One of the conundrums presented by COVID-19 is that it is not uncommon for patients to present with remarkably low oxygen saturation levels but with very little breathlessness. Additionally, it has been noted that there is an additional risk of stroke in COVID-19 patients (and COVID-19-recovered patients) and that they seem to have what is being referred to as ‘sticky blood’. A recent paper by Professor Yost (Department of Pediatrics, University of Utah School of Medicine, UT, USA) and colleagues ‘Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome’ discusses the role that neutrophil extracellular traps (NETs) can play in the development of these symptoms and prothrombotic disease outcomes. CLI was very fortunate to catch up with Professor Yost to learn more about NETs and their effects in COVID-19 and other conditions.
For those of us for whom ‘Immunology 101’ was a number of years ago, can you give us a brief overview of neutrophils, what they are and how they contribute to our immune system?
For some, ‘Immunology 101’ may have described poly morphonuclear leukocytes (PMNs; neutrophils) as cells pre-programmed and equipped for one task – to respond to potential microbial invasion. Neutrophils represent the most numerous circulating white blood cell (WBC), and if unchallenged with inflammatory stimuli, they circulate for 1 to 2 days before undergoing apoptosis and being replaced by new neutrophils continuously produced by the bone marrow. When challenged, however, with inflammatory stimuli produced by microbial invasion or sterile inflammatory tissue damage, neutrophils respond first as the primary effector WBC of the innate immune response. Neutrophils rapidly chemotax to areas of inflammation, release pro-inflammatory mediators through degranulation, and phagocytize offending microbes with intent to kill engulfed pathogens intracellularly. Thus, in the phagolysosome, microbes encounter high concentrations of microbicidal factors contained in the neutrophil granules coupled with superoxide anion and hydrogen peroxide generated through intracellular NADPH-oxidase activity. The antimicrobial activities of neutrophils are absolutely essential as evidenced by the lifethreatening and recurrent infections evident in patients with syndromes of neutrophil dysfunction such as chronic granulomatous disease or leukocyte adhesion deficiency.
Early descriptions of neutrophils, however, failed to recognize other neutrophil activities such as cytokine synthesis and secretion or the clearance of DNA-containing debris in sterile inflammatory injury. Both of these activities require de novo transcription and translation suggesting that the neutrophil participates in nuanced ways within the inflammatory milieu. Thus, the neutrophil represents much more than a vehicle for preformed microbicidal factors to areas of infection.
In your recent paper  you discuss neutrophil extracellular traps – what are these, how are they created/regulated and what is their significance to our immune response?
In 2004, Brinkmann et al. described for the first time a novel cell death pathway leading to the formation of neutrophil extracellular traps (NETs) . This neutrophil cell death process, termed NETosis, is distinct from apoptosis and necrosis and can be induced by various inflammatory stimuli. These inflammatory stimuli include not only pathogen-associated molecular patterns [PAMPs; lipopolysaccharide (LPS), lipoteichoic acid (LTA), fungal elements, etc.] recognized by toll-like and other receptors, but also inflammatory cytokines, markers of endothelial cell damage [von Willebrand factor (vWF), etc.], activated platelets, and damage-associated molecular patterns [DAMPs; heme, high mobility group box protein 1 (HMGB1), histones, etc.]. In neutrophils primed for NETosis, inflammatory stimuli trigger nuclear decondensation and dissolution of the nuclear membrane. Granule membranes also dissolve and allow for complete mixing of granule contents – degradative enzymes and microbicidal factors – with decondensed nuclear chromatin. With subsequent rupture of the plasma membrane, neutrophils release the decondensed chromatin decorated with the antimicrobial factors into the extracellular space to form NETs. These webs of sticky chromatin and antimicrobial factors then trap and putatively kill microbes in the extracellular space. In addition, others have described ‘vital NETosis’ where the neutrophil decondenses only part of its tightly packed chromatin and releases a NET into the extracellular space without undergoing cell death. Furthermore, the process of releasing extracellular traps in the inflammatory response is not limited to neutrophils; other granulo-cytes (such as eosinophils, macrophages, and mast cells) form extracellular traps in response to a variety of inflammatory signals.
