Neutrophil extracellular traps (NETs) kill pathogens via the release of chromatin and anti-microbial granule enzymes in an extracellular mesh or “net”. NET release represents a novel form of cell death (NETosis), distinct from apoptosis and necrosis, that can be induced by inflammatory agents (LPS, TNFα, IFNα) and micro-organisms. During NETosis, reactive oxygen species (ROS) production and protein-arginine deiminase (PAD)-4 activation induces histone citrullination, chromatin disruption and disintegration of the nuclear membrane. Granule enzymes are released from membrane-bound organelles into the cytoplasm, whereupon they mix with histones and decondensed chromatin, before the protein-loaded DNA is released via disruptions in the cell membrane. NETs are also a source of autoantigens in autoimmune diseases, including systemic lupus erythematosus (SLE) where they contribute to organ damage and nephritis.
Rheumatoid arthritis (RA) is a chronic, inflammatory arthritis that affects ~1% of the adult UK population. RA is heterogeneous in terms of severity, pathology and response to therapy. Neutrophils contribute to RA pathology via secretion of collagen-degrading enzymes, ROS, cytokines and chemokines. Emerging evidence implicates NETs as a source of citrullinated neoepitopes in RA, causing loss of immune tolerance and development of autoantibodies to citrullinated proteins (ACPA). Citrulline residues in aggrecan and vimentin are preferentially recognised by antigen-presenting cells in individuals carrying the HLA-DRB1*04:01/04 allele providing a molecular explanation for the strong association between this allele and the development of RA. NETosis is enhanced in neutrophils from RA patients, compared to healthy controls, and RA sera, particularly those with high ACPA titres, stimulate NETosis and cross-react with citrullinated histone-H4 derived from neutrophil NETs.
Recent work in our group has identified over 300 proteins found in NETs from patients with RA and SLE using quantitative proteomics, and several of these are significantly different depending on the agonist used to stimulate NETosis. This suggests that NETosis is a carefully orchestrated, agonist-specific process, rather than just a rapid non-specific response to inflammation. Many of these proteins have now been visualised in RA and SLE NETs using immuno-fluorescence antibody staining, and interestingly we have identified some proteins that are differentially expressed in RA and SLE NETs.
This studentship will significantly extend our current analysis of NET proteomics data from RA and SLE patients, and carry out further work to explore the molecular regulation of NET production in RA and SLE neutrophils. The broad aims of the project will be to:
1. Carry out further analysis of the NET proteomics data, including identification of post-translationally modified peptides (e.g. citrullination, methylation etc).
2. Collect new patient samples and visualise NET proteins using immuno-fluorescence microscopy to confirm proteomics data.
3. Carry out experiments to investigate the signalling pathways involved in NET production in response to different agonists, including investigating the effect of therapeutic inhibitors such as JAK inhibitors and APPA (a new treatment for arthritis currently in clinical trial in Liverpool).
4. Determine whether NET production in RA and SLE is different in patients at different stages of disease (early, late) and also whether NET production is different depending on auto-antibody status of patients (e.g. ACPA positive/negative RA).
The student will be provided with training in personal and professional development skills, by learning the following: planning their PhD, presenting research at seminars/conferences, creative thinking, problem solving and project management.
The Institute of Ageing and Chronic Disease is fully committed to promoting gender equality in all activities. In recruitment we emphasize the supportive nature of the working environment and the flexible family support that the University provides. The Institute holds a silver Athena SWAN award in recognition of on-going commitment to ensuring that the Athena SWAN principles are embedded in its activities and strategic initiatives.
The successful candidate should have, or expect to have an Honours Degree at 2.1 or above (or equivalent). Candidates whose first language is not English should have an IELTS score of 6.5 or equivalent. This project is most suitable to a student with an undergraduate degree or Master’s degree in the Life Sciences (Biology, Genetics, Biochemistry, Biomedical Sciences, Cell Biology and Medicine).
To apply: please send your CV and a covering letter to [email protected]
with a copy to [email protected]
Corsiero E, Bombardieri M, Carlotti E, Pratesi F, Robinson W, Migliorini P, Pitzalis C. Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs. Annals of the Rheumatic Diseases 2016; 75(10) 1866-75
Wright HL, Moots RJ, Edwards SW. The multifactorial role of neutrophils in rheumatoid arthritis. Nature Reviews Rheumatology 2014; 10(10) 593-601.
Wright HL, Makki F, Moots RJ, Edwards SW. Low density granulocytes: functionally distinct, immature neutrophils in rheumatoid arthritis with altered properties and defective TNF signalling. Journal of Leukocyte Biology 2017; 101 599-611
Wright HL, Moots RJ, Bucknall RC, Edwards SW. Neutrophil function in inflammation and inflammatory disease. Rheumatology 2010; 49(9), 1618-31.