Centers for Disease Control and Prevention (CDC)
Centers for Disease Control and Prevention (CDC) activated its Emergency Operations Center (EOC) to assist public health partners in responding to the coronavirus disease 2019 (COVID-19) outbreak first identified in Wuhan, China. [CDC; photo credit, James Gathany]

While recent research has shown how the body can mount an effective immune response against SARS-CoV-2 in mild to moderate cases of COVID-19, scientists are now considering how to tackle the effects of immune system overreaction, such as the cytokine release syndrome (CRS), or cytokine storm, which can trigger severe lung damage and potentially lead to acute respiratory distress syndrome (ARDs) and death, as well as the less well know phenomenon of neutrophil extracellular traps (NETs).

A Massachusetts Institute of Technology (MIT) research team has developed a decoy receptor approach to tackling CRS that would effectively mop up the excess cytokines, while a report by a separate, international consortium of scientists points to evidence suggesting that a little known function of neutrophils may also be linked with the severity of COVID-19 infection.

Reporting in the Quarterly Review of Biophysics (QRB) Discovery, the MIT researchers described the development of modified, water-soluble receptor proteins that are similar in structure to antibodies, and which they believe could soak up the cytokine overload produced during CRS.

“The idea is that they can be injected into the body and bind to the excessive cytokines as generated by the cytokine storm, removing the excessive cytokines and alleviating the symptoms from the infection,” said Rui Qing, an MIT research scientist who is one of the senior authors of the team’s published study. The researchers hope to begin testing their proteins in human cells and in animal models of cytokine release and coronavirus infection. Shuguang Zhang, PhD, principal research scientist in the MIT Media Lab’s Laboratory of Molecular Architecture, is also a senior author of the paper, which is titled, “QTY code-designed water-soluble Fc-fusion cytokine receptors bind to their respective ligands.” The paper’s lead author is Shilei Hao, a visiting scientist at MIT, and David Jin, CEO and president of Avalon GloboCare, is a co-author on the paper.

In a separate, unrelated study reported in the Journal of Experimental Medicine, an international consortium of researchers headed by Betsy Barnes, PhD, a professor at the Feinstein Institutes, considers whether the severity of COVID-19 may be linked to the production of neutrophil extracellular traps (NETs) that can damage the lungs and other organs. In their paper, titled, “Targeting potential drivers of COVID-19: Neutrophil extracellular traps,” the authors concluded, “If our hypothesis is correct, targeting NETs directly and/or indirectly with existing drugs may reduce the clinical severity of COVID-19.”

In their paper describing a new strategy against CRS, the MIT authors explained that while the phenomenon of cytokine storm is a recognized, and potentially fatal side effect of CAR-T cell immunotherapy, it can also be triggered by viral infections, such as hepatitis, flu, and now, it seems, COVID-19. “The current COVID-19 (Coronavirus Disease 2019) triggers CRS in many stages of its pathological course that causes lung fibrosis, acute respiratory distress syndrome [ARDS], and eventually leads to multi-organ failure,” they wrote. The team’s work on blocking cytokine storms has grown out of a project started 10 years ago by Zhang, which aimed to develop modified versions of membrane-embedded proteins that are usually hard to study because once extracted from the cell membrane, their structure can only be maintained if they are suspended in special types of detergents.

After years of research, Zhang and Qing developed a method for modifying the hydrophobic regions of these membrane proteins, making them soluble in water and so easier to study. Their method, called the QTY code, involves replacing some hydrophobic amino acids with hydrophilic amino acids that have similar structures. Leucine is converted to glutamine, isoleucine and valine are converted to threonine, and phenylalanine is converted to tyrosine. “We have previously devised a novel tool called ‘QTY code’ which regulates the water solubility of redesigned membrane proteins through pairwise substitution of hydrophobic amino acids by hydrophilic ones,” the investigators wrote.

The QTY code concept has now been extended to the design of water-soluble versions of cell surface receptors for cytokines, which are signaling molecules that stimulate inflammation and other immune responses. Jin reasoned that these proteins could feasibly be injected to mop up the excess cytokines produced during a cytokine storm. Water-soluble proteins can travel efficiently through the human bloodstream, whereas the original, hydrophobic versions of the proteins would likely stick to cells that they encountered.

The researchers also attached an antibody Fc domain to their water-soluble cytokine receptor proteins, which helps to further stabilize the proteins in the bloodstream, and makes them less susceptible to attack by the immune system. “The receptors were fused with Fc domain of mouse IgG2a protein to form an antibody-like structure,” they wrote. “These Fc-fusion receptors can be expressed and purified in an E. coli system …”

The team applied the technology to the design of proteins that mimic six different cytokine receptors that bind to cytokines such as interferon and interleukin, as well as to a class of cytokines called chemokines. “It has been shown that this protein design approach enables the solubilization of many types of chemokine receptors with tunable functionality,” they wrote.

In laboratory tests the researchers found that their modified proteins bound to cytokines with similar strength as did the naturally occurring cytokine receptors. “These QTY code-designed receptors show ligand-binding properties similar to their native receptor counterparts without the presence of hydrophobic patches,” they commented. “These QTY code designs of functional, water-soluble Fc-fusion cytokine receptors can potentially be used clinically as a decoy therapy to rapidly remove excessive cytokines in the setting of hyperactive immune reactions during CRS including current COVID-19 severely infected patients … When the QTY-designed water-soluble Fc-receptors bind to excessive cytokines, they may inhibit excessive cytokine interaction with target cells, thereby reducing the organ damage and toxicity.”

