Neutrophil hyperexocytosis and hypochlorous acid exposure in early cystic fibrosis lung disease

早期囊性纤维化肺病中的中性粒细胞胞吐过多和次氯酸暴露

• 批准号: 10586308
• 负责人: Joshua D Chandler
• 金额: $ 38.58万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586308
• 关键词: Acute; Address; Age; Anti-Inflammatory Agents; Antioxidants; Binding; Biological; Birth; Bronchiectasis; Caring; Cell Survival; Cells; Chemotactic Factors; Chlorides; Chlorine; Clinical; Clinical Research; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cystic Fibrosis sputum; Cytoplasmic Granules; Data; Defect; Disease; Disease Progression; Enzymes; Epithelial Cells; Epithelium; Event; Exhibits; Exocytosis; Experimental Designs; Generations; Glucose; Glutathione; Goals; Human; Hydrogen Peroxide; Hypochlorous Acid; Immunobiology; In Vitro; Incubated; Inflammation; Inflammatory; Intervention; Knowledge; Label; Leukotriene B4; Longevity; Lung; Lung diseases; Mediating; Membrane Proteins; Metabolic; Metabolism; Modeling; Molecular; Monitor; Mus; Nutrient; Organ; Oxidants; Oxidation-Reduction; Oxygen; Pathway interactions; Patients; Peroxidases; Pharmaceutical Preparations; Phenotype; Physiological; Prevalence; Production; Proteins; Proteomics; Pseudomonas aeruginosa; Pulmonary Cystic Fibrosis; Pulmonary Inflammation; Pulmonary Pathology; Reaction; Reactive Oxygen Species; Research; Resistance; Role; Sampling; Series; Signal Transduction; Specimen; Sputum; Stains; Staphylococcus aureus; Testing; Therapeutic; Thiocyanates; Tissues; VX-770; Validation; Vesicle; airway epithelium; airway inflammation; analytical method; antimicrobial drug; antioxidant therapy; autooxidation; cell injury; chlorination; clinical development; cystic fibrosis airway; cystic fibrosis airway epithelia; cystic fibrosis patients; cytotoxicity; design; detection method; drug development; early cystic fibrosis; experimental study; extracellular; extracellular vesicles; glucose production; healthspan; immunoregulation; improved; inflammatory lung disease; inhibitor; lung injury; metabolomics; methionine sulfoxide; migration; mortality; mutant; neutrophil; next generation; novel; oxidation; pathogen; pharmacologic; rational design; research and development; response; standard of care; targeted biomarker; targeted treatment; trafficking; translational model

PROJECT SUMMARY / ABSTRACT Current standard of care for cystic fibrosis (CF) does not include drugs unequivocally effective at curbing airway inflammation, in contrast to crucial gains made in correcting defects in the CF transmembrane conductance regulator (CFTR) via highly effective modulator therapy (HEMT) and an array of antimicrobial drugs. Effectively treating exuberant neutrophil-dominated inflammation in CF, particularly in the earliest stages of the disease, is a critical goal because doing so would increase patient lifespan and health span. This project is designed to test the hypothesis that neutrophil hyperexocytosis, which occurs in CF from a very young age, is a critical event that releases active myeloperoxidase (MPO) from neutrophil granules and enables the generation of extracellular hypochlorous acid (HOCl), a strong and promiscuous oxidant that can damage cells. We further hypothesize that the released MPO becomes associated with extracellular vesicles (EVs), providing MPO with sustained metabolism to fuel its HOCl-generating activity and conferring resistance to inhibitors. Ultimately, we hypothesize that increased airway HOCl injures airway epithelial cells (AECs) and promotes maladaptive cellular responses that contribute to bronchiectasis. Research from our group has shown that MPO is active in CF airways from the earliest stages of disease and is associated with initial manifestations of bronchiectasis. Furthermore, we have identified molecular products of HOCl exposure, such as methionine sulfoxide, that can be monitored by LC-MS across a broad range of biological specimens, including basic and translational models and clinical samples. Using state-of-the-art LC-MS, rigorous cell-, protein-, and EV- detection methods, highly translational models of neutrophils and EVs, and clinical sample validation, this project sets out a series of parallel experiments designed to identify the sequence of events leading from neutrophil transmigration into CF airways, to active MPO release and extracellular HOCl generation, to CF AEC injury and maladaptation to the potent oxidative insult. These experiments are enumerated in the following Specific Aims: (1) Determine the impact of hyperexocytosis on extracellular HOCl and innate defense; (2) Determine mechanisms of granule-releasing, immunomodulatory, and metabolically active (GRIM) neutrophil EV-based HOCl production; and (3) Determine mechanisms of CF airway epithelial cytotoxicity mediated by MPO and HOCl. Furthermore, these experiments account for potential impacts of novel HEMT therapy elexacaftor-tezacaftor-ivacaftor (ETI) in inflammatory pathways by comparing CF sputum samples with HEMT to the same samples from donors not receiving ETI. The ultimate goal of the proposed studies is to generate knowledge informing clinical and drug development research and forestall CF lung disease progression at the earliest stages and before end organ damage. Furthermore, owing to the prevalence of GRIM neutrophils in a number of lung diseases beyond CF, information pertinent to other lung diseases including neutrophil inflammation will also be gained by the completion of this project.

