Lung-derived complement in pneumonia

肺炎中的肺源性补体

基本信息

批准号：
10568567
负责人：
Hrishikesh Satish Kulkarni
金额：
$ 54.37万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-19 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10568567
关键词：
Acute Acute Pneumonia Address Anabolism Area Attenuated Bacteremia Bacteria Bacterial Infections Bacterial Pneumonia Binding Biochemical Biological Assay Bronchopneumonia CRISPR screen Cause of Death Cell Death Cell Death Induction Cell Survival Cell secretion Cells Cessation of life Circulation Clustered Regularly Interspaced Short Palindromic Repeats Complement Complement 3 Convertase Complement 3a Complement 3b Complement Activation Complement Factor B Cytoprotection Data Deposition Enzymes Epithelial Cells Epithelium Gene Delivery Genetic Goals Host Defense Host Defense Mechanism Human Immune response In Vitro Individual Infection Inflammatory Response Knock-out Knowledge Libraries Ligands Liquid substance Liver Lung Mediating Modeling Molecular Morbidity - disease rate Mucous Membrane Mus National Heart, Lung, and Blood Institute Pathway interactions Pattern Plasmids Pneumonia Predisposition Proteins Proteomics Pseudomonas aeruginosa Pseudomonas aeruginosa infection Pseudomonas aeruginosa pneumonia Reporting Research Research Priority Role Severities Site Source Stress Structure-Activity Relationship Surface Testing Tissues Transfection Transgenic Mice Work airway epithelium alveolar epithelium arm cell injury cellular imaging complement system design gene therapy human model humoral immunity deficiency improved in vivo knowledge integration lung injury mortality mouse model mutant novel novel therapeutics overexpression oxidant stress pathogen pathogenic bacteria pharmacologic pneumonia treatment protective effect protein transport pulmonary function receptor resilience uptake working group

项目摘要

PROJECT ABSTRACT The objective of this R01 proposal is to investigate the function of lung-derived complement proteins and harness their activities to mitigate the severity of pneumonia. Complement proteins C3 and Factor B (FB) comprise an early arm of the host immune response. They are primarily derived from the liver and function in the circulation by killing pathogens such as bacteria. However, our recent work has demonstrated the importance of local C3 expression in lung epithelial cell survival during stress. This proposal focuses on an emerging role for lung- derived FB, a ligand for C3, in reducing excessive tissue damage in the setting of an acute bacterial pneumonia. Our goal is to determine how FB promotes pulmonary host defense. We will investigate sources of FB in the lung and establish its putative protective effect relative to C3. A major hurdle for investigating tissue-specific roles of complement has been the limited availability of models and assays. We have developed novel transgenic mouse models and functional assays that distinguish the roles of liver- and lung-derived complement proteins, and specifically identify the role of lung-derived FB in pneumonia. Our mouse models are supplemented with data from in vitro human models that demonstrate the role of FB to protect against stress-induced epithelial cell death. Additionally, CRISPR-induced deletion of cell-derived complement proteins suggests active internalization of exogenous complement proteins. These combined results support our central hypothesis that lung-derived FB promotes host defense by mitigating epithelial cell death. This proposal will test our hypothesis by achieving two Specific Aims. Aim 1 compares global FB-deficient, targeted liver FB-deficient, and lung epithelial cell-derived FB-deficient mice to assess how lung-derived FB mitigates acute bronchopneumonia severity and cell death. We also will assess if augmenting FB in the lung using pharmacological and gene delivery approaches protects against pneumonia. Aim 2 analyzes whether lung-derived, intracellular FB activity mitigates cell death in vitro by leveraging a combination of human primary lung epithelial cells and FB-deficient cells to dissect the molecular and biochemical mechanisms responsible for complement function in the lung. The proposal integrates knowledge of pulmonary complement activation, intracellular complement protein trafficking, and structure-function relationships with gene therapy, cell imaging, proteomics, and CRISPR screens to determine how lung-derived FB promotes epithelial cytoprotection during stress. These approaches are independent but complementary for investigating the immunobiological role of lung-derived FB in mucosal barrier protection of the lung. The proposed work is important because understanding how the early host immune response modulates tissue damage is essential for designing and implementing urgently needed, new therapies for pneumonia. Thus, we will assess how lung-derived FB facilitates host defense at the site of infection and promotes tissue resilience. These efforts will help our long-term goal of developing a novel host-focused therapy for pneumonia, thus aligning with a priority area of the NHLBI Working Group Report on pneumonia research.

项目摘要该R01提案的目的是研究肺衍生的补体蛋白质和线束的功能他们的活动以减轻肺炎的严重性。补充蛋白质C3和因子B（FB）包括宿主免疫反应的早期手臂。它们主要源自肝脏，并在循环中功能通过杀死病原体，例如细菌。但是，我们最近的工作证明了本地C3的重要性应激期间肺上皮细胞存活中的表达。该提案着重于肺的新兴作用在急性细菌性肺炎的情况下，衍生的Fb是C3的配体，可减少组织过多的组织损伤。我们的目标是确定FB如何促进肺部宿主防御。我们将研究FB的来源肺并建立相对于C3的推定保护作用。研究组织特异性的主要障碍补充的作用是模型和测定的有限供应。我们已经开发了新颖的转基因小鼠模型和功能测定法，区分肝和肺部补体蛋白的作用，并特别识别肺衍生的FB在肺炎中的作用。我们的鼠标模型补充了来自体外人类模型的数据证明了FB在防止应力诱导的上皮细胞中的作用死亡。此外，CRISPR诱导的细胞来源补体蛋白缺失表明活跃外源补体蛋白质的内在化。这些结合的结果支持我们的中心假设肺衍生的FB通过减轻上皮细胞死亡来促进宿主防御。该建议将测试我们的通过实现两个具体目标来假设。 AIM 1比较全球FB缺陷，靶向肝FB缺陷和肺上皮细胞衍生的FB缺陷小鼠，以评估肺衍生的FB如何减轻急性支气管肿瘤严重性和细胞死亡。我们还将评估使用药理学和基因递送在肺中增加FB 接近可预防肺炎。 AIM 2分析肺衍生的细胞内FB活性是否会减轻通过利用人类原发性肺上皮细胞和FB缺陷细胞的组合来在体外死亡剖析负责肺部补体功能的分子和生化机制。这建议整合了肺补体激活，细胞内补体蛋白运输的知识，与基因疗法，细胞成像，蛋白质组学和CRISPR筛选的结构功能关系确定肺衍生的FB如何促进应激期间的上皮细胞保护。这些方法是独立但互补的，用于研究肺源性FB在粘膜屏障中的免疫生物学作用保护肺。拟议的工作很重要，因为了解早期宿主免疫如何响应调节组织损伤对于急需需要的新疗法至关重要对于肺炎。因此，我们将评估肺衍生的FB如何在感染部位促进宿主防御和促进组织弹性。这些努力将有助于我们的长期目标，即开发一种以宿主为中心的新型疗法对于肺炎，因此与NHLBI工作组的优先区域保持一致。