Regulation of neutrophil functions by cell cycle proteins

Neutrophils are the most common immune cells circulating in the blood stream; they account for 50-70% of all white blood cells and are essential for defending us from bacteria, viruses and other parasites. They are found in all vertebrates, including fish, birds and mammals, and are the 'first-responders' to infections and wounding - they move rapidly into the affected tissues to deal with the microbial threat. If neutrophils do not arrive to the correct place, or if they arrive too late, the body remains defenceless.
Once they have located the invaders, neutrophils have several ways of destroying them to prevent the infection from spreading. They can engulf microbes and kill them by producing toxic chemicals such as bleach. Neutrophils can also release "NETs" - Neutrophil Extracellular Traps. NETs are made when neutrophils release webs of DNA that are adhesive and contain toxic antimicrobial proteins; NETs trap microbes and prevent them from spreading.
The toxic antimicrobials that neutrophils use against microbes are a dangerous cargo. Their release via NETs must be carefully controlled because they also have the potential to damage our own cells and tissues. In fact, neutrophils are often incorrectly activated in many non-infectious diseases. These include various autoimmune diseases, such as lupus, rheumatoid arthritis and asthma, but also cancer and atherosclerosis (which leads to stroke or heart attack).
Despite their relevance for disease, we know very little about how neutrophils 'decide' to react one way or another. The aim of this Fellowship is to understand which genes control neutrophil behaviour and how signals from the environment regulate the way in which a neutrophil acts. I found that genes which normally control cell division are also involved in regulating neutrophil functions. This is an exciting hypothesis because it would give us a major insight into how neutrophils carry out their tasks and how things go awry. Since cell division genes are extensively studied in the context of cancer, it might also mean that certain cancer drugs could be re-purposed to treat diseases where neutrophils show faulty behaviour.
The research outlined in this MRC CDA Fellowship is interdisciplinary and combines concepts from different areas of biology (immunology, cell division and microbiology). The University of Bristol will be an ideal place to be immersed with researchers from all of these fields, and to have access to unique technical expertise, all of which will be invaluable throughout my Fellowship.

Neutrophils are essential immune cells that migrate to wounds and sites of infection and engage effector functions such as release of neutrophil extracellular traps (NETs). NETs consist of extruded chromatin released via a cell death pathway called 'NETosis'. NETs are antimicrobial; they bind microbes and prevent them from disseminating but can also be detrimental in inflammatory diseases.
Neutrophils are carefully controlled at the levels of activation and localisation, yet in both cases our understanding of mechanisms is incomplete. This project will investigate regulation of NETosis and neutrophil migration with a focus on the role of cell cycle and DNA repair factors.
Aim 1 will investigate NET formation and the role of cyclin dependent kinase 6 (CDK6), a cell cycle regulator that is required for NETs. Aim 1a will characterize how CDK6-mediated signalling triggers a checkpoint kinase 1 (CHK1) DNA damage response and leads to NET release. Aim 1b will examine a role for CDK6 in regulating NETs via a metabolic switch to the pentose phosphate pathway. I will use patient samples and a zebrafish in vivo NET model to genetically validate these pathways, as well as phosphoproteomics to identify downstream targets.
In Aim 2, I will investigate how DNA repair factors regulate migration of neutrophils in vivo. Specifically, I will examine the role of ataxia telangiectasia mutated (ATM), which I previously showed to be a regulator of neutrophil chemokines. In Aim 2a, I will genetically deplete atm in a zebrafish model that allows tracking of individual neutrophils to wounds and E. coli infections. In Aim 2b, I will test the hypothesis that ATM is a target for bacterial virulence factors. E. coli and other Gram (-) pathogens, produce genotoxins - bacterial molecules that induce DNA damage and activate ATM. I will use the zebrafish model to test the ability of two genotoxins (colibactin and cytolethal distending factor) to modulate neutrophil migration via ATM.

My project will benefit several non-academic sectors and has the potential for real impact in health policy and development of therapeutics:

1) Public beneficiaries via NHS and policymakers

a. Guiding treatments of common inflammatory diseases.
Neutrophils contribute to many inflammatory and autoimmune disorders. These include diseases with alarmingly rising rates in the UK such as atherosclerosis, diabetes and cancer. Similarly, several lines of evidence (including my own), have implicated neutrophil migration and NETs in pathogenesis of malaria which is responsible for the death of approximately 800,000 people in developing countries every year. Knowledge derived from this proposal will be directly applicable towards developing treatment strategies for both inflammatory diseases of developed countries, as well as for malaria which exerts a tremendous burden on the African continent.


b. Guiding treatment of ataxia telangiectasia and Nijmegen's Breakage syndrome
This project will directly investigate primary neutrophils from individuals with genetic deficiencies in DNA damage signalling. There is currently no therapy for Nijmegen breakage syndrome and ataxia telangiectasia, and treatment is based on palliative care only. If neutrophil responses are found to be dysregulated in these patients, it will have immediate consequences for their treatment options and hopefully enhance their quality of life.

2) Private sector beneficiaries

a. Pharmaceutical product development by the private sector

Neutrophils are important therapeutic targets in inflammatory diseases. My project will investigate cell cycle pathways in regulation of migration and NETs. Importantly, pharmaceuticals targeting these pathways are already in development for cancer by several companies with operations in the UK. Some of these products have even completed clinical trials, meaning they could easily be repurposed for treatment of these additional inflammatory disorders. Results from this project will thus have important ramifications for pharmaceutical product development and design of clinical trials, leading to increased competitiveness for the UK pharmaceutical sector.

b. Biotechnology product development by the private sector

In recent years, there has been considerable interest in harnessing the health benefits of probiotics and several products have come on the market. Probiotic bacteria are proposed to dampen inflammation; interestingly, the beneficial component of the commonly used probiotic E. coli Nissle 1917 (marketed as Mutaflor in the EU) was shown to be the genotoxin colibactin, which I will investigate for its ability to modulate neutrophil migration. My work on genotoxins will therefore be of direct benefit to the UK private sector in informing design and manufacture of biotechnologies based on probiotics, which may contribute to improved overall economic performance.