Neutrophil released NETs to control microbial/viral infections, could serve as a therapeutic target in coronavirus infections.

In March this year, Dr. Knight and Kanthy of Michigan University observed a striking similarity between an autoimmune disease known as antiphospholipid syndrome (APS) and COVID-19. Both conditions appeared to involve blood clots in arteries, veins, and the microvasculature. 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, , they decided to study whether NETs could be relevant to COVID-19 as well.

The results, published April 24 inJCI Insight, show increased levels of these biomarkers in sera of COVID-19 patients with severe disease compared to healthy controls. 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. 

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. 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.

In a perspective piece published in the Journal of Experimental Medicine in April,  Mikala Egeblad 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.

The authors  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.

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.

Isolated human neutrophils forming NETs (neutrophil elastase labeled green and chromatin labeled red) 

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.

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 and they may be critical in this emerging connection between blood clots, NETs, and COVID-19.

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.

NETS and proteolytic storm

Neutrophils, through Neutrophil Elastase Traps (NETS), excessively activate STING, inducing further recruitment and the TRL4 activation, with TNFalfa and IL1 release, contributes to subsequent endothelitis, with overexpression of P-Selectin and local prothrombotic conditions.

Furthermore, neutrophil degranulation releases high levels of serine proteases activators, causing a “proteolytic storm” due to significant imbalance of four major proteolytic cascades (coagulation, complement, fibrinolysis and kallikrein) with activators prevalence over SERPIN family inhibitors. This results in uncontrolled proteolytic activation in the lung, in the endothelium and in organs with unhinibited activation of platelets, extrinsic and intrinsic pathway of blood coagulation, complement and fibrinolysis cascades, with extensive cross talk mutually fine-tuning their activation status.

NETS reinforce “Proteolytic storm”, in a feedback loop, disrupting epithelial lining, inducing platelet aggregation, activating new neutrophils recruitment, the intrinsic and extrinsic pathways of coagulation and forming a scaffold for thrombus formation by promoting platelet adhesion, by binding factor XII and supporting its activation through NETosis. At endothelial level, inflammatory vasculopathy, FXIa and α-thrombin reduce ADAMTS13, increasing the release of Ultra Large von Willebrand Factor multimers (ULVWF) by endothelial cells, resulting in persistence of ULVWF strands and causing a further increase in platelet adhesion. 

The hypercoagulability, as imbalance of proteases/antiproteases cascades, decrease of ADAMTS13, endotheliopathy, increased platelet activation, ULVWF multimers and NETosis together create a severe thromboembolic environment with microthrombi formation. These microthrombi can become sufficiently large to be released from endothelial cells into the circulation, resulting in embolism or can be physically disrupted (as in forced ventilation) disseminating through general circulation to organs, causing MOF.

The clinical evolution to critical conditions is thus triggered by a “proteolytic storm”, due to serine proteases cascades imbalancing.

The potential efficacy of convalescent plasma cannnot be related to NAbs, but to its ability to reintegrate a physiological balance.  

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