It remains unknown why only a of subset of neutrophils seem to be primed for NETosis at any given time. This represents perhaps a determination of cellular senescence mediated by regulators of the cell cycle in neutrophils. Once triggered for NETosis, several mechanistic pathways come into play, which may vary depending on the factors inducing NETosis. Clear evidence exists for neutrophil elastase and myeloperoxidase release from granules with translocation to the nucleus leading to histone degradation and chromatin decondensation. Similarly, clear evidence exists for peptidyl-arginine-deiminase 4 (PAD4) enzymatic activity in converting histone arginine amino acid residues to citrulline and thus releasing electrostatic forces which help maintain the tightly compacted chromatin structure. Others have implicated additional signalling cascades such as the mammalian Target of Rapamycin/ Hypoxia-inducible Factor 1 alpha pathway, the pathway leading to autophagy, and regulatory molecules in control of cell cycle.
Although the importance of NET formation in vivo is often hotly debated, clear evidence exists demonstrating that NETS, at the very least, trap pathogens and prevent the spread of an infection from its original nidus. However, ample evidence now exists linking dysregulated NET formation with inflammatory tissue damage and patient morbidity and mortality. NETs formed in an inappropriate magnitude, the wrong location, or at the wrong time within the inflammatory response now predict pathology in inflammatory syndromes such as systemic lupus erythematosus, sepsis, stroke, atherosclerosis, and cancer.
Typically, when we have a disease or an infection, our symptoms are caused by the way our immune system reacts – what symptoms do NETs produce?
NETs represent one potent facet of the innate immune response to tissue injury and/or infection. The potential for NETs to trap microbes and limit the spread of pathogens represents a real advantage selected for over evolutionary time. However, in settings of overwhelming infection, chronic infection, cancer, or autoimmune diseases, NETs can participate pathogenically in the disease process. In sepsis, NETs can create endothelial tissue damage leading to capillary leak and hypotension. In both infectious and sterile acute lung injury, NETs in the pulmonary vasculature and alveoli can impair lung function, potentiate ventilation/perfusion mismatch, and increase the risk for ‘acute respiratory distress syndrome’ (ARDS). NETs also serve as scaffolds for activated platelets and procoagulant factors such as vWF and platelet factor 4 (PF4) leading to thrombosis. This process of thrombosis resulting from the interaction of procoagulant factors with elements of the immune response is termed immunothrombosis, and may also play a role in ischemic stroke. NETs can also serve as a safe harbour for some microbes potentiating the risk for chronic infection. NET formation in the lungs of cystic fibrosis patients following microbial colonization correlates with airway obstruction, endothelial and epithelial cell damage, and persistent colonization with microbes, most notably of Pseudomonas aeruginosa and Staphylococcus aureus. NETs also appear to participate in cancer, and researchers now implicate tumour-associated neutrophils with protection of the tumour from the host immune defences. The formation of cancerinduced NETs throughout the body may also serve as potential safe landing sites for metastatic tumour cells. In autoimmune diseases such as systemic lupus erythematosus, NET formation not only damages host tissues leading to potential multi-organ failure in severe cases, but also serves as the source of autoantigens leading to the anti-self antibodies at the heart of so many autoimmune diseases. Thus, dysregulated NET formation can play a variety of pathogenic roles in different disease processes.
If NETs are a factor in immunothrombosis, are they linked in any way to the symptoms we are seeing in COVID-19 patients?
Our recent work does suggest that immunothrombosis plays a dramatic role in severe COVID-19 disease, contributing not only to the severe ARDS experienced by many patients, but also to the increased risk of thrombotic disorders encountered by many. While our study failed to look at thrombotic complications in our cohort, others have demonstrated that COVID-19 patients can present anywhere along the spectrum of thrombotic disease. Some COVID-19 patients remain asymptomatic until they develop a thrombotic complication such as a myocardial infarction or ischemic stroke. Others present with pronounced coagulopathy, pulmonary embolism, thrombosis-associated acute kidney disease, or microvascular clots.
