In the early 2000s, Arturo Zychlinsky at the Max Planck Institute for Infection Biology in Berlin found that mammalian immune cells called neutrophils use an enzyme called neutrophil elastase (NE) to cleave bacterial virulence factors. When Zychlinsky and his colleagues delved deeper into this defense mechanism, they realized that when activated by bacteria, human neutrophils release NE in what, under the microscope, looked like a fibrous structure. This structure turned out to be a meshwork of NE, other proteins, and copious amounts of DNA. In cultured human neutrophils, the webs were able to trap the bacteria that had triggered their formation, thereby limiting infection, so Zychlinsky and colleagues dubbed them neutrophil extracellular traps, or NETs.
The fact that neutrophils used their nuclear material to catch pathogens was intriguing to immunologists and cell biologists alike. The work of the Zychlinsky lab suggested that the release of NETs was an active process, and that the material wasn’t simply released by passive lysis. This has motivated a new line of research devoted to characterizing these unique structures, delineating the mechanisms that prompt their formation, and identifying their relevance in mammalian biology.
As more and more researchers join the burgeoning field, the spectrum of pathogens known to induce NET release from neutrophils has expanded from a variety of bacteria to fungi and, most recently, to viruses. However, it has also become clear that NETs can have negative consequences for the organisms that produce them—by activating autoimmune pathways or encouraging tumor cells to metastasize, for example.
Today, it is widely accepted that NETs have both a protective and a pathological impact on the host.
Today, it is widely accepted that NETs have both a protective and a pathological impact on the host. In 2012, Mariana Kaplan, now of the National Institutes of Health, and the University of Tennessee’s Marko Radic termed NETs a “double-edged sword of immunity” and suggested that healthy organisms must tightly control their release to minimize negative consequences for the host. The details of NET regulation and function are now a very active area of research.
Neutrophils are essential for immune defense and prevention of microbial overgrowth. They are very abundant—around 100 billion are produced in a human’s bone marrow in a single day—and they circulate in the bloodstream to quickly infiltrate tissues if the neutrophils detect a microbial threat. Belonging to a class of white blood cells called granulocytes, they are characterized by a cytoplasm packed with granules containing antimicrobial proteins. Neutrophils can engulf pathogens and then fuse their granules with their phagosomes, which contain the internalized microbes. Alternatively, the cells can fuse their granules with the plasma membrane to release antimicrobials to attack extracellular parasites.
Research on neutrophils is complicated by the fact that they are short-lived cells. For instance, unlike some other human cell types, neutrophils cannot be cultured for more than a few hours, and they are not amenable to gene editing. For this reason, we still lack a detailed mechanistic picture of how exactly NETs are formed. Early reports confirmed the original hypothesis that NETs do not result from passive necrosis of neutrophils. Later studies added complexity by demonstrating that different inflammatory triggers induce various pathways that all lead to the release of NETs, and that NET release doesn’t always result in lysis of the neutrophil.
That said, most pathways of NET formation do kill the immune cell, typically as a result of the production of reactive oxygen species (ROS). Bacterial or fungal pathogens cause neutrophils to activate kinases that induce assembly of an enzyme complex called nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. NADPH oxidase then produces large amounts of superoxide—a highly reactive oxygen compound that carries an extra electron—during a process called the neutrophil oxidative burst. ROS resulting from the oxidative burst trigger disintegration of a multiprotein complex to release active NE, a primary component of NETs, into the cytoplasm. (See illustration on page 45.)
NE then migrates to the neutrophil’s nucleus, where it cleaves histones and other proteins to decondense the chromatin. Eventually, the chromatin fills up the entire cell until the cell lyses and extrudes the NET into the extracellular space, a process known as NETosis. We recently identified an important role for the pore-forming protein gasdermin D in both the nuclear expansion and the lysis processes, although the mechanisms aren’t yet clear. In the extracellular space, the webs are thought to trap and kill the triggering pathogens.
NETs in immune defense
NETs’ ability to trap microbial invaders has been demonstrated in vitro for a variety of pathogens. The structures appear to be particularly important in defense against pathogenic fungi, suggesting NETs may have evolved as a way to trap large organisms that cannot be engulfed via phagocytosis. But a lack of specific genetic tools has made it difficult to nail down the exact contribution of NETs to antimicrobial defense in vivo. Most of the molecules that have so far been found to regulate NETs are also involved in other immune responses, so a “NET knockout” organism is still beyond our reach.
