New research finds a connection between destructive white blood cells and a more severe disease course in patients with COVID-19.
“We found that patients with COVID-19 infection have higher blood levels of neutrophil extracellular traps, also called NETs, which are a product of an inflammatory type of neutrophil cell death called NETosis,” says first author Yu (Ray) Zuo, M.D., a Michigan Medicine rheumatologist.
Zuo worked on the study with Yogen Kanthi, M.D., a cardiologist and vascular medicine specialist at the Michigan Medicine Frankel Cardiovascular Center, and Jason Knight, M.D., Ph.D., a rheumatologist at Michigan Medicine, who study inflammation and neutrophils. The researchers analyzed blood samples from 50 patients with COVID-19 for this publication.
Zuo and colleagues say, in light of the COVID-19 pandemic, there is an urgent need to better understand what causes the inflammatory storm and blood clots triggered by SARS-CoV-2 infection – a storm that leads to respiratory failure and a requirement for mechanical ventilation in many patients.
They believe NETs may be relevant to many aspects of COVID-19 research, given that thrombosis and inflammation are hallmarks of severe infection.
This is the first publication to come out of the Frankel CVC’s CV Impact Research Ignitor Grant program, which was created to address COVID-19 from both basic science and clinical perspectives.
Patients with severe coronavirus disease 2019 (COVID-19)–associated pneumonitis and/or acute respiratory distress syndrome (ARDS) have increased pulmonary inflammation, thick mucous secretions in the airways, elevated levels of serum pro-inflammatory cytokines, extensive lung damage, and microthrombosis.
This late stage of the disease is difficult to manage, and a large number of patients die (Chen et al., 2020a,Preprint; Wang et al., 2020; Zhao et al., 2020,Preprint; Zheng et al., 2020). The severity of COVID-19, combined with its pandemic spread, has placed unprecedented pressure on our healthcare system, and treatment strategies are urgently needed.
Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, but it is an exacerbated and poorly understood host response involving a cytokine storm that drives severe COVID-19 (Mehta et al., 2020).
It is unclear what initiates and propagates the cytokine storm. We propose that the exacerbated host response in patients with severe COVID-19 centers around the aberrant activation of the most common leukocyte in peripheral blood: the neutrophil. Neutrophilia predicts poor outcomes in patients with COVID-19 (Wang et al., 2020), and the neutrophil-to-lymphocyte ratio is an independent risk factor for severe disease (Liu et al., 2020,Preprint).
Furthermore, in autopsy samples from the lungs of three COVID-19 patients at Weill Cornell Medicine, we observed neutrophil infiltration in pulmonary capillaries, acute capillaritis with fibrin deposition, extravasation of neutrophils into the alveolar space, and neutrophilic mucositis (Fig. 1).
Neutrophil infiltration was also noted in two recent reports on the pathological findings from autopsied COVID-19 patients (Fox et al., 2020,Preprint; Yao et al., 2020). Although leukocytosis and neutrophilia are hallmarks of acute infection, in the case of COVID-19, we propose that neutrophilia could also be a source of excess neutrophil extracellular traps (NETs).
NETs and disease
Neutrophils are recruited early to sites of infection where they kill pathogens (bacteria, fungi, and viruses) by oxidative burst and phagocytosis (Schönrich and Raftery, 2016). However, neutrophils have another much less recognized means of killing pathogens: the formation of NETs (Brinkmann et al., 2004).
NETs are web-like structures of DNA and proteins expelled from the neutrophil that ensnare pathogens (Fig. 2). Expelling DNA to the extracellular space is not widely recognized as a critical immune function. Yet, even plants have specialized cells that kill soil pathogens by this mechanism (Wen et al., 2009).
NET formation is a regulated process, although the signals involved are incompletely understood. Key enzymes in the formation of NETs are: neutrophil elastase (NE), which degrades intracellular proteins and triggers nuclear disintegration; peptidyl arginine deiminase type 4 (PAD4), which citrullinates histones to facilitate the decondensation and release of the chromosomal DNA; and gasdermin D, which generates pores in the membrane of the neutrophil, thereby facilitating cell membrane rupture and the expulsion of DNA and the associated molecules (Chen et al., 2018; Kaplan and Radic, 2012; Papayannopoulos, 2018; Papayannopoulos et al., 2010; Rohrbach et al., 2012; Sollberger et al., 2018).
