In March this year, two University of Michigan physicians spotted a striking similarity between an autoimmune disease they had been studying and COVID-19. Both conditions appeared to involve blood clots in arteries, veins, and the microvasculature, rheumatologist Jason Knight and cardiologist and vascular medicine specialist Yogen Kanthi observed. Because a release of neutrophil extracellular traps (NETs), webs of chromatin and proteins flung from immune cells, underlies the excessive clotting seen in the autoimmune disease, known as antiphospholipid syndrome (APS), they decided to study whether NETs could be relevant to COVID-19 as well.
The research team used serum samples collected at admission from 50 patients with a confirmed diagnosis of COVID-19 based on a PCR test, and measured their levels of NET biomarkers—specifically, cell-free DNA, myeloperoxidase-DNA, and citrullinated histone 3.
The results, published April 24 in JCI Insight, show increased levels of these biomarkers in sera of COVID-19 patients with severe disease compared to healthy controls. Kanthi tells The Scientist that the amount of NET biomarkers at admission was predictive of which patients progressed to respiratory failure. The team also found that sera from COVID-19 patients triggered NET formation from control neutrophils in vitro.
Sampling these biomarkers could aid decision-making regarding the allocation of precious resources, such as ventilators, he says.
Paul Kubes, an immunologist at the University of Calgary who works on NETs and is currently studying their function in COVID-19, says that the study was largely correlative, but “they provided pretty good evidence that there are NETs” in COVID-19. Now researchers want to determine whether NETs can be used not only to predict disease symptoms, but stop them as well.
Neutrophils in COVID-19
NETs are extracellular DNA fibers studded with histones and antimicrobial proteins and enzymes, such as neutrophil elastase and myeloperoxidase, which form a meshwork that ensnares and kills pathogens. NETs were first described in bacterial infections by Volker Brinkmann and Arturo Zychlinsky at the Max Planck Institute for Infection Biology in Germany in 2004. The role of neutrophils and NETs in viral infections was recognized by Kubes and others in 2012.
For Harvard Medical School’s Denisa Wagner, who has been working on NETs for the past 12 years and studying their role in COVID-19 since February, the presence of NETs in COVID-19 was a foregone conclusion. She tells The Scientist that almost every virus induces NETosis, as the process of NET formation is called.
NETs are released by neutrophils, which often die during the process. Along with macrophages, neutrophils are the prime phagocytic cells or scavengers of the body, and are part of the innate immune system—the first line of defense against pathogens.
Although NETs evolved as part of the innate immune response by the host, they can cause significant collateral damage, such as inflammation and blood clots. “I would not be surprised if it turned out that NETs have clinical relevance in severe COVID-19,” says Brinkmann.
In a perspective piece published in the Journal of Experimental Medicine in April, Betsy Barnes, an immunologist at the Feinstein Institutes for Medical Research, Mikala Egeblad, a breast cancer researcher at Cold Spring Harbor Laboratory, and others report extensive neutrophil infiltration and inflammation in the capillaries of the lungs as well as neutrophils in the alveoli, or air spaces, of the lungs in autopsy specimens from three patients who succumbed to COVID-19. Knight, who is also a coauthor of the report, tells The Scientist in an email, “It looks like NETs can be found at the scene of crime. Of course, further mechanistic studies will be needed to prove causality”—that is, whether NETs are indeed behind the damage.
The authors of this report and the study in JCI Insight are part of a consortium called the NETwork to Target Neutrophils in COVID-19, which has about 25 members located in five countries. The NETwork was started by Egeblad with the goal to rapidly determine whether neutrophils and NETs play a role in COVID-19.
“We really need more data to know what is going on there,” says Barnes, who studies NETs in the context of autoimmune disease.
Egeblad, who has been working on the role of NETs in cancer metastasis, says that the next step would be to establish causality. “This will probably not be known for several months as causality can only be shown either directly through COVID-19 experimental model systems that are only just emerging or indirectly by targeting NETs with drugs in clinical trials,” she adds.
