Intrapulmonary itaconate as a host-protective metabolite during ER Stress and Klebsiella pneumoniae infection

项目摘要 革兰氏阴性菌是危重病患者医院获得性肺炎最常见的病原体 患者特别是肺炎克雷伯氏菌（KP）随着肺炎克雷伯氏菌（KP）的增加而在世界范围内变得突出。 抗生素耐药性、高毒力菌株和侵袭性临床综合征的流行。宿主机制 在易感宿主中，负责有效清除肺KP感染的防御机制仍不清楚。 顺乌头酸脱羧酶1（Acod 1）是一种在小鼠和人肺泡上皮细胞中强烈诱导的线粒体酶， 巨噬细胞催化衣康酸的产生。衣康酸通过抑制炎症反应， 一个建议的Nrf 2依赖机制。该提案的目的是明确骨髓源性 衣康酸在KP感染期间肺宿主防御中的作用，并确定它如何保护局部 组织损伤在使用KP作为病原体的肺炎小鼠模型中，我们已经表明Acod 1缺陷 与存活率降低和细菌传播增加有关。我们还发现衣康酸盐 消耗减少ER期间细胞整合应激反应（ISR）途径的关键蛋白的表达 压力，这表明它可以调节这一途径。我们的建议将针对以下具体目标：1） 确定骨髓细胞衍生的衣康酸盐是否限制局部KP感染并防止肺外败血症; 和2）为了确定衣康酸盐是否通过ISR具有宿主保护性，并减少细胞损伤， KP感染。我们假设Acod 1和衣康酸的产生对脓毒症有保护作用， 相关的组织损伤，并增加主机的弹性。我们还预测巨噬细胞来源的 衣康酸盐在体外和体内增加巨噬细胞和肺上皮细胞对ER应激的保护。 这些研究将利用体外和转化工具，包括体内肺内KP感染模型 使用野生型和Acod 1-/-小鼠、骨髓嵌合体、RAW 264.7巨噬细胞系（野生型和 Acod 1-/-）、人和小鼠精确切割的肺组织切片以及人肺泡巨噬细胞来解决我们的 研究问题。此外，我们将在受影响的组织中进行转录组学和代谢组学分析。 在播散性KP感染期间，免疫表型骨髓亚群，并执行互补细胞生物学 在存在或不存在衣康酸盐的情况下，研究整合应激反应途径的关键靶标 在骨髓和上皮细胞中。这项工作将阐明新的免疫机制，可利用 降低与该病原体相关的死亡率。该提案包括一个强大的导师团队，由博士领导。 Janet Lee担任急性肺损伤卓越中心主任，并制定了强大的职业发展计划。 通过K 08职业发展奖支持这一提案将为成功奠定基础， 独立的候选人作为一个医生，科学家在肺部和重症监护医学。

PROJECT ABSTRACT Gram negative bacteria are the most common pathogens implicated in nosocomial pneumonia in critically ill patients. Klebsiella pneumoniae (KP) in particular has grown in prominence worldwide with increasing prevalence of antibiotic resistance, hypervirulent strains, and invasive clinical syndromes. Mechanisms of host defense responsible for effective clearance of KP infection from the lung in susceptible hosts are still unclear. Cis-aconitate decarboxylase 1 (Acod1) is a mitochondrial enzyme robustly induced in murine and human alveolar macrophages that catalyzes the production of itaconate. Itaconate suppresses inflammatory responses through a proposed Nrf2-dependent mechanism. The goal of this proposal is to define the role of myeloid-derived itaconate in pulmonary host defense during KP infection and to determine how it confers protection from local tissue injury. In a murine model of pneumonia using KP as the pathogen, we have shown that Acod1 deficiency is associated with decreased survival and increased bacterial dissemination. We have also shown that itaconate depletion reduces expression of key proteins of the cellular integrated stress response (ISR) pathway during ER stress, suggesting that it may regulate this pathway. Our proposal will address the following specific aims: 1) To determine if myeloid cell-derived itaconate limits local KP infection and protects against extrapulmonary sepsis; and 2) To determine whether itaconate is host-protective through the ISR and decreases cellular injury during KP infection. We hypothesize that Acod1, and thus itaconate production, is protective against sepsis and associated tissue injury in this model and increases host resilience. We also anticipate that macrophage-derived itaconate increases protection from ER stress both in macrophages and lung epithelial cells in vitro and in vivo. These studies will utilize in vitro and translational tools including an in vivo intrapulmonary KP infection model using wild-type and Acod1-/- mice, bone marrow chimeras, RAW 264.7 macrophage cell lines (wild-type and Acod1-/-), human and mouse precision-cut lung tissue slices, and human alveolar macrophages to address our research questions. In addition, we will perform transcriptomic and metabolomic profiling across affected tissues during disseminated KP infection, immunophenotype myeloid subsets, and execute complementary cell biology studies to examine key targets of the integrated stress response pathway in the presence or absence of itaconate in myeloid and epithelial cells. This work will elucidate novel immune mechanisms that may be exploited to reduce mortality associated with this pathogen. The proposal incorporates a strong mentorship team led by Dr. Janet Lee as Director of the Acute Lung Injury Center of Excellence and a robust career development plan. Support of this proposal with a K08 Career Development Award will establish a foundation for success and independence for the candidate as a physician-scientist in pulmonary and critical care medicine.