Biosensor based approach to measure release of Nociceptin/Orphanin FQ from live single immune cells and consequences for immune cell function.

基于生物传感器的方法可测量活体单个免疫细胞中痛敏肽/孤啡肽 FQ 的释放以及对免疫细胞功能的影响。

基本信息

批准号：
BB/N000188/1
负责人：
David Lambert
金额：
$ 43.12万
依托单位：
University of Leicester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2016
资助国家：
英国
起止时间：
2016 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FN000188%2F1
关键词：
Biosensor based approach measure release

项目摘要

Context: Classical opioid receptors are classified as MOP(mu), DOP(delta) and KOP(kappa). In addition to these three receptor types there is a fourth non-classical type, the receptor for nociceptin/orphaninFQ (N/OFQ) or NOP. The peptide N/OFQ is produced from a precursor pre-pro-N/OFQ (ppNoc). Opioids cause immune suppression but the mechanisms are poorly understood; in particular for the N/OFQ-NOP system. In this regard all immune cells we have tested to date express mRNA for NOP but variably express mRNA for N/OFQ. Little is known about the release of N/OFQ from immune cells and nothing at the single cell level. We have a Chinese Hamster Ovary (CHO) cell clone that expresses a chimeric Galpha-i/q protein enabling Gi coupled receptors to increases in intracellular Calcium; this can be measured fluorimetrically. This cell line is co-transfected with NOP; effectively allowing receptor NOP activation to be monitored as a Calcium signal; a biosensor for N/OFQ. If we overlay Galpha-i/q cells with polymorphonuclear leucocytes and stimulate them to degranulate with fMLP we have been able to measure N/OFQ release at the single cell level for the first time. This release was sensitive to NOP antagonists but not purinergic antagonists. There are two hypotheses to our application; 1-Leucocytes differentially release N/OFQ and we will develop a novel cell based Galpha-i/q biosensor system to measure this at the single cell level; 2-NOP activation by N/OFQ will modulate leucocyte function at the cellular physiological level and we will use the intracellular signalling pathways and migration as readouts. Aims and objectives: We will develop and characterise a Galpha-i/q chimera based biosensor assay for use with cells expressing recombinant human NOP in confocal microscopy. Volunteer human blood will be separated into individual immune cell populations (monocytes, B and T lymphoctyes and basophil, neutrophil and eosinophil granulocytes) with each assessed for release capability. Based on preliminary data showing differential ppNoc expression we predict that of the granulocyte population, eosinophils will be the source of N/OFQ. In addition we predict that monocytes and lymphocytes will also release N/OFQ and that we can measure this from single cells. We will assess antagonist sensitivity to confirm NOP as the target, explore Calcium dependence of the release process and confirm each cell immune cell type by immunofluoresence. Cellular content of N/OFQ peptide will also be measured by immunofluoresence with specific N/OFQ antibodies. Of note, there are no NOP reliable antibodies for use in Western blotting.We will characterise the effects of immune cell NOP activation with peptide and non-peptide agonists by measuring ERK1/2 phosphorylation and JNK using Western blot. Activation of ERK1/2 has been linked to apoptosis so we will also measure cytochrome c and cleaved caspase 3 as we have done previously in neuronal cells. As Rho GTPases are linked with cell migration and we will be assessing this we will examine the effects of NOP activation on Rho GTPase using the Active RHO pull down and detection kit. We will also measure the cytokine release profile from cell populations in response to degranulation stimuli in the absence and presence of N/OFQ using Luminex xMAP technology and the Human 25-Plex panel. We will use transwell migration assays to assess autocrine and paracrine control of migration and the effects of NOP activation.Applications/Benefits: Data from this project will provide: (1) a novel technique for measuring single-cell release, (2) the first detailed release profile of N/OFQ from immune cells, currently lacking in the literature and (3) detailed information of the consequences of that release at a biochemical and functional level. Furthermore, we believe that these experimental techniques and data will have applicability to other cells and systems (e.g. neuronal transmitter release).