Having decided to publish the results they have generated with their technology so far, the team is now planning additional tests in human cells and in animal models of COVID-19 infection. “The cytokine receptors that we designed will soak up the majority of the excessive cytokines that are released during the cytokine storm,” Jin stated.

The potential utility of the team’s approach underscores the importance of curiosity-driven research, Zhang believes. “As it turns out, our research initiated in April 2019 is directly relevant to the treatment of COVID-19 infected patients. Curiosity-driven, or even proactive research often leads to preparedness, which is key to preventing future disasters.”

The researchers have filed for patents on their proteins, as well as for the overall approach to creating water-soluble cytokine receptors. “Obviously this approach will need further animal studies, and potentially human clinical studies,” Jin said. “But we have confidence that this discovery will contribute to clinical applications to address viral diseases that involve cytokine storms.”

In an independent paper in the Journal of Experimental Medicine, the NETwork consortium of eleven international medical research organizations looked at existing research to reason whether the release of NETs may be responsible for the most severe cases of COVID-19. The NETwork includes research teams at Cold Spring Harbor Laboratory, the Feinstein Institutes for Medical Research, and the Research Institute of the McGill University Health Centre (RI-MUHC).

While severe COVID-19 disease has been linked with CRS, what causes this cytokine storm isn’t understood, the NETwork researchers pointed out. “Patients with severe COVID-19–associated pneumonitis and/or ARDS have increased pulmonary inflammation, thick mucous secretions in the airways, elevated levels of serum proinflammatory cytokines, extensive lung damage, and microthrombosis.” This late stage of the disease is difficult to manage and many patients die. “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.”

Looking at existing patient data, the NETwork researchers suggest that the exacerbated host response in patients with severe COVID-19 may be linked with overactivation of immune system neutrophils and NET formation. There have already been reports that neutrophilia is a predictor of poor outcome in patients with COVID-19, and neutrophil-to-lymphocyte ratio is an independent risk factor for severe disease, the team noted. Neutrophil infiltration of the lungs has also been reported in autopsied patients. “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.”

Part of the body’s immune system, neutrophils detect bacteria and can expel their DNA (see arrows) to attack the bacteria with a gauzy web of DNA laced with toxic enzymes, called a NET. [Egeblad lab/CSHL]
Neutrophils are usually recruited early to the sites of infection, where they kill pathogenic bacteria, fungi, and viruses. But in addition to their well-recognized function, NET formation by neutrophils is another, less well-recognized means of killing pathogens. “NETs are web-like structures of DNA and proteins expelled form the neutrophil that ensnare pathogens,” the scientists explained. And while NETs can ensnare and digest unwanted pathogens, in cases of ARDS, they damage the lungs and other organs. “… 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. Importantly, these are three commonly affected organ systems in severe COVID-19,” the investigators pointed out.

“NETs were identified 2004, but many scientists have never heard of them,” commented Cold Spring Harbor Laboratory cancer biologist Mikala Egeblad, PhD, who set up the NETwork research group around COVID-19, and is senior and corresponding author of the Journal of Experimental Medicine paper. “Most of the researchers in the NETwork have worked on NETs in other diseases, and when we started hearing about the symptoms of the COVID-19 patients, it sounded familiar.”

 NETs
Excess NET formation can drive a variety of severe pathologies. In the lungs, NETs drive the accumulation of mucus in CF patients’ airways. NETs also drive ARDS after a variety of inducers, including influenza. In the vascular system, NETs drive atherosclerosis and aortic aneurysms, as well as thrombosis (particularly microthrombosis), with devastating effects on organ function.

Jonathan Spicer, MD, PhD, a clinician scientist at the RI-MUHC and assistant professor of surgery at McGill University is a thoracic surgeon who has witnessed the effects of COVID-19 infection at the bedside. “We see in these patients severe lung damage known as ARDS, another serious problem caused by excess NETs and seen in cases of severe influenza,” he said. “In addition, their airways are often clogged with thick mucus and unlike most severe lung infections, these patients tend to form small clots throughout their body at much higher rates than normal. NETs have also been found in the blood of patients with sepsis or cancer, where they can facilitate the formation of such blood clots.”

Barnes says that the clear similarities between the clinical presentation of severe COVID-19 and other known diseases driven by NETs, such as ARDS, is an indication that excess NETs may play a major role in the disease. Researchers at the NETwork institutions are now following research lines to investigate whether NETs are a common feature in COVID-19 cases. “As samples from patients become available, it will be important to determine whether the presence of NETs associates with disease severity and/or particular clinical characteristics of COVID-19,” Barnes added. If the researchers’ findings show that excess NETs cause the severe symptoms of COVID-19, then it may be possible to devise new therapeutic options. Current treatments used in other NET- and neutrophil-driven diseases, such as cystic fibrosis, gout, and rheumatoid arthritis, might dampen the activity of NETs in COVID-19 patients, reducing the need for invasive mechanical ventilation.

“Though treatments targeting NETs would not directly target the SARS-CoV-2 virus, they could dampen the out-of-control host response, thereby reducing the number of patients who need invasive mechanical ventilation, and importantly, reducing mortality,” the investigators concluded. “Targeting NETs in COVID-19 patients should therefore be considered by the biomedical community.”

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