Targeting NETs in COVID-19 clinical trials
DNase—the enzyme that degrades DNA—is the only FDA-approved NET inhibitor; this seems to be well tolerated by cystic fibrosis patients, some of whom have NETs in their mucus. “If the mucus in the lungs of COVID-19 patients contains NETs, this treatment should fluidify the mucus and allow better gas exchange,” says Brinkmann.
A recent paper proposes the use of DNase against runaway levels of NETs in severe SARS-CoV-2 infection. Brinkmann explains that DNA-containing chromatin is the backbone of NETs. Attached to this backbone are protein complexes that contain cytoplasmic enzymes and peptides. “If you destroy the DNA, NETs fall apart like a torn pearl necklace.”
Currently, two clinical trials in the UK and France are testing DNase in COVID-19 patients. Barnes is leading a retrospective study in collaboration with Cold Spring Harbor Laboratory on COVID-19 patients who were treated with DNase while on mechanical ventilation. In Canada, Kubes is exploring the possibility of running a clinical trial with DNase on COVID-19 patients admitted to the ICU.
NETs can also be targeted by experimental drugs that block their formation. Kubes says that neutrophil elastase inhibitors, which block an enzyme required for NETosis, would be very effective “because you prevent NET release rather than just destroying NETs that have already been made.” Wagner is also in talks with an Indian company that is testing peptidylarginine deiminase 4 (PAD4) inhibitors, which interfere with another enzyme that is essential during NET formation.
The link between NETs and thrombosis
Blood clots are a frequent complication of COVID-19, and scientists are beginning to suspect that NETs might be the culprit.
Wagner’s team first showed in 2010 that NETs are linked to thrombosis. “NETs are terribly thrombogenic,” she says. So, whenever there is neutrophilia, an excess of neutrophils in the blood circulation, there is an associated predisposition to thrombosis. Neutrophilia has been a constant theme across all cohorts of COVID-19 patients, and complications related to clotting, ranging from pulmonary embolism to swollen, painful, discolored toes, are increasingly being reported.
Adrian Newland, a hematologist at Barts and The London NHS Trust, tells The Scientist in an email that globally among hospitalized patients with COVID-19, there is a 33 percent incidence of venous thromboembolism (VTE), a condition where blood clots clog the veins and may break off and lodge in the lungs. “This reflects a disordered clotting system as a reaction to the infection, and is well described, although we learn more about the condition on an almost daily basis,” he says.
In a preprint posted on May 5, Kanthi and Knight demonstrated significantly elevated NET remnants in the sera of three COVID-19 patients presenting with VTE. The authors say that given the strong association between NETs and thrombosis in other diseases, the potential link between NETs and VTE in COVID-19 warrants exploration.
Intrinsic to VTE are platelets, the chief blood cells that stop bleeding, and they may be critical in this emerging connection between blood clots, NETs, and COVID-19. “Platelets activate neutrophils, which form NETs,” Wagner explains, “but NETs bind platelets and activate them, so it snowballs.”
Targeting platelets could indirectly lead to reduced NET formation. In fact, the Michigan group led by Kanthi is currently planning a clinical trial to test whether the anti-platelet drug dipyridamole could be used to treat COVID-19 patients. They recently showed that the drug protects against NETosis and thrombosis in APS.
“Whether anti-platelet agents have a part to play here is up for discussion,” says Newland. He adds that there is also the counterargument that if used in patients with low platelets (mildly reduced platelet counts are consistently seen in COVID-19), they may lead to more bleeding events.
Y. Zuo et al., “Neutrophil extracellular traps in COVID-19,” JCI Insight, doi:10.1172/jci.insight.138999, 2020.
B.J. Barnes et al., “Targeting potential drivers of COVID-19: Neutrophil extracellular traps,” J Exp Med, doi:10.1084/jem.20200652, 2020.