Analysis of PI3K dependent Control of Immunosenescence in the Neutrophil

尽管在骨髓中不断更新，但中性粒细胞在老年时变得不那么有效;一个重要的变化是趋化性变差[1]。磷酸肌醇3-激酶（PI 3 K）显示在老年供体的中性粒细胞中上调，虽然有数据表明PI 3 K在中性粒细胞免疫衰老中的作用[1]，但负责中性粒细胞免疫衰老的PI 3 K上游和下游蛋白质的身份仍不清楚。我们的目标是解决这个重要的问题。我们将确定PI 3 K的下游信号是如何参与调节老年人中性粒细胞功能的，重点是ARAP 3。SV的实验室在遗传学上表明，ARAP 3是中性粒细胞功能的重要调节因子。与许多其他PI 3 K效应物不同，ARAP 3在中性粒细胞趋化性中至关重要[2]。其中ARAP 3与PI 3 K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性缺陷[3]。我们假设PI 3 K下游的ARAP 3信号传导参与了健康老年人中性粒细胞的趋化缺陷，将分析健康老年人和年轻人中性粒细胞的ARAP 3表达，并测定中性粒细胞刺激后ARAP 3底物的活性。我们将比较来自健康、年轻和老年捐赠者的中性粒细胞中受体刺激后整体ARAP 3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化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。作为短寿命的终末分化细胞，中性粒细胞不适合转染或转导。因此，磷酸化事件对ARAP 3活性的影响将在更易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。这将鉴定可用作PI 3 K/ARAP 3活性的生物标志物的ARAP 3磷酸化位点。将培养并验证磷酸特异性抗体，旨在报告年轻和老年健康捐赠者中性粒细胞中的PI 3 K/ARAP 3激活状态。这可能与不良的趋化方向性相关，并可能被证明是一个非常宝贵的工具试剂，支持未来对健康老龄化生理学的研究。B计划。如果上述实验早期证实我们的假设是错误的，我们将研究PI 3 K依赖的信号传导和基因调控网络在老年时是如何改变的。对来自健康年轻和老年供体的中性粒细胞的受体刺激后的蛋白质组和磷酸化蛋白质组调节的时间依赖性定量分析将使得能够进行无偏的差异分析。生物信息学数据分析，包括Incidity Pathway和Metacore等工具，将识别年轻和老年中性粒细胞中的PI 3 K依赖性网络和信号动力学。将对少数特别有希望的变化（例如，提示参与已知为PI 3 K信号网络一部分的信号传导酶的事件）进行验证和进一步研究。在可行的情况下，将使用任何现成的药理学试剂对原代中性粒细胞进行检测。此外，在时间允许的情况下，将使用替代的、更易处理的生物系统进行基于瞬时转染的生物化学/细胞生物学实验。我们利用这些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础性的发现。

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.