毒和细菌病原体。

Project Summary Bacterial pneumonia causes significant morbidity and mortality worldwide despite the availability of antibiotics. The immune system must balance the competing requirements of clearing the pathogen while avoiding local organ damage. There remains an unmet need for host-directed therapeutics that limit injury or promote resolution without impairing pathogen control. Neutrophils constitute the initial response to bacterial pneumonia and control infection through direct killing of bacteria and production of mediators including chemokines, cytokines, proteases and reactive oxygen species (ROS). This R03 application aims to define novel functions for the innate immune kinase TANK-binding kinase 1 (TBK1) in neutrophils during pneumonia. TBK1 promotes the expression of type 1 interferons (IFN) by phosphorylating transcription factors from the interferon regulatory family (IRF) during viral infection, but its functions in bacterial pneumonia and in neutrophils remain undefined. My K08 Award investigates how TBK1 functions in monocytes and macrophages during influenza infection. Data from that project and from our previous publication show that during S. pneumoniae pneumonia, lung neutrophils upregulate genes coding for TBK1 and its accessory proteins, but fail to express the common downstream product, type 1 IFN, suggesting that neutrophils use the TBK1 signaling module in a unique way. We find that in S. pneumoniae pneumonia, TBK1 is required for multiple aspects of neutrophil function, including elaboration of multiple cytokines, production of reactive oxygen species (ROS), and bacterial clearance. We hypothesize that neutrophil TBK1 regulates both IRF-dependent and IRF-independent transcriptional programs as well as other cellular programs such as metabolism. The proposed aims focus on identifying cellular and molecular details of TBK1 signaling in neutrophils in response to infection by S. pneumoniae. In Aim 1, we will determine the transcriptional programs driven by TBK1 in lung neutrophils, characterizing in particular 1) the activation of transcription factors by assessing the expression of their target genes and 2) the expression of genes that regulate metabolic processes. In Aim 2, we will determine how TBK1 promotes neutrophil ROS production, with a focus on NADPH oxidase assembly and neutrophil metabolism. We will assess glucose uptake, autophagy, and mitochondrial function in lung neutrophils from WT and TBK1 KO mice in the setting of pneumonia. The proposed studies will explore new functions for the kinase TBK1 with the ultimate goal of improving patient outcomes in this common form of lung injury. These studies diverge from my K08 Award through their focus on neutrophils and bacterial pneumonia and their need for technologies unfamiliar to me. They will increase the breadth and depth of my research program to engage both myeloid cell lineages and both viral and bacterial pathogens.