Analysis of PI3K dependent Control of Immunosenescence in the Neutrophil

中性粒细胞免疫衰老的 PI3K 依赖性控制分析

• 批准号: 1923603
• 金额: --
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1923603
• 关键词: Analysis; PI3K; dependent; Control; Immunosenescence

Despite being constantly renewed in the bone marrow, neutrophils become less efficacious in old age; one important change is poorer chemotaxis [1]. Phosphoinositide 3-kinase (PI3K) was shown to be upregulated in neutrophils from elderly donors and whilst there are data suggesting a role of PI3K in neutrophil immunosenescence [1], the identities of proteins involved up- and downstream of PI3K that are responsible neutrophil immunosenescence remain obscure. We aim to tackle this important question.We will determine how signalling downstream of PI3K is involved in regulating neutrophil function in the elderly, placing an emphasis on ARAP3. SV's lab showed genetically that ARAP3 is an important regulator of neutrophil function. Unlike many other PI3K effectors, ARAP3 is critical in neutrophil chemotaxis [2]. Neutrophils in which ARAP3 is uncoupled from activation by PI3K have a chemotaxis defect that resembles that of immunosenescent neutrophils [3]. We hypothesize that ARAP3 signalling downstream of PI3K is involved in the chemotactic defect of neutrophils from healthy, elderly donors.Neutrophils from healthy, elderly and young donors will be analysed for ARAP3 expression, and the activity of ARAP3's substrates upon neutrophil stimulation will be assayed. We will compare differences in global ARAP3 phosphorylation and of individual phosphorylation sites upon receptor stimulation in neutrophils from healthy, young and elderly donors. This will be done by combining co-immunoprecipitation with phospho-proteomics in collaboration with colleagues at Cellzome (GSK Heidelberg, Germany), an innovative method for analysing post-translational changes that is complementary to genomic analyses.Next, we will establish how individual phosphorylation events affect ARAP3 activity. As short-lived, terminally differentiated cells neutrophils are not amenable to transfection or transduction. Effects of phosphorylation events on ARAP3 activity will therefore be characterised in a more tractable system, employing site-directed mutagenesis and transient transfection-based assays. This will identify ARAP3 phosphorylation site(s) that could serve as a biomarker for PI3K/ARAP3 activity. Phosphospecific antibodies will be raised and validated with the aim to report on the PI3K/ARAP3 activation status in neutrophils from young and elderly healthy donors. This may enable a correlation with poor chemotactic directionality, and could prove to be an invaluable tool reagent underpinning future research on the physiology of healthy ageing. Plan B. If the above experiments establish early on that our hypothesis was incorrect, we will investigate instead how PI3K-dependent signalling and gene-regulatory networks are altered in old age. A time-dependent quantitative analysis of proteome and phosphoproteome regulation upon receptor stimulation of neutrophils from healthy young and elderly donors will enable an unbiased differential analysis. Bioinformatical data analysis including tools such as Ingenuity Pathway and Metacore will identify PI3K-dependent networks and signalling dynamics in the young and old neutrophil. A small number of particularly promising changes (e.g. events suggesting the involvement of a signalling enzyme that is already known to be part of the PI3K signalling web) will be validated and further investigated. Where feasible, this will be done with primary neutrophils, making use of any readily available pharmacological reagents. In addition, and time-permitting, transient transfection-based biochemical/cell biology experiments will be carried out with alternative, more tractable biological systems. Our ability to exploit such data driven Biology should enable us to contribute fundamental discoveries in the area of ageing physiology.

尽管中性粒细胞在骨髓中不断更新，但在老年时其功效会降低。一项重要的变化是趋化性较差[1]。研究表明，老年捐献者的中性粒细胞中磷酸肌醇 3 激酶 (PI3K) 上调，虽然有数据表明 PI3K 在中性粒细胞免疫衰老中发挥作用 [1]，但参与 PI3K 上下游的、负责中性粒细胞免疫衰老的蛋白质的身份仍然不清楚。我们的目标是解决这个重要问题。我们将确定 PI3K 下游信号传导如何参与调节老年人的中性粒细胞功能，重点关注 ARAP3。 SV 的实验室从遗传学角度表明 ARAP3 是中性粒细胞功能的重要调节因子。与许多其他 PI3K 效应器不同，ARAP3 在中性粒细胞趋化作用中至关重要 [2]。 ARAP3 与 PI3K 激活解偶联的中性粒细胞具有类似于免疫衰老中性粒细胞的趋化缺陷 [3]。我们假设 PI3K 下游的 ARAP3 信号传导与来自健康、老年供体的中性粒细胞的趋化缺陷有关。将分析来自健康、老年和年轻供体的中性粒细胞的 ARAP3 表达，并测定 ARAP3 底物在中性粒细胞刺激后的活性。我们将比较来自健康、年轻和老年供体的中性粒细胞受体刺激后整体 ARAP3 磷酸化和个体磷酸化位点的差异。这将通过与 Cellzome（GSK 海德堡，德国）的同事合作将免疫共沉淀与磷酸蛋白质组学相结合来完成，这是一种分析翻译后变化的创新方法，与基因组分析互补。接下来，我们将确定单个磷酸化事件如何影响 ARAP3 活性。作为短命的终末分化细胞，中性粒细胞不适合转染或转导。因此，磷酸化事件对 ARAP3 活性的影响将在更易于处理的系统中进行表征，采用定点诱变和基于瞬时转染的测定。这将鉴定可作为 PI3K/ARAP3 活性生物标志物的 ARAP3 磷酸化位点。将产生并验证磷酸特异性抗体，目的是报告来自年轻和老年健康捐赠者的中性粒细胞的 PI3K/ARAP3 激活状态。这可能与较差的趋化方向性相关，并且可能被证明是支撑健康衰老生理学未来研究的宝贵工具试剂。 B 计划。如果上述实验尽早证明我们的假设是错误的，我们将研究 PI3K 依赖性信号传导和基因调控网络在老年时如何改变。对健康年轻和老年供体的中性粒细胞受体刺激后的蛋白质组和磷酸化蛋白质组调节进行时间依赖性定量分析，将能够进行无偏的差异分析。生物信息数据分析（包括 Ingenuity Pathway 和 Metacore 等工具）将识别年轻和年老中性粒细胞中的 PI3K 依赖性网络和信号动态。少数特别有希望的变化（例如，表明已知属于 PI3K 信号网络一部分的信号酶参与的事件）将得到验证和进一步研究。在可行的情况下，这将利用原代中性粒细胞来完成，利用任何现成的药理学试剂。此外，在时间允许的情况下，将使用更易于处理的替代生物系统进行基于瞬时转染的生化/细胞生物学实验。我们利用这种数据驱动的生物学的能力应该使我们能够在衰老生理学领域做出基本发现。

项目成果

The 5-Phosphatase SHIP2 Promotes Neutrophil Chemotaxis and Recruitment.

• DOI: 10.3389/fimmu.2021.671756
• 发表时间: 2021
• 期刊: Frontiers in immunology
• 影响因子: 7.3
• 作者: Michael M;McCormick B;Anderson KE;Karmakar U;Vermeren M;Schurmans S;Amour A;Vermeren S
• 通讯作者: Vermeren S