Neutrophil Plasticity and H. pylori Pathogenesis

 描述(申请人提供)：幽门螺杆菌是一种细菌病原体，感染全球50%的人的胃粘膜，并引起胃炎，可发展为消化性溃疡或胃癌，每年导致超过65万人死亡。目前还没有预防或治疗幽门螺杆菌感染的疫苗，而且抗生素耐药性是一个日益严重的问题，破坏了治疗效果。幽门螺杆菌感染的一个显著特征是以中性粒细胞(PMN)为主的慢性炎症反应。患者的活检样本显示，中性粒细胞到达胃上皮上的粘液层，并吞噬了该场所的大量细菌。然而，幽门螺杆菌不会被杀死，NADPH氧化酶衍生的活性氧物种(ROS)释放到细胞外环境中损害宿主组织。尽管中性粒细胞在幽门螺杆菌的发病机制中起着核心作用，但我们对细菌-中性粒细胞相互作用的了解还很初级。因此，我们开展了这项研究，以解决关于受感染的PMN及其细菌载体的表型和命运的关键知识空白。为此，我们创建了一组缺乏主要基因的同基因细菌突变体 毒力因子单独或联合使用，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反应中的作用的理解，其免疫调节能力和在体内经历亚型分化的能力表明了这一点。我们的中心假设是，幽门螺杆菌利用PMN的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，表明幽门螺杆菌与中性粒细胞的相互作用比以前认识到的要复杂得多，并定义了三个不同的感染阶段。在早期感染期间，幽门螺杆菌通过 吞噬小体成熟和颗粒靶向的操作。几小时后，诱导PMN亚型分化。同时，PMN的凋亡显著受损，细胞寿命延长。大约3天后，感染的PMN屈服，不是通过延迟的凋亡，而是通过一种非典型的死亡机制，这种死亡机制支持在死亡的PMN身体上和周围强劲的细胞外幽门螺杆菌生长。为了验证这一感染模型，我们将从三个特定的目标分析细菌的运输和脱颗粒，定义PMN的表型和功能的变化，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微流控芯片对细胞因子生产的高通量分析。最后，我们还将确定临床批准的PMN凋亡诱导剂是否可以加速PMN的死亡或破坏HP的存活，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.