Impact of macrophage differentiation on adenosine receptor expression and function

腺苷受体在全身许多组织中表达，并在组织稳态中起重要作用。有四种腺苷受体（A1、A2 A、A2 B和A3），具有组织特异性表达，其在多种疾病状态中改变。我们和其他人已经证明，不同的腺苷受体相互作用，这种相互作用导致下游细胞功能的改变。然而，这些相互作用的机制和确切效果知之甚少。鉴于这些受体的普遍表达及其在许多不同疾病中的重要性，了解这些机制是了解其在疾病状态中的作用并实现有效的腺苷受体靶向的关键目标。腺苷受体在细胞功能中发挥作用的重要细胞是免疫细胞，特别是巨噬细胞。本项目旨在：1）确定巨噬细胞极化如何影响腺苷受体表达2）评估腺苷受体在各种条件下在巨噬细胞中的相互作用，降解和加工3）探索巨噬细胞中腺苷受体的差异表达如何影响下游信号传导和细胞功能项目计划：将在作为人单核细胞-巨噬细胞模型系统的THP 1细胞和原代人单核细胞衍生的巨噬细胞中进行工作。我们已经开发了多种新技术来可视化原代细胞中的腺苷受体表达，包括配体指导的共价标记、受体选择性纳米抗体和荧光标记的配体（Comeo等人，2020; Soave等人，2020 a，B; Stoddart等人，2020年）。这些技术将用于定义腺苷受体在不同类型的单核细胞衍生的巨噬细胞中的表达。目的2和3：我们还将使用CRISPR/Cas9基因组编辑结合基于核转染剂的电穿孔，将纳米荧光素酶片段HiBiT或SmBiT引入到THP 1细胞中内源表达的每个腺苷受体的N或C末端。在添加纯化的LgBiT后，纳米荧光素酶可以被重新补充以形成生物发光蛋白。该方法将用于使用生物发光共振能量转移（BRET）动态研究配体-受体和受体-受体相互作用，以及分化成巨噬细胞后的受体降解和加工。可在表达胞质LgBiT的细胞中使用C-末端HiBiT或SmBiT标签跟踪真实的-时间受体降解。通过监测cAMP积累、CREB磷酸化和细胞因子/趋化因子的产生，研究具有不同腺苷受体表达的巨噬细胞功能（包括转染细胞和原代巨噬细胞）。该项目是诺丁汉大学膜蛋白和受体中心（COMPARE）几个学院的多学科合作，利用尖端技术剖析基本生物过程。除了进一步了解腺苷受体药理学，该项目的结果将对多种疾病产生影响。COMPARE有一个著名的团队科学方法进行研究生培训，学生将受益于在这种环境中工作。

Adenosine receptors are expressed in many tissues throughout the body and play an important role in tissue homeostasis. There are four adenosine receptors (A1, A2A, A2B and A3), with tissue-specific expression which is altered in a wide variety of disease states. We and others have demonstrated that different adenosine receptors interact with each other and this interaction leads to altered downstream cellular function. However, the mechanisms and exact effects of these interactions are poorly understood. Given the ubiquitous expression of these receptors and their importance in many different diseases, understanding these mechanisms is a key goal to understand their role in disease states and enable effective adenosine-receptor targeting. Important cells where adenosine receptors have been shown to be differentially expressed and play a role in cellular function are immune cells, specifically macrophages.This project aims to:1) Define how macrophage polarisation affects adenosine receptor expression2) Assess adenosine receptor interactions, degradation and processing in macrophages under a variety of conditions3) Explore how differential adenosine receptor expression in macrophages affects downstream signalling and cellular functionProject plan:Work will be carried out in THP1 cells as a human monocyte-macrophage model system and primary human monocyte-derived macrophages.Aim 1: We have developed a variety of novel techniques to visualise adenosine receptor expression in primary cells including ligand-directed covalent labelling, receptor-selective nanobodies and fluorescently-tagged ligands (Comeo et al., 2020; Soave et al., 2020a,b; Stoddart et al., 2020). These techniques will be used to define adenosine receptor expression in different types of monocyte-derived macrophages.Aims 2 &3: We will also use CRISPR/Cas9 genome editing in conjunction with nucleofector based electroporation to introduce the nanoluciferase fragments HiBiT or SmBiT onto the N or C terminus of each adenosine receptor endogenously expressed in THP1 cells. Following addition of purified LgBiT, the nanoluciferase can be re-complemented to form a bioluminescent protein. This approach will be used to dynamically study ligand-receptor and receptor-receptor interactions using bioluminescence resonance energy transfer (BRET), and receptor degradation and processing following differentiation into macrophages. Real- time receptor degradation can be followed using C-terminal HiBiT or SmBiT tags in cells expressing cytosolic LgBiT. Macrophage function with differential adenosine receptor expression (both in transfected cells and primary macrophages) will be studied by monitoring cAMP accumulation, CREB phosphorylation and cytokine/chemokine production.This project is a multi-disciplinary collaboration across several Schools within the Centre of Membrane Proteins and Receptors (COMPARE) University of Nottingham, utilising cutting-edge techniques to dissect fundamental biological processes. As well as furthering our understanding ofadenosine receptor pharmacology, the results from this project will have implications in a wide variety of diseases. COMPARE has a renowned Team Science approach for postgraduate training and the student will benefit from working within this environment.