Neutrophil Plasticity and H. pylori Pathogenesis

中性粒细胞可塑性和幽门螺杆菌发病机制

• 批准号: 9109153
• 负责人: Lee-Ann H Allen
• 金额: $ 17.47万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-11 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9109153
• 关键词: Accounting; Activities of Daily Living; Address; Antibiotic Resistance; Apoptosis; Bacteria; Biochemical; Biopsy Specimen; Carcinogens; Cell Death; Cells; Cessation of life; Chronic; Coculture Techniques; Collection; Complex; Confocal Microscopy; Cytoplasmic Granules; DNA; Data; Development; Disease; Electron Microscopy; Enzymes; Epithelium; Evaluation; Gastric mucosa; Gastritis; Gene Expression; Goals; Growth; Health; Helicobacter Infections; Helicobacter pylori; Hour; Human; Immune response; Infection; Inflammatory Response; Inhibition of Apoptosis; Knowledge; Link; Locales; Longevity; Malignant neoplasm of lung; Methods; Microfluidic Microchips; Modeling; Molecular; Mucous body substance; NADPH Oxidase; Pathogenesis; Pathway interactions; Patients; Peptic Ulcer; Phagosomes; Phenotype; Process; Production; Proteins; Reactive Oxygen Species; Resolution; Role; Staging; Stomach; T-Lymphocyte; Testing; Therapeutic; Time; Tissues; Treatment Efficacy; Vaccines; Variant; Virulence; Virulence Factors; arginase; base; cytokine; extracellular; high throughput analysis; in vivo; killings; malignant stomach neoplasm; mutant; neutrophil; novel; novel therapeutics; pathogen; prevent; roscovitine; trafficking; transcriptome sequencing

 DESCRIPTION (provided by applicant): Helicobacter pylori is a bacterial pathogen that infects the gastric mucosa of 50% of humans worldwide and elicits gastritis that can progress to peptic ulcers or gastric cancer, which accounts for more than 650,000 deaths each year. No vaccine is available to prevent or treat H. pylori infection, and antibiotic resistance is an ever-increasing problem that undermines treatment efficacy. A distinguishing feature of H. pylori infection is the chronic, polymorphonuclear leukocyte (PMN, neutrophil)-dominant inflammatory response. Patient biopsy samples demonstrate that PMNs reach the mucus layer over the gastric epithelium and engulf large numbers of bacteria in this locale. However, H. pylori is not killed and NADPH oxidase-derived reactive oxygen species (ROS) released into the extracellular milieu damage host tissue. Despite the central role of neutrophils in H. pylori pathogenesis, our understanding of bacteria-PMN interactions is rudimentary. Thus, we undertook this study to address critical knowledge gaps regarding the phenotype and fate of infected PMNs and their bacterial cargo. To this end, we created a collection of isogenic bacterial mutants that lack major virulence factors alone or in combination, and also exploited recent discoveries that have revolutionized our understanding of the role of neutrophils in the immune response, as indicated by their immunomodulatory capacity and ability to undergo subtype differentiation in vivo. Our central hypothesis is that H. pylori exploits PMN phenotypic plasticity as part of its virulence strategy. Consistent with this, we present extensive, convincing preliminary data to suggest that H. pylori-neutrophil interactions are significantly more complex than previously appreciated, and which define three distinct stages of infection. During early infection H. pylori evades killing by manipulation of phagosome maturation and granule targeting. This is followed a few hours later by induction of PMN subtype differentiation. In parallel, PMN apoptosis is significantly impaired, and cell lifespan is prolonged. After about 3 days infected PMNs succumb, not by delayed apoptosis, but rather by an atypical mechanism of death that supports robust extracellular H. pylori growth upon and around dying PMN carcasses. To test this infection model we will in three specific aims analyze bacterial trafficking and degranulation, define changes in PMN phenotype and functional capacity, elucidate the effects of H. pylori on PMN lifespan and mechanism of cell death, and begin to determine the roles of major bacterial virulence factors in these aspects of disease. Methods utilized will include but are not limited to super-resolution confocal microscopy, electron microscopy, RNA-Seq, and high-throughput analysis of cytokine production using Fluidigm microfluidic chips. Finally, we will also determine if clinically approve PMN apoptosis-inducing agents can accelerate PMN death or undermine Hp survival as a first step toward evaluation of their therapeutic potential.

 描述（由应用提供）：幽门螺杆菌是一种细菌病原体，可感染全球50％的人类胃粘膜，并引起胃胃癌，可以发展为消化性溃疡或胃癌，每年占死亡人数超过650,000。没有可用于预防或治疗幽门螺杆菌感染的疫苗，抗生素耐药性是一个不断增加的问题，会破坏治疗效率。幽门螺杆菌感染的区别是慢性，多形核白细胞（PMN，中性粒细胞）炎性反应。患者活检样品表明，PMN在该地区的胃皮细胞上到达粘液层，并吞噬了大量细菌。然而，幽门螺杆菌未被杀死，氧化氧化物衍生的活性氧（ROS）释放到细胞外环境破坏宿主组织中。尽管中性粒细胞在幽门螺杆菌发病机理中的核心作用，但我们对细菌-PMN相互作用的理解是基本的。因此，我们进行了这项研究，以解决有关感染PMN及其细菌货物的表型和命运的关键知识差距。为此，我们创建了一个缺乏主要的等源细菌突变体的集合 单独或合并毒力因素，还探讨了最近的发现，这些发现彻底改变了我们对中性粒细胞在免疫响应中的作用的理解，如它们的免疫调节能力和体内经历亚型分化的能力所表明的那样。我们的中心假设是，幽门螺杆菌利用PMN表型可塑性作为其病毒策略的一部分。与此相一致，我们提供了广泛的，令人信服的初步数据，以表明幽门螺杆菌中的嗜性相互作用明显比以前所欣赏的要复杂得多，并且定义了三个不同的感染阶段。在早期感染期间，幽门螺杆菌逃避了 操纵吞噬体成熟和颗粒靶标。几个小时后，通过诱导PMN亚型分化。同时，PMN的凋亡显着受损，细胞寿命延长。大约3天后感染了PMN，而不是通过延迟细胞凋亡，而是通过非典型的死亡机制，该机制支持垂直的PMN尸体上及其周围的幽默细胞外幽门螺杆菌生长。为了测试这种感染模型，我们将在三个特定的目的中分析细菌的运输和脱粒化，定义PMN表型和功能能力的变化，阐明幽门螺杆菌对PMN寿命和细胞死亡机制的影响，并开始确定主要细菌病毒因子在这些疾病的这些方面的作用。所使用的方法将包括但不限于使用流体微流体芯片对细胞因子产生的超分辨率共聚焦显微镜，电子显微镜，RNA-Seq和高通量分析。最后，我们还将确定临床批准的PMN凋亡诱导的药物是否可以加速PMN死亡或破坏HP存活，这是评估其治疗潜力的第一步。