Here, we report for the first time to our knowledge elevated levels of serum NETs in many hospitalized patients with COVID-19. We measured 3 markers commonly used to detect NET remnants in blood (cell-free DNA, MPO-DNA, and Cit-H3) and found significant elevations in all 3. We also found that COVID-19 sera are potent stimulators of NETosis when added to control neutrophils. Taken together, these data provide evidence that COVID-19, at least in hospitalized patients, is a pro-NETotic state. The triggers of NETosis in COVID-19 are potentially myriad and will require further investigation. Possibilities include virus-damaged epithelial cells (14, 30), activated platelets (31, 32), activated endothelial cells (33), and inflammatory cytokines, such as IL-1β (20, 34), IL-8 (35, 36), granulocyte colony-stimulating factor (37, 38), and likely others (18).

Of the markers we tested, cell-free DNA was most closely aligned with traditional inflammatory markers used to track COVID-19, including C-reactive protein, D-dimer, and lactate dehydrogenase. Notably, although cell-free DNA is not a highly specific marker for NETs, it was strongly correlated with absolute neutrophil count, as was the more specific marker of NETs, MPO-DNA. Somewhat unexpectedly, Cit-H3 did not correlate well with the other 2 markers but did associate strongly with platelet levels. It is believed that the predominant driver of histone citrullination (i.e., production of Cit-H3) in NETs is the enzyme peptidylarginine deiminase 4 (PAD4) (39). However, neutrophils can be triggered to undergo NETosis by a variety of stimuli, and in vitro studies demonstrate that not all pathways to NETosis are equally reliant on PAD4 activity (40); for example, stimuli that lead to robust reactive oxygen species production may be relatively PAD4 independent (41). The dichotomy between MPO-DNA and Cit-H3 levels in the COVID-19 sera tested here potentially suggests that 2 or more pathways to NETosis are active in patients with COVID-19, with the pathway leading to Cit-H3 perhaps having some relationship to platelets (42). It should also be noted that neutrophils are relatively short-lived cells that may experience cell death through many pathways, including apoptosis, necrosis, pyroptosis, NETosis, and others. Markers such as lactate dehydrogenase, cell-free DNA, and Cit-H3 may therefore also be produced by neutrophil cell death that is independent of NETosis (43, 44). The activation of other cell death programs, and their relationship to the inflammatory storm, certainly warrant further investigation in COVID-19.

NETs were first described in 2004 as a novel pathogen eradication strategy that could function as an alternative to phagocytosis (36), but it is now recognized that NETs have double-edged-sword properties and likely exacerbate (and in some cases even initiate) autoimmune and vascular diseases (45). NETs present and stabilize a variety of oxidant enzymes in the extracellular space, including MPO, NADPH oxidase, and nitric oxide synthase (46), while serving as a source of extracellular histones that carry significant cytotoxic potential (47, 48). In light of these toxic cargo, it is not surprising that NETs play a role in a variety of lung diseases, including cystic fibrosis (where they occlude larger airways) (49),smoking-related lung disease (50), and, with particular relevance here, pathogen-induced acute lung injury and ARDS (14, 51, 53). NETs have also been very well studied in the setting of cardiovascular disease, where they infiltrate and propagate inflammation in the vessel wall (53) and, when formed intravascularly, occlude arteries (54), veins (55), and microscopic vessels (56). Early studies of COVID-19 suggest a high risk of morbid arterial events (57), and one can speculate that the risk of venous thrombosis will increasingly reveal itself as more data become available (58).

Severe COVID-19 appears to be defined by neutrophilia, as well as elevations in IL-1β, IL-6, and D-dimer (18), the latter suggesting hyperactivity of the coagulation system. All these findings have significant potential for crosstalk with NETs. NETs are linked to IL-1β (both upstream and downstream) in cardiovascular and pulmonary diseases (19–22), including as described by our group for venous thrombosis (9). The same is true for IL-6, either directly (23), or perhaps with IL-1β as an intermediary (24). Of course, as discussed above, examples of NETs as drivers of thrombosis are myriad because intravascular NETosis is responsible for initiation and accretion of thrombotic events in arteries, veins, and — particularly pertinent to COVID-19 — the microvasculature, where thrombotic disease can drive end organ damage in lungs, heart, kidneys, and other organs (59, 60). Mechanistically, NETs, via electrostatic interactions, activate the contact pathway of coagulation (61), while presenting tissue factor to activate the intrinsic pathway (62). Simultaneously, serine proteases in NETs dismantle natural brakes on coagulation, such as tissue factor pathway inhibitor and antithrombin (63). Bidirectional interplay between NETs and platelets may also be critical for COVID-19–associated microvascular thrombosis, as has been characterized in a variety of disease models (60, 61).

Of interest, a recent small study performed in China suggested potential efficacy of the adenosine receptor agonist, dipyridamole, in severe cases of COVID-19 (64). Dipyridamole is an FDA-approved drug that our group recently discovered to inhibit NET formation by activation of adenosine A 2A receptors (7). In the aforementioned trial, patients with COVID-19–associated bilateral pneumonia were treated with oral dipyridamole for 7 days, in addition to treatment with antiviral agents (64). As compared with controls, dipyridamole-treated patients demonstrated improvements in platelet counts and D-dimer levels (64). Given the urgent need for effective treatments for COVID-19, a randomized study to characterize the impact of dipyridamole on COVID-19–related NETosis, thrombo-inflammatory storm, and, of course, outcomes may be warranted. Other approaches to combating NETs have been reviewed (65, 66) and include the dismantling of already-formed NETs (deoxyribonucleases) and strategies that might prevent initiation of NET release, including inhibitors of neutrophil elastase and PAD4.

This study is not without limitations, including the use of serum samples retrieved from the clinical laboratory, rather than samples drawn specifically for research purposes. Here, it is certainly possible that NETs were partially degraded over time, thereby lowering our measurements. It should also be emphasized that it is not clear whether the NET remnants described here are drivers of disease severity or a mere consequence of acute inflammation in patients. Indeed, the definitive accounting of COVID-19 pathophysiology and answering questions of causality will likely await the development of model systems. Our hope, though, is that these findings will ignite further research into the role of neutrophil effector functions in the complications of COVID-19 (67). As a first step, future studies should investigate the predictive power of circulating NETs in well-phenotyped longitudinal cohorts. Furthermore, given the dichotomy we found here between MPO-DNA and Cit-H3, investigators should be encouraged to continue to include diverse markers of NETosis in future studies. As we await definitive antiviral and immunological solutions to the current pandemic, we posit that antineutrophil therapies may be part of a personalized strategy for some individuals affected by COVID-19 who are at risk for progression to respiratory failure.