Our findings of diffuse NET-associated microvascular clots in the lungs of COVID-19 patients at autopsy strongly supports a role for NETs contributing to the severity of the ARDS and hypoxia noted in many COVID-19 patients. In addition, some patients with COVID-19 present without respiratory distress but significant hypoxia, and these microvascular clots may indeed generate significant ventilation/ perfusion mismatch resulting in hypoxia without hypercapnia and respiratory distress seen in some patients.
If NETs contribute to the severity of COVID-19, can this knowledge be used to create a test or screen to identify the most at-risk patients?
Data from our prospective cohort of COVID-19 patients demonstrate a clear correlation of increasing NET levels with increasing mortality, ICU hospitalizations, and disease severity scores. We assessed plasma NET levels in these patients using a myeloperoxidase (MPO)- DNA ELISA. Basically, MPO, a granule protein in neutrophils and an antimicrobial factor, is complexed with decondensed chromatin during the process of NETosis. When we detect MPO-DNA complexes in plasma, we can use them as a surrogate for NET formation as MPO does not normally complex with chromatin outside of the process of NETosis. While we feel that this assay may be developed for clinical risk stratification down the road, significant uncertainty remains regarding assays for NETs in plasma. It is entirely possible that plasma NET levels measured via MPO-DNA ELISA may not fully capture the degree of NET formation in some patients due to rapid NET degradation or organ specific NET formation not represented in the plasma sample. Use of NET quantitation in plasma from COVID-19 patients will require additional study before plasma NET levels can be added to the risk stratification calculation for our clinical colleagues on the front lines of this pandemic.
How do you envisage that this knowledge will help with treatment and prognosis of COVID-19 patients and can it also be transferred to other conditions known to be caused/aggravated by NETs?
Clearly you saved the hardest question for last! I remain fascinated by the role that NETs play in COVID-19 and participate actively in the ‘NETwork to Target Neutrophils in COVID-19’, a group led by Mikala Egeblad at Cold Spring Harbor Laboratory. The NETwork seeks to further characterize the role of NETs and neutrophils in COVID-19. One component of my work in this area suggests that activated platelets play a significant role in the coagulopathy associated with COVID-19. Such platelet hyperreactivity may respond to anti-platelet medications like aspirin in addition to the standard of care anticoagulant treatment with heparin or low molecular weight heparin. Further study is warranted. Additional work from our laboratory group also hints at the potential for NET inhibitors to improve outcomes in COVID-19. However, such compounds remain investigational, and the testing of novel therapeutic agents lags behind the need as we wait for the development of pre-clinical models of SARS-CoV-2 infection leading to experimental COVID-19-like disease. The ‘NETwork to Target Neutrophils in COVID-19’ also facilitates COVID-19 clinical trials of off-label use of drugs currently available in the clinic. One trial is currently recruiting patients to evaluate the potential therapeutic efficacy of dornase alpha (Pulmozyme) in COVID-19 with the hypothesis that degrading NETs will improve outcomes. Other trials for medications such as dipyridamole, which may target NETs and neutrophils in COVID-19, are under development.
With regard to NET inhibition in other inflammatory syndromes, my laboratory recently discovered NET-inhibitory peptides in the cord blood of newborn infants. These peptides robustly inhibit NET formation in vitro and in vivo. Use of these peptides in translational, pre-clinical models of sepsis significantly inhibit NET formation and improve survival, suggesting that, at least in models of sepsis, NETs play a pathogenic role and that NET inhibition may ameliorate the manifestations of disease in other non-COVID-19 inflammatory diseases of neonates, children, and adults.
Program Director – Neonatal ECMO Therapy, Primary Children’s Hospital,
SLC, UT, USA
Principle Investigator – Molecular Medicine Program, University of
Utah School of Medicine, SLC, UT, USA
Professor – Department of Pediatrics, University of Utah School
of Medicine, SLC, UT, USA