背景：经典阿片受体分为 MOP(mu)、DOP(delta) 和 KOP(kappa)。除了这三种受体类型之外，还有第四种非经典类型，即伤害感受肽/孤啡肽 FQ (N/OFQ) 或 NOP 受体。肽 N/OFQ 由前体 pre-pro-N/OFQ (ppNoc) 产生。阿片类药物会导致免疫抑制，但其机制尚不清楚；特别是对于 N/OFQ-NOP 系统。在这方面，我们迄今为止测试过的所有免疫细胞都表达 NOP 的 mRNA，但表达 N/OFQ 的 mRNA 不同。关于免疫细胞释放 N/OFQ 的情况知之甚少，在单细胞水平上也一无所知。我们有一个中国仓鼠卵巢 (CHO) 细胞克隆，它表达嵌合 Galpha-i/q 蛋白，使 Gi 偶联受体能够增加细胞内钙；这可以通过荧光测定来测量。该细胞系与 NOP 共转染；有效地允许受体 NOP 激活作为钙信号进行监测；用于 N/OFQ 的生物传感器。如果我们用多形核白细胞覆盖 Galpha-i/q 细胞并用 fMLP 刺激它们脱颗粒，我们就能够首次在单细胞水平上测量 N/OFQ 的释放。该释放对 NOP 拮抗剂敏感，但对嘌呤能拮抗剂不敏感。我们的应用有两个假设； 1-白细胞差异性地释放 N/OFQ，我们将开发一种基于细胞的新型 Galpha-i/q 生物传感器系统，以在单细胞水平上测量这一点； N/OFQ 激活 2-NOP 将在细胞生理水平上调节白细胞功能，我们将使用细胞内信号传导途径和迁移作为读数。目的和目标：我们将开发并表征基于 Galpha-i/q 嵌合体的生物传感器测定，用于在共聚焦显微镜中表达重组人 NOP 的细胞。志愿者的人类血液将被分成单独的免疫细胞群（单核细胞、B 和 T 淋巴细胞以及嗜碱性粒细胞、中性粒细胞和嗜酸性粒细胞），并对每个细胞群的释放能力进行评估。根据显示 ppNoc 表达差异的初步数据，我们预测粒细胞群中的嗜酸性粒细胞将是 N/OFQ 的来源。此外，我们预测单核细胞和淋巴细胞也会释放 N/OFQ，并且我们可以从单个细胞中测量这一点。我们将评估拮抗剂敏感性以确认 NOP 作为靶点，探索释放过程的钙依赖性，并通过免疫荧光确认每种细胞的免疫细胞类型。 N/OFQ 肽的细胞含量也将通过特定 N/OFQ 抗体的免疫荧光来测量。值得注意的是，没有可靠的 NOP 抗体可用于蛋白质印迹。我们将通过使用蛋白质印迹测量 ERK1/2 磷酸化和 JNK 来表征肽和非肽激动剂对免疫细胞 NOP 激活的影响。 ERK1/2 的激活与细胞凋亡有关，因此我们还将测量细胞色素 c 和裂解的 caspase 3，就像我们之前在神经元细胞中所做的那样。由于 Rho GTP 酶与细胞迁移相关，我们将对此进行评估，我们将使用活性 RHO 下拉和检测试剂盒检查 NOP 激活对 Rho GTP 酶的影响。我们还将使用 Luminex xMAP 技术和 Human 25-Plex panel 测量在不存在和存在 N/OFQ 的情况下细胞群响应脱颗粒刺激的细胞因子释放情况。我们将使用 Transwell 迁移测定来评估迁移的自分泌和旁分泌控制以及 NOP 激活的影响。应用/优点：该项目的数据将提供：(1) 一种测量单细胞释放的新技术，(2) 第一个N/OFQ 从免疫细胞中的详细释放曲线，目前文献中缺乏；(3) 在生化和功能水平上释放后果的详细信息。此外，我们相信这些实验技术和数据将适用于其他细胞和系统（例如神经元递质释放）。