Immune function of bitter taste receptors in human macrophages

项目摘要 T2 R苦味受体是最初在舌头上鉴定的G蛋白偶联受体（GPCR）， 而且在其他组织中也发挥不同的作用。“口外”（舌头外）味觉受体可能是 在呼吸道感染、哮喘、早产甚至癌症中起重要作用。在气道上皮细胞中，T2 Rs 4，14， 16和38识别革兰氏阴性细菌产生的群体感应分子，包括 条件致病菌铜绿假单胞菌这些受体的刺激改变了 抗菌分子，包括一氧化氮和防御素。一些多态性降低了功能性 T2 R的增加患者对上呼吸道感染和慢性鼻窦炎的易感性。靶向T2 R 因为治疗需要更好地阐明它们的表达和下游效应。这一点必须在 原代人类细胞，因为小鼠T2 R在数量和激活它们的激动剂上不同。 我们在原代人单核细胞和巨噬细胞中鉴定了几种T2 R受体。巨噬 是重要的先天性免疫细胞，吞噬和杀死细菌，以及分泌促或抗- 炎性细胞因子以改变免疫应答。刺激这些受体激活钙释放 和急性（5-15分钟内）增强细菌吞噬作用的一氧化氮产生。初步数据 表明T2 R可能是刺激先天免疫应答以杀死细菌的靶点，而不使用 传统抗生素，减少抗生素耐药性的压力。这可能是特别有用的上部 呼吸道疾病。急性和慢性鼻窦炎占美国所有抗生素处方的20%以上。 美国，因此是抗生素耐药微生物的重要驱动力。慢性鼻窦炎和 相关的鼻息肉还在许多患者中引起嗅觉障碍、嗅觉减退和/或嗅觉丧失。 我们将进一步确定增加巨噬细胞吞噬作用的信号通路，并测试它是如何影响巨噬细胞吞噬作用的。 影响细菌杀灭（目标1）。由于细菌如铜绿假单胞菌可以侵入细胞并在细胞内存活， 病原体逃避进一步的免疫检测，我们将测试T2 Rs如何影响巨噬细胞杀菌活性。 这项工作也将是第一次使用活细胞成像和荧光生物传感器来研究巨噬细胞 GPCR信号在真实的时间。接下来（目标2），我们将研究T2 R激活如何减少炎症反应， Toll样受体（TLR）。我们的初步数据表明T2 R-TLR串扰的新途径 可能与阿克特有关最后（目的3），我们将比较表型，T2 R表达，和T2 R功能， 炎症性慢性鼻窦炎鼻息肉的巨噬细胞与来自 同样的病人。我们将检查息肉的巨噬细胞浸润是否与T2 R多态性有关， 苦味感知我们还研究了鼻息肉中巨噬细胞数量的增加是否会影响慢性炎症， 鼻窦炎疾病表型。

PROJECT SUMMARY T2R bitter taste receptors are G-protein coupled receptors (GPCRs) originally identified on the tongue, but which also serve diverse roles in other tissues. “Extraoral” (outside the tongue) taste receptors are likely important in airway infection, asthma, pre-term labor, and even cancer. In airway epithelial cells, T2Rs 4, 14, 16, and 38 recognize quorum-sensing molecules produced by gram-negative bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. Stimulation of these receptors modifies production of antibacterial molecules, including nitric oxide and defensins. Some polymorphisms reducing the functionality of T2Rs increase patient susceptibility to upper respiratory infection and chronic rhinosinusitis. Targeting T2Rs as therapies requires better elucidation of their expression and downstream effects. This must be studied in primary human cells, as mouse T2Rs differ in number and agonists that activate them. We identified several T2R receptors in primary human monocytes and macrophages. Macrophages are important innate immune cells that phagocytose and kill bacteria as well as secrete pro- or anti- inflammatory cytokines to modify immune responses. Stimulation of these receptors activates calcium release and nitric oxide production that acutely (within 5-15 min) enhances phagocytosis of bacteria. Preliminary data suggest that T2Rs may be targets for stimulation of innate immune responses to kill bacteria without the use of conventional antibiotics, reducing pressures for antibiotic resistance. This may be especially useful in upper respiratory diseases. Acute and chronic rhinosinusitis account for >20% of all antibiotic prescriptions in the US, and thus are important drives of antibiotic resistant micro-organisms. Chronic rhinosinusitis and associated nasal polyps also cause dysosmia, hyposmia, and/or anosmia in many patients. We will further define the signaling pathway that increases macrophage phagocytosis and test how it affects bacterial killing (Aim 1). As bacteria such as P. aeruginosa can invade cells and live as intracellular pathogens to evade further immune detection, we will test how T2Rs affect macrophage bactericidal activity. This work will also be among the first to use live cell imaging and fluorescent biosensors to study macrophage GPCR signaling in real time. Next (Aim 2), we will examine how T2R activation reduces inflammatory responses to toll-like receptors (TLRs). Our preliminary data suggest novel pathways of T2R-TLR crosstalk possibly involving Akt. Finally (Aim 3) we will compare phenotype, T2R expression, and T2R function in macrophages from inflamed chronic rhinosinusitis nasal polyps with naïve serum-derived macrophages from the same patients. We will examine if macrophage infiltration of polyps is altered with T2R polymorphisms or bitter taste perception. We also examine if increased macrophage numbers in nasal polyps affects chronic rhinosinusitis disease phenotypes.