MRC TS Award: Regulation of neutrophil functions by cell cycle proteins

Neutrophils are the most abundant immune cells in the blood and they are essential for defending us against disease-causing microbes. They are considered the 'first line of defence' in response to infection or wounding. Neutrophils have several ways of destroying microbes to prevent the infection from spreading. They can engulf microbes or produce antimicrobial chemicals. Neutrophils can also release "NETs" - Neutrophil Extracellular Traps. NETs consist of webs of DNA that are adhesive and also contain toxic antimicrobial proteins; NETs trap microbes and prevent them from spreading away from the site of infection. They also help to amplify inflammation and to recruit additional immune cells.
NET release must be carefully controlled because excessive NET formation can also damage our own cells and tissues. In fact, neutrophils are often hyperactivated in many non-infectious inflammatory diseases. Excessive NET formation is thought to contribute to autoimmune diseases such as lupus, rheumatoid arthritis and asthma, as well as cancer and atherosclerosis (which can cause stroke and heart attack).
If we could understand how NETs formation is regulated, we could potentially develop therapies to boost NET formation when the body needs to prevent infections or, conversely, to block their release when they are causing pathological inflammation. The aim of this proposal is to complete undergoing studies that aim to understand which proteins control NET release. We discovered that some proteins that normally control cell division are also involved in regulating NET formation. We now propose to undertake detailed studies to understand exactly how these proteins function in NET formation. We will do this by analysing which additional proteins the regulators interact with, and what aspects of cellular biology they affect.
Completing these aims will give us a better understanding of the molecular mechanism of NETs. It would also help to establish my group as international experts in the field of neutrophil biology, allowing us to tackle important global health issues.

Neutrophils are abundant blood cells that form the first line of defence against pathogens. They suppress proliferation of microbes via effector functions such as release of neutrophil extracellular traps (NETs). NETs consist of extruded chromatin bound to antimicrobial proteins; they are released by a type of regulated cell death called 'NETosis'. NETs trap microbes and prevent them from disseminating; however excessive or dysregulated NET formation can promote inflammatory diseases, including autoimmunity, cardiovascular disease, malaria and cancer.
Our understanding of NETosis remains rudimentary, with only a few established regulatory factors. These include cell cycle proteins such as CDK6, despite the fact that neutrophils are terminally differentiated, post-mitotic cells. This proposal builds on findings from my CDA fellowship to investigate the mechanism of NET formation, with a focus on the role of cell cycle and DNA repair factors. We hypothesise that NET formation results from an aborted cell cycle re-entry attempt.
Objective 1 is to investigate how DNA damage response (DDR) signalling regulates NETs. We will use shRNA and CRISPR/Cas9 gene knockdown in neutrophil-like cells to test the involvement of DDR pathways in NETosis. We will validate in vitro findings with malaria and fungal in vivo infection models, as well as using primary neutrophils from patients with mutations in DDR genes. We will use phosphoproteomics to identify downstream targets of essential DDR kinases.
Objective 2 is to characterise the mechanism of CDK6 control of NETosis. We demonstrated that CDK6 regulates NETs by altering metabolic pathways, particularly the pentose phosphate shunt. We will use metabolic profiling and phosphoproteomics in neutrophils from wildtype and CDK6 -/- mice to test whether CDK6 controls a switch in cell death pathways by controlling glucose catabolism.
The two objectives will lead to a comprehensive view of how NET formation is regulated.