Investigating a role for activity-regulated cytoskeleton-associated protein in the mast cell response to external stimuli, including hydrogen peroxide

在综合博士学位的第一年，安娜威利斯将参加一个诱导和介绍性培训课程，然后是生物医学科学，定量细胞生物学和环境因素，如病原体，过敏原或污染已被证明会引起哮喘恶化，并有助于呼吸系统疾病的发展。肥大细胞（MC）位于粘膜表面，它们是抵御环境挑战的第一道防线的一部分。在哮喘患者的肺中，具有活化表型的MC的数量增加，其响应于这些刺激而释放促炎介质。MC介导的炎症反应可导致慢性炎症、气道高反应性和支气管收缩，因此MC参与了哮喘的发病机制。暴露于污染物产生的过氧化氢（H2 O2）可以通过促进气道重塑和诱导白细胞募集和细胞因子表达而促进哮喘发病机制。类似地，鼻病毒（RV）感染是哮喘恶化的主要原因，通过其诱导免疫应答，其涉及促炎细胞因子和趋化因子的释放，以及加剧炎症的免疫细胞的募集。转录组学分析确定活性调节的细胞因子相关蛋白（ARC）作为RV 16感染的LAD 2 MC中最高度上调的基因。ARC调节细胞骨架重排，在神经可塑性和形成突触连接中具有明确的作用。除了在突触传递中的作用外，对ARC功能的研究有限。然而，最近的研究表明ARC在上皮细胞对热休克和H2 O2的反应中，以及在单纯疱疹病毒1型（HSV-1）复制从神经元的复制和释放中起作用。本项目研究ARC在MC对H2 O2和RV 16感染的反应中的作用。我已经显示了在HeLa细胞和MC中响应于H2 O2处理的ARC基因和蛋白表达的诱导，以及在LAD 2 MC中响应于H2 O2的HSPA 1A、HSPA 1B和HSPB 1基因表达的诱导。我还进行了转录组学分析，结果表明CACNG 3和ARC是RV 16感染的LAD 2 MC中上调最高的基因。使用siRNA转染在LAD 2 MC中敲低ARC需要进一步优化，然后才能用于研究ARC在MC对H2 O2和RV感染的反应中的作用。

In the first year of the Integrated PhD, Anna Willis will attend an induction and introductory training programme followed by taught modules in Research Skills for Biomedical Sciences, Quantitative Cell Biology anEnvironmental factors such as pathogens, allergens, or pollution have been shown to induce asthma exacerbations and contribute to the development of respiratory disease. Mast cells (MCs) are localised at mucosal surfaces where they are part of the first line of defence against environmental challenges. There are increased numbers of MCs with an activated phenotype in the lungs of asthma patients, which release proinflammatory mediators in response to these stimuli. MC mediators can induce chronic inflammation, airway hyperresponsiveness and broncho-constriction in those with pre-existing inflammation, therefore MCs have been implicated in asthma pathogenesis. Hydrogen peroxide (H2O2) generated by exposure to pollutants can contribute to asthma pathogenesis through promotion of airway remodelling and the induction of leukocyte recruitment and cytokine expression. Similarly, Rhinovirus (RV) infection is a major cause of asthma exacerbations through its induction of an immune response which involves release of proinflammatory cytokines and chemokines, and recruitment of immune cells which exacerbate inflammation. A transcriptomic analysis identified activity-regulated cytoskeleton-associated protein (ARC) as the most highly upregulated gene in RV16-infected LAD2 MCs. ARC regulates cytoskeleton rearrangement, with a well-defined role in neuroplasticity and forming synaptic connections. There is limited research on the function of ARC outside of its role in synaptic transmission. However, recent studies show a role for ARC in the response to heat shock and H2O2 in epithelial cells, as well as in the replication and release of herpes simplex virus 1 (HSV-1) replication from neurons. This project investigates a role for ARC in the MC response to H2O2 and RV16 infection. I have shown an induction of ARC gene and protein expression in response to H2O2 treatment in HeLa cells and MCs, as well as induction of HSPA1A, HSPA1B and HSPB1 gene expression in response to H2O2 in LAD2 MCs. I also carried out a transcriptomic analysis which shows that CACNG3 and ARC are the most highly upregulated genes in RV16-infected LAD2 MCs. Knock down of ARC in LAD2 MCs using siRNA transfection required further optimisation before it can be used to investigate a role for ARC in the MC response to H2O2 and RV infection.