Analysis of PI3K dependent Control of Immunosenescence in the Neutrophil

尽管中性粒细胞在骨髓中不断更新，但到了老年，中性粒细胞的效率会降低；一个重要的变化是趋化性变差[1]。磷脂酰肌醇3-激酶(PI3K)被证明在老年供者的中性粒细胞中上调，虽然有数据表明PI3K在中性粒细胞免疫衰老中起作用[1]，但涉及PI3K上下游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的问题。我们将确定PI3K下游的信号如何参与调节老年人的中性粒细胞功能，重点是ARAP3。SV的实验室从遗传学上证明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在中性粒细胞趋化中起关键作用[2]。ARAP3被PI3K解偶联的中性粒细胞具有类似于免疫衰弱的中性粒细胞的趋化缺陷[3]。我们假设PI3K下游的ARAP3信号参与了健康、老年捐赠者中性粒细胞的趋化缺陷。我们将分析健康、老年和年轻捐赠者的中性粒细胞ARAP3的表达，并分析ARAP3‘S底物对中性粒细胞刺激的活性。我们将比较来自健康、年轻和老年供者的中性粒细胞在受体刺激下整体ARAP3磷酸化和单个磷酸化位点的差异。这将通过与Cellzome(GSK Heidelberg，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，是对基因组分析的补充。接下来，我们将确定个别磷酸化事件如何影响ARAP3活性。中性粒细胞是一种寿命较短的终末分化细胞，不能被转基因或转导。因此，磷酸化事件对ARAP3活性的影响将在一个更容易处理的系统中得到表征，采用定点突变和基于瞬时转染的分析。这将确定ARAP3磷酸化位点(S)可以作为PI3K/ARAP3活性的生物标志物。将提出并验证磷酸特异性抗体，目的是报告年轻和老年健康捐赠者中性粒细胞中PI3K/ARAP3的激活状态。这可能与较差的趋化方向性相关，并可能被证明是未来健康老龄化生理学研究的宝贵工具试剂。B计划B如果上述实验在早期证实我们的假设是错误的，我们将转而研究依赖PI3K的信号和基因调控网络在老年是如何改变的。对来自健康的年轻和老年供者的中性粒细胞受体刺激的蛋白质组和磷蛋白质组调节的时间依赖的定量分析将使无偏见的差异分析成为可能。生物信息数据分析，包括Invenity Path和MetaCore等工具，将识别PI3K依赖的网络和年轻和老年中性粒细胞中的信号动力学。少数特别有希望的变化(例如，表明涉及已知的PI3K信号网一部分的信号酶的事件)将得到验证和进一步研究。在可行的情况下，将利用任何现成的药理试剂，对初级中性粒细胞进行这项工作。此外，在时间允许的情况下，基于瞬时转基因的生化/细胞生物学实验将使用替代的、更易处理的生物系统进行。我们利用这种数据驱动生物学的能力应该使我们能够在衰老生理学领域做出基础性发现。

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.