项目总结/摘要 目前囊性纤维化（CF）的护理标准不包括明确有效抑制 气道炎症，与纠正CF跨膜缺陷的关键进展相反， 电导调节器（CFTR）通过高效调制器疗法（HEMT）和抗微生物 毒品有效治疗CF中旺盛的嗜中性粒细胞为主的炎症，特别是在早期阶段 这是一个关键的目标，因为这样做可以延长患者的寿命和健康寿命。这个项目 旨在检验中性粒细胞分泌过多的假设，这发生在CF从很小的年龄， 是从中性粒细胞颗粒释放活性髓过氧化物酶（MPO）并使 细胞外次氯酸（HOCl）的产生，一种可以损伤细胞的强且混杂的氧化剂。 我们进一步假设释放的MPO与细胞外囊泡（EV）相关， MPO具有持续的代谢，以促进其HOCl生成活性并赋予对抑制剂的抗性。 最后，我们假设增加的气道HOCl损伤气道上皮细胞（AEC）并促进气道上皮细胞的增殖。 导致支气管扩张的适应不良的细胞反应。我们小组的研究表明， MPO从疾病的最早阶段开始在CF气道中活跃，并且与肺纤维化的初始表现相关。 支气管扩张此外，我们已经确定了HOCl暴露的分子产物，如蛋氨酸 亚砜，可以通过LC-MS对广泛的生物样本进行监测，包括基础和 转化模型和临床样本。使用最先进的LC-MS，严格的细胞，蛋白质和EV- 检测方法，中性粒细胞和EV的高度翻译模型，以及临床样本验证， 该项目提出了一系列平行实验，旨在确定事件的顺序， 中性粒细胞迁移到CF气道，激活MPO释放和细胞外HOCl生成，CF AEC损伤和对强氧化损伤的适应不良。这些实验列举在 以下具体目的：（1）确定过度分泌对细胞外HOCl和先天性HOCl的影响。 （2）确定颗粒释放，免疫调节和代谢活性（GRIM）的机制 嗜中性粒细胞基于EV的HOCl产生;和（3）确定CF气道上皮细胞毒性的机制 通过MPO和HOCl介导。此外，这些实验解释了新型HEMT的潜在影响 通过比较CF痰液样本和 将HEMT与来自未接受ETI的供体的相同样品进行比较。拟议研究的最终目标是 为临床和药物开发研究提供信息，并预防CF肺病 在最早阶段和终末器官损伤之前进展。此外，由于 在CF以外的许多肺部疾病中的GRIM中性粒细胞，与其他肺部疾病相关的信息 包括中性粒细胞炎症也将通过该项目的完成而获得。