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院内肺炎最常见的病原菌 病人。肺炎克雷伯氏菌(Kp)在世界范围内日益突出，日益增多。 抗生素耐药性、超强毒力菌株和侵袭性临床症状的流行。寄主机制 负责有效清除易感宿主肺部KP感染的防御措施仍不清楚。 顺式附子酸脱羧酶1(Acod1)是一种在小鼠和人肺泡内强效诱导的线粒体酶 催化衣康酸产生的巨噬细胞。依他康酸通过抑制炎症反应 提出了一种NRF2依赖的机制。这项提议的目标是定义髓系衍生细胞的作用 衣康酸在肺炎克雷伯菌感染时的肺宿主防御中的作用及其对局部感染的保护作用 组织损伤。在以KP为病原体的肺炎小鼠模型中，我们已经证明Acod1缺乏 与存活率下降和细菌传播增加有关。我们还证明了衣康酸 内质网耗竭降低细胞综合应激反应(ISR)途径关键蛋白的表达 压力，这表明它可能调节这一途径。我们的建议将针对以下具体目标：1) 确定髓系细胞来源的衣康酸是否限制局部KP感染并预防肺外脓毒症； 2)确定衣康酸是否通过ISR保护宿主，并减少细胞损伤 KP感染。我们假设Acod1，从而衣康酸的生产，对脓毒症和 在这个模型中相关的组织损伤，并增加宿主的韧性。我们还预计巨噬细胞来源的 衣康酸在体外和体内均可增强巨噬细胞和肺上皮细胞对ER应激的保护作用。 这些研究将利用体外和翻译工具，包括体内肺内KP感染模型 利用野生型和Acod1-/-小鼠、骨髓嵌合体、RAW 264.7巨噬细胞系(野生型和 Acod1-/-)、人和小鼠精密切割的肺组织切片和人肺泡巨噬细胞，以解决我们的 研究问题。此外，我们还将对受影响的组织进行转录和代谢组学分析。 在播散性KP感染期间，免疫表型髓系亚群，并进行互补细胞生物学 在衣康酸存在或不存在的情况下检测综合应激反应通路关键靶点的研究 在髓系细胞和上皮细胞中。这项工作将阐明新的免疫机制，可能被利用来 降低与这种病原体相关的死亡率。该提案纳入了一个强大的导师团队，由Dr。 珍妮特·李担任急性肺损伤卓越中心主任，并制定了稳健的职业发展计划。 通过K08职业发展奖支持这项提议将为成功和 作为一名肺部和重症监护医学的内科科学家，候选人的独立性。

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.