The role of surface-bound ectonucleotidase CD73 in modulation of Porphyromonas gingivalis infection in gingival epithelial cells

项目总结/摘要 危险信号分子细胞外ATP（eATP）由感染细胞释放并代谢为 腺苷通过多个酶促步骤，其中一个步骤由CD 73催化，CD 73是一种表面结合酶， 将腺苷一磷酸（AMP）转化为腺苷。CD 73对AMP的水解是不可逆的。 腺苷信号传导中的限制步骤，通常被称为从促炎性细胞因子转化为细胞因子的“免疫开关”。 （eATP）转化为抗炎（腺苷）介质。牙龈卟啉单胞菌是一种重要的致病菌， 与严重的牙周病相关，并且可以使用多种机制成功地侵入，复制， 并通过人类牙龈上皮细胞（GECs）传播， 在粘膜表面。尽管在包括癌症在内的各种病理状态中越来越重要， 炎症性疾病和最近的细胞感染，CD 73调节宿主免疫应答的作用， 口腔中的细菌感染还不太清楚。我们的实验室先前表明，牙龈卟啉单胞菌 可以抑制eATP介导的活性氧（ROS）的产生NADPH氧化酶2和细菌， 生长通过GEC中腺苷2a受体偶联的腺苷信号传导来调节和增强。我们的新型 初步发现还揭示了GECs中牙龈卟啉单胞菌的CD 73表达显著增加。 此外，CD 73介导的信号传导特异性地影响牙龈卟啉单胞菌的细胞内存活，抑制牙龈卟啉单胞菌的细胞内存活。 ROS的产生，并抑制白细胞介素-6（IL-6）的基因表达，IL-6的加入导致细胞凋亡减少。 GEC中牙龈卟啉单胞菌的水平。因此，我们的总体假设是牙龈卟啉单胞菌可以选择性地 调节宿主CD 73介导的免疫信号传导途径，以实现丰富的细胞内生长和存活 在牙龈上皮中。我们将通过完成以下具体目标来检验我们的假设。具体 目的1通过进一步研究CD 73在牙龈卟啉单胞菌胞内感染中的调控作用 支持GEC中牙龈卟啉单胞菌/CD 73偶联信号传导的功能重要性。具体目标2将 阐明CD 73调节牙龈卟啉单胞菌细胞内生长的细胞和分子机制 和生存，特别是通过干扰促炎性IL-6信号。这两个目标都将使用主要的 GECs模型系统功能解剖出机制和定义分子事件。所以这 该提案将表征口腔细菌感染的新的生理学相关调节剂， 揭示了宿主CD 73/牙龈卟啉单胞菌/IL-6轴之间的多向串扰，用于调节 牙龈卟啉单胞菌在宿主细胞内的感染。这项研究的结果将提供从根本上新颖的 牙龈卟啉单胞菌在口腔粘膜中的持久性机制的分子理解， 有助于将来开发用于控制牙龈卟啉单胞菌相关感染的有效治疗剂。这 研究金还将提供口腔微生物学/免疫学方面的培训，并将促进受训者的发展 成为一个独特的和急需的口腔健康学术临床医生/科学家。

PROJEST SUMMARY/ABSTRACT Danger signal molecule extracellular ATP (eATP) is released by infected cells and becomes metabolized to adenosine by multiple enzymatic steps, one of which is catalyzed by CD73, a surface-bound enzyme that converts adenosine monophosphate (AMP) to adenosine. This AMP hydrolysis by CD73 is an irreversible rate- limiting step in adenosine signaling that is often referred to as “an immunological switch” from pro-inflammatory (eATP) to anti-inflammatory (adenosine) mediator. Porphyromonas gingivalis is a keystone pathogen strongly associated with severe periodontal disease and can use multiple mechanisms to successfully invade, replicate, and disseminate within and through the human gingival epithelial cells (GECs), a major arm of initial defenses in mucosal surfaces. Despite the growing significance in various pathological states including cancer, inflammatory diseases, and recently cellular infection, the role of CD73 for regulating host immune response and bacterial infection in the oral cavity is not well understood. Our laboratory previously showed that P. gingivalis can inhibit eATP-mediated reactive oxygen species (ROS) generation by NADPH oxidase 2 and that the bacterial growth is modulated and enhanced by adenosine 2a receptor-coupled adenosine signaling in GECs. Our novel preliminary findings also revealed significantly increased CD73 expression with P. gingivalis in GECs. Furthermore, CD73-mediated signaling specifically impacted on the P. gingivalis intracellular survival, inhibited ROS generation, and dampened the gene expression of interleukin-6 (IL-6) whose addition led to decreased levels of P. gingivalis in GECs. Thus, our overarching hypothesis is that P. gingivalis can selectively regulate host CD73-mediated immune signaling pathways for affluent intracellular growth and survival in the gingival epithelium. We will test our hypothesis through completion of the following specific aims. Specific Aim 1 will establish the regulatory role of CD73 for P. gingivalis intracellular infection by further investigating functional importance supporting the P. gingivalis/CD73 coupled signaling in the GECs. Specific Aim 2 will elucidate select cellular and molecular mechanisms by which CD73 modulates P. gingivalis intracellular growth and survival, specifically through interfering with pro-inflammatory IL-6 signaling. Both aims will use primary GECs model system to functionally dissect out mechanisms and define the molecular events. Hence, this proposal will characterize novel physiologically relevant modulator(s) of oral bacterial infection and reveal the multidirectional cross-talk between host CD73/P. gingivalis/IL-6 axis for the modulation of intracellular P. gingivalis infection in the host cells. The results of this study will provide fundamentally novel molecular understanding of P. gingivalis persistence mechanisms in the oral mucosa and may ultimately contribute to future development of effective therapeutics for controlling P. gingivalis associated infections. This fellowship will also provide training in oral microbiology/immunology and will foster the development of the trainee into a unique and critically needed oral health academic clinician/scientist.