Analysis of PI3K dependent Control of Immunosenescence in the Neutrophil

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.