Although NETs are beneficial in the host defense against pathogens, collateral damage from sustained NET formation also stimulates many disease processes, including those that occur during viral infections (Schönrich and Raftery, 2016).
Indeed, excessive NET formation can trigger a cascade of inflammatory reactions that promotes cancer cell metastasis, destroys surrounding tissues, facilitates microthrombosis, and results in permanent organ damage to the pulmonary, cardiovascular, and renal systems (Jorch and Kubes, 2017; Kessenbrock et al., 2009; Papayannopoulos, 2018; Fig. 3). Importantly, these are three commonly affected organ systems in severe COVID-19 (Bonow et al., 2020; Chen et al., 2020b).
NETs and ARDS
Prior reports extensively link aberrant NET formation to pulmonary diseases, particularly ARDS. Indeed, NET levels in plasma are higher in patients with transfusion-associated ARDS than in subjects without ARDS (Caudrillier et al., 2012).
Furthermore, neutrophils from patients with pneumonia-associated ARDS appear “primed” to form NETs, and both the extent of priming and the level of NETs in blood correlate with disease severity and mortality (Adrover et al., 2020; Bendib et al., 2019; Ebrahimi et al., 2018; Lefrançais et al., 2018; Mikacenic et al., 2018).
Extracellular histones, likely partly originating from NETs, are elevated in the bronchoalveolar lavage fluid and plasma of ARDS patients (Lv et al., 2017). Naked histones are toxic to cells, and there is strong experimental evidence supporting a role for histones in ARDS and sepsis (Wygrecka et al., 2017; Xu et al., 2015).
It is therefore likely that NETs, as a source of extracellular histones, contribute to ARDS and sepsis (Chaput and Zychlinsky, 2009; Lefrançais and Looney, 2017; Xu et al., 2009). In animal models of lung injury, NETs develop in response to a variety of ARDS-inducing stimuli, and preventing or dissolving NETs reduces lung injury and increases survival (Caudrillier et al., 2012; Lefrançais et al., 2018; Liu et al., 2016; Narasaraju et al., 2011).
NETs and cystic fibrosis (CF)
The mucous secretions found in the airways of COVID-19 patients (Mao et al., 2020,Preprint) are reminiscent of those seen in CF patients (Martínez-Alemán et al., 2017). The cause and origin of these secretions are unclear.
However, in CF, mucous secretions impair gas exchange and have been shown to contain extracellular DNA, in part originating from NETs released in response to persistent lung infections. Furthermore, the excessive formation of NETs with increased NE makes the mucus thick and viscous (Manzenreiter et al., 2012), not only impairing ventilation but also facilitating the colonization of bacteria.
Such colonization further promotes neutrophil recruitment and NET formation, increasing mucus viscosity and consequently lowering the patient’s respiratory function. If the mucous secretions in COVID-19 contain NETs, they may play similar roles as they do in CF: impairing gas exchange and facilitating secondary infections.
NETs and excessive thrombosis
Acute cardiac and kidney injuries are common in patients with severe COVID-19 and contribute to the mortality of this disease (Bonow et al., 2020). D-dimer (a fibrin degradation product indicative of hyperactive coagulation) has emerged as a reliable marker of severe COVID-19 (Zhou et al., 2020).
High blood levels of NETs may explain these findings: intravascular NETs have been shown to play a vital role in initiating and accreting thrombosis in arteries and veins (Fuchs et al., 2012). For example, in severe coronary artery disease, complexes of NETs are elevated, and NET levels positively associate with thrombin levels, which predict adverse cardiac events (Borissoff et al., 2013).
In addition, autopsy samples collected from septic patients show that NETs infiltrate microthrombi (Jiménez-Alcázar et al., 2017). Thus, when NETs circulate at high levels in blood, they can trigger the occlusion of small vessels, leading to damage in the lungs, heart, and kidneys (Cedervall et al., 2015; Fuchs et al., 2010; Laridan et al., 2019; Martinod and Wagner, 2014).
