Neutrophil Plasticity and H. pylori Pathogenesis

 描述（由申请人提供）：幽门螺杆菌是一种细菌病原体，可感染全球50%的人的胃粘膜，并可发展为消化性溃疡或胃癌，每年导致超过650，000人死亡。没有疫苗可用于预防或治疗H。幽门螺杆菌感染和抗生素耐药性是一个日益严重的问题，破坏了治疗效果。H.幽门螺杆菌感染是慢性、多形核白细胞（PMN，嗜中性粒细胞）主导的炎症反应。患者活检样本表明，中性粒细胞到达胃上皮上的粘液层，并在该部位吞噬大量细菌。然而，H.幽门螺杆菌不被杀死，并且NADPH氧化酶衍生的活性氧（ROS）释放到细胞外环境中损伤宿主组织。尽管中性粒细胞在H. pylori的发病机制，我们对细菌-中性粒细胞相互作用的理解是初步的。因此，我们进行了这项研究，以解决关键的知识差距，表型和命运的感染的中性粒细胞和他们的细菌货物。为此，我们创建了一系列同基因细菌突变体， 毒力因子单独或组合，还利用了最近的发现，这些发现彻底改变了我们对中性粒细胞在免疫应答中的作用的理解，如它们的免疫调节能力和在体内经历亚型分化的能力所示。我们的中心假设是H. pylori利用PMN表型可塑性作为其毒力策略的一部分。与此一致，我们提出了广泛的，令人信服的初步数据表明，H。幽门-嗜中性粒细胞相互作用比以前认识到的要复杂得多，并且定义了感染的三个不同阶段。在感染H. pylori通过以下方式逃避杀死 操纵吞噬体成熟和颗粒靶向。几小时后，诱导PMN亚型分化。同时，PMN凋亡显著受损，细胞寿命延长。大约3天后，受感染的PMN死亡，不是由于延迟的凋亡，而是由于非典型的死亡机制，支持强大的细胞外H。幽门螺杆菌生长在死亡的PMN尸体上和周围。为了验证这一感染模型，我们将从三个方面分析细菌的运输和脱粒，确定PMN表型和功能能力的变化，阐明H。pylori对PMN寿命和细胞死亡机制的影响，并开始确定主要细菌毒力因子在这些疾病方面的作用。使用的方法将包括但不限于超分辨率共聚焦显微镜、电子显微镜、RNA-Seq和使用Fluidigm微流体芯片的细胞因子产生的高通量分析。最后，我们还将确定临床批准的中性粒细胞凋亡诱导剂是否可以加速中性粒细胞死亡或破坏Hp生存，作为评估其治疗潜力的第一步。

 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.