Neutrophil Extracellular Traps: not just for catching pathogens.

The most abundant white blood cell type in humans are neutrophils, comprising 50-70% of all circulating leukocytes. Neutrophils are the first leukocytes to accumulate at sites of infection/inflammation and play a vital role in eliminating pathogens. Traditionally, this role consisted of engulfing pathogens via phagocytosis, generation of reactive oxygen species (ROS), and the release of microbicidal molecules (proteinases, reactive oxygen species, and/or anti-bacterial proteins) from granules to kill microbes. However, a 2004 study identified another method used by neutrophils to prolong their anti-microbial function: Neutrophil Extracellular Traps (NETs) (1).

NETs are exactly as they sound: a web-like structure released from activated neutrophils that can trap and kill pathogens. The web-like structure is comprised primarily of decondensed chromatin, with histones and granular proteinases (i.e. Neutrophil elastase (NE), myeloperoxidase, proteinase 3) dispersed throughout the web. NETs are typically induced at sites of infection through pattern recognition receptor (i.e. TLR) activation by bacterial components like lipopolysaccharide (LPS), inflammatory cytokine (IL-8, TNF-?) signaling, and/or activation of Fc receptors (1). An alternative cell death mechanism that produces NETs, termed NETosis, has several distinct features. First, the nucleus delobulates and granular membranes disappear, allowing for membrane vesiculation. As a result, chromatin decondenses and mixes with the granular components. As the chromatin continues to decondense, the cell plasma membrane ruptures and the NET is released into the extracellular milieu where it can ensnare pathogens and exert it’s anti-microbial effects through modified hitsone and granular proteinases contained within it (2). Released MPO and peptidylarginine deiminase 4 (PAD4) help mediate chromatin decondensation that drives the rupturing of nuclear and plasma membrane in NETosis (3,4,5). The involvement of protein citrullination by PAD4 suggests a possible link between NETs and rheumatoid arthritis (RA), where anti-citrullinated protein antibodies (ACPAs) are a key marker (6).

Indeed, netting neutrophils have been found in peripheral blood, synovial fluid/tissue, skin, and RA nodules of RA patients (7). Synovial fluid from RA patients also typically has an abundance of neutrophil granule derived proteins (NE, MPO, etc.) that can damage joint tissue, further strengthening the linkage between NETs and RA. In fact, NETs appear to play a role in a variety of autoimmune diseases including systemic lupus erythematosus (SLE) (8), gout (9), and vasculitis (10). For instance, degraded NETs in blood may provide autoantigens for the development of anti-DNA antibodies seen in SLE. Accumulation of NET immune complexes in the kidneys could also be involved in the pathogenesis of lupus nephritis (1). Further research of NETs in inflammatory diseases could lead to new treatment modalities for complex autoimmune diseases.

The influence of NETs may even extend beyond immune-mediated diseases. A 2013 study looking at NETs in Ewing sarcoma patients found that patients with tumor-associated neutrophils (TANs) in tumor biopses had a poorer prognosis than patients without netting neutrophils (11), although NETs were not directly identified. This hypothesis was further tested by evaluating cancer metastases in a mouse sepsis model to elucidate a linkage of postsurgical infections and poor cancer prognosis (12). Sepsis was induced in mice through cecal ligation and puncture (CLP) while a control group was given a sham surgery. The CLP group showed significantly higher levels of extracellular DNA deposition in liver than control groups. Both groups were then intrasplenically injected with H59 Lewis lung carcinoma cells to mimic the increase in circulating tumor cells associated with non-small cell lung cancer surgery. The CLP group receiving H59 cells showed significantly higher hepatic metastases compared to the control group. Treatment of these mice with DNase or NE inhibitors attenuated metastasis, indicating that NETs may promote cancer cells adhesion and enhance tumor cell metastasis (12). Additionally, NETs may be involved in local cancer growth and progression by promoting angiogenesis, as well as promoting  tumor-associated thrombosis, which is the second leading cause of death in cancer patients (13). 

It appears that NETs can have a drastic effect on a variety of disease states. While there has been a plethora of research studying NETs since their discovery in 2004, there is still much we do not know about NETs. In fact, recent studies have found extracellular net structures are not unique to neutrophils and are found in mast cells, eosinophils, and monocytes/macrophages (14). Basic science research using animal models of inflammatory diseases like rheumatoid arthritis, sepsis, systemic lupus erythematosus, and cancer will further deepen our understanding of how NETs (and other extracellular traps) influence disease states and how these pathways can be exploited for therapeutic use. 

References

1. M. Kaplan, M. Radic, Neutrophil Extracellular Traps: double-edged sword of innate immunity. J Immunol 189, 2689-2695 (2012).
2. G. Sollberger, D. Tilley, A. Zychlinsky, Neutrophil extracellular traps: the biology of chromatin externalization. Dev Cell 44, 542-553 (2018).
3. H. Hussein, N. Palaniyar, Post-translational modifications in NETosis and NETs-mediated diseases. Biomolecules 9, (2019).
4. E. Neubert et al., Chromatin swelling drives neutrophil extracellular trap release. Nature Comm 9, (2018).
5. Q. Remijsen et al., Dying for a cause: NETosis, mechanisms behind an anti-microbial cell death modality. Cell Death Differ 18, 581-588 (2011).
6. F. Pratesi et al., Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps. Ann Rheum Dis 73, 1414-1422 (2014).
7. F. Apel, A. Zychlinsky, E. Kenny, The role of neutrophil extracellular traps in rheumatic diseases. Nat Rev Rheumatol 14, 467-475 (2018).
8. E. A. Chapman et al., Caught in a trap? Proteomic Analysis of neutrophil extracellular traps in rheumatoid arthritis and systemic lupus erythematosus. Front Immunol, (2019).
9. R. Balázs, Neutrophil extracellular traps and microcrystals. J Immunol Res,  (2017).
10. D. Söderberg, M. Segelmark, Neutrophil extracellular traps in vasculitis, friend or foe? Curr Opin Rheumatol 30, 16-23 (2018).
11. S. Berger-Achituv et al., A proposed role for neutrophil extracellular traps in cancer immunoediting. Front Immunol 4, (2013).
12. J. Cools-Lartigue et al., Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest 123, 3446-3458 (2013).
13. L. Erpenbeck, M. Schön, Neutrophil extracellular traps: protagonists of cancer progression? Oncogene 36, 2483-2490 (2016).
14. O. Goldmann, E. Medina, The expanding world of extracellular traps: not only neutrophils but much more. Front Immunol 3,  (2012).