In mouse models of septicemia, intravascular NETs form microthrombi that obstruct blood vessels and cause damage to the lungs, liver, and other organs (Jiménez-Alcázar et al., 2017). Mechanistically, NETs activate the contact pathway of coagulation (also called the plasma kallikrein–kinin system) via electrostatic interactions between the NET histones and platelet phospholipids (Oehmcke et al., 2009).
Histones can also promote platelet activation by acting as ligands for the Toll-like receptors on platelets (Semeraro et al., 2011). At the same time, NE (which is bound in its active form to NETs) likely also plays an important role by digesting the major coagulation inhibitors antithrombin III and tissue factor pathway inhibitor (Massberg et al., 2010). Furthermore, there is almost surely a feedback loop whereby pro-coagulant activity (e.g., that of thrombin) leads to platelet activation, and activated platelets then further enhance NET formation (Caudrillier et al., 2012; Clark et al., 2007; Fuchs et al., 2010; Massberg et al., 2010; Sreeramkumar et al., 2014; von Brühl et al., 2012).
Dissolving NETs with DNase I restores normal perfusion of the heart and kidney microvasculature in animal models (Cedervall et al., 2015; Jansen et al., 2017; Nakazawa et al., 2017; Raup-Konsavage et al., 2018).
Based on the above findings, we argue that targeting intravascular NETs may similarly reduce thrombosis in patients with severe COVID-19.
NETs and the cytokine storm
Severe COVID-19 is associated with a cytokine storm characterized by increased plasma concentrations of IL1β, IL2, IL6, IL7, IL8, IL10, IL17, IFNγ, IFNγ-inducible protein 10, monocyte chemoattractant protein 1 (MCP1), G-CSF, macrophage inflammatory protein 1α, and TNFα (Huang et al., 2020; Mehta et al., 2020; Ruan et al., 2020; Wu et al., 2020; Wu and Yang, 2020; Zhang et al., 2020).
These inflammatory mediators regulate neutrophil activity and induce the expression of chemoattractants (molecules that increase the trafficking of neutrophils to sites of inflammation). Moreover, cytokine storms lead to acute lung injury, ARDS, and death (Channappanavar and Perlman, 2017; Chousterman et al., 2017).
It is especially noteworthy that NETs can induce macrophages to secrete IL1β and that IL1β enhances NET formation in various diseases, including aortic aneurysms and atherosclerosis (Kahlenberg et al., 2013; Meher et al., 2018; Sil et al., 2017; Warnatsch et al., 2015).
Together, these data suggest that under conditions in which the normal signals to dampen inflammation are lost, such as during a cytokine storm, a signaling loop between macrophages and neutrophils can lead to uncontrollable, progressive inflammation.
Indeed, a correlation between NETs and IL1β exists in severe asthma (Lachowicz-Scroggins et al., 2019). If a NET–IL1β loop is activated in severe COVID-19, the accelerated production of NETs and IL1β could accelerate respiratory decompensation, the formation of microthrombi, and aberrant immune responses. Importantly, IL1β induces IL6 (Dinarello, 2009), and IL6 has emerged as a promising target for COVID-19 treatment (Mehta et al., 2020; Xu et al., 2020,Preprint).
IL6 can signal via classic and trans-signaling (Calabrese and Rose-John, 2014). In classic signaling, IL6 binds to a complex of the transmembrane receptor IL6Rα with the common cytokine receptor gp130. In trans-signaling, soluble IL6Rα (sIL6Rα) binds IL6 to initiate signaling via gp130.
Trans-signaling is strongly associated with pro-inflammatory states (Calabrese and Rose-John, 2014), and lower levels of sIL6Rα are associated with better lung function in, e.g., asthma (Ferreira et al., 2013; Hawkins et al., 2012).
Neutrophils can shed sIL6Rα in response to IL8 (Marin et al., 2002), which is abundant in the COVID-19–associated cytokine storm (Wu and Yang, 2020; Zhang et al., 2020). Together, these findings lead us to speculate that antagonizing IL-6 trans-signaling and/or IL1β could be effective indirect strategies for targeting neutrophils and NETs in severe COVID-19.
University of Michigan