Impact of macrophage differentiation on adenosine receptor expression and function

巨噬细胞分化对腺苷受体表达和功能的影响

• 批准号: 2594721
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2594721
• 关键词: Impact; macrophage; differentiation; adenosine; receptor

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.

腺苷受体在人体许多组织中表达，在组织稳态中起重要作用。有四种腺苷受体（A1, A2A， A2B和A3），具有组织特异性表达，在多种疾病状态下发生改变。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，人们对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它们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。腺苷受体差异表达并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)定义巨噬细胞极化如何影响腺苷受体表达2)评估各种条件下巨噬细胞中腺苷受体的相互作用、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THP1细胞作为人类单核细胞-巨噬细胞模型系统和原代人类单核细胞来源的巨噬细胞进行研究。目标1：我们开发了多种新技术来可视化原代细胞中腺苷受体的表达，包括配体定向共价标记、受体选择性纳米体和荧光标记配体（Comeo等人，2020；Soave等人，2020a,b； Stoddart等人，2020）。这些技术将用于定义腺苷受体在不同类型的单核细胞源性巨噬细胞中的表达。目标2和3：我们还将使用CRISPR/Cas9基因组编辑与基于核因子的电穿孔相结合，将纳米荧光素酶片段HiBiT或SmBiT引入到THP1细胞中内源性表达的每个腺苷受体的N或C端。加入纯化的lgb后，纳米荧光素酶可以被重新补充，形成生物发光蛋白。该方法将用于动态研究配体-受体和受体-受体相互作用的生物发光共振能量转移（BRET），以及受体分化为巨噬细胞后的降解和加工。在表达细胞质LgBiT的细胞中，可以使用c端HiBiT或SmBiT标签跟踪实时受体降解。通过监测cAMP积累、CREB磷酸化和细胞因子/趋化因子的产生，将研究巨噬细胞功能与腺苷受体表达差异（在转染细胞和原代巨噬细胞中）。该项目是诺丁汉大学膜蛋白和受体中心（COMPARE）内几所学校的多学科合作，利用尖端技术来解剖基本的生物过程。除了进一步加深我们对腺苷受体药理学的理解外，这个项目的结果将对各种各样的疾病产生影响。COMPARE拥有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。