In spite of these technical challenges, researchers are slowly elucidating the function of NETs in immunity. In 2012, Paul Kubes of the University of Calgary and colleagues depleted NETs in mice with skin infections by injecting recombinant DNase 1, an enzyme that chops up the DNA backbone of NETs after they are released. This led to increased dissemination of Staphylococcus aureus from the skin to the bloodstream, demonstrating that NETs participate in containing bacteria at the site of entry. This experimental strategy of NET destruction by nucleases is also used naturally by pathogenic bacteria as a way to escape from NETs: Staphylococcus and Streptococcus species both secrete DNases, and experimental deletion of the genes that encode these NET-degrading enzymes in those microbes leads to reduced bacterial virulence.
When a neutrophil encounters a pathogen, it can respond in several ways: phagocytosis, degranulation, or by releasing neutrophil extracellular traps (NETs). In NET release, shown here, the enzyme complex NADPH oxidase generates reactive oxide species (ROS), which in turn initiate the disintegration of granules, releasing neutrophil elastase (NE). NE then migrates to the neutrophil’s nucleus, where it cleaves proteins that package the cell’s DNA as chromosomes. The chromatin expands until it fills up the entire cell, which breaks open and extrudes the NET into the extracellular space. There, the webs are thought to trap and kill the triggering pathogens.
The idea that NETs form barriers at mucosal surfaces was recently strengthened by additional work from Kubes and Ajitha Thanabalasuriar, now at MedImmune, showing that NETs form an exclusionary microbicidal “dead zone” in infected corneas. This confines bacteria to the outer surface of the eye and prevents them from entering the eye and spreading to the brain.
The role of NETs in infections is not limited to trapping microbes, however. The structures contain multiple anti-microbial molecules. Histones, for example, are major components of the chromatin in NETs, and these proteins have important bactericidal and immunostimulatory functions. After discovering NETs, Zychlinsky proposed antimicrobial defense as the alternative, less-studied function of histones, whose more prominent and established function is to organize and package DNA in the nucleus.
NETs also contain several alarmins, molecules that activate the immune system and help propagate the inflammatory response. Release of alarmin-containing NETs alerts the rest of the immune system to the presence of microbes or foreign substances. Twenty-five years ago, immunologist Polly Matzinger of the National Institute of Allergy and Infectious Diseases proposed the “danger theory” of immunity, postulating that the immune system is concerned with limiting virulent bacteria and tolerating nontoxic ones. NET release may serve as one mechanism of flagging intolerable bacteria. Instead of engulfing the microbes and initiating the immunologically silent apoptotic pathway, the NET-producing form of cell death utilizes proinflammatory pathways. Bacteria that are toxic enough to lead to NETosis will send a powerful inflammatory signal via the alarmins in NETs. This is illustrated nicely in the case of Pseudomonas aeruginosa, an opportunistic pathogen that is able to colonize the lungs of cystic fibrosis patients. P. aeruginosa only triggers NETs when it expresses virulence factors such as flagella or the toxin pyocyanin. In the absence of these virulence factors, neutrophils mostly respond to these bacteria by attempting to engulf them.
Triggers of NET formation
It’s not just pathogens that can initiate NET formation. A variety of host molecules can also activate neutrophils to extrude their innards into the extracellular space. One example is crystals of cholesterol, a form of the lipid that causes inflammation. And in 2017, we and our colleagues found that mitogenic signaling, which induces cell division in some immune cells, also triggers various cell cycle–related phenomena in neutrophils, but with the consequence of NET formation rather than division.
Notably, NETs can form independently of the canonical pathway involving ROS-producing NADPH oxidase, stimulated by pathogens or compounds such as ionophores, agents that lead to ion fluxes through the cell membrane. It is unclear whether NADPH oxidase–independent pathways rely on ROS produced by other means or whether these are truly ROS-independent processes. Interestingly, ionophore-driven activation of neutrophils triggers strong calcium fluxes, which activate the enzyme peptidyl arginine deiminase 4 (PAD4). PAD4 converts the amino acid arginine to citrulline and acts on various substrates, including histones. Citrullination of histones is thus a good biomarker for NETs in the extracellular space.
Perhaps the most surprising mechanism of NET formation involves DNA release by living neutrophils, a process termed vital NETosis by Kubes and colleagues. It is unclear which molecules mediate DNA release in this case—or how the release even occurs—but it seems that neutrophils remain viable, phagocytic, and are even able to “push” the NET forward as they migrate. These findings demonstrate that neutrophils do not necessarily die after they form NETs.
NETS IN HEALTH
The exact contribution of NETs to antimicrobial defense has been difficult to nail down, but researchers are slowly elucidating their roles in protecting the body from invaders and other threats, including runaway inflammation. Those roles include:
Downsides of NETs
Unfortunately, as with other neutrophil responses, NETs have a dark side that makes them dangerous when inappropriately deployed. This is the case with malaria, which is caused by Plasmodium parasites that invade and proliferate in red blood cells. Infected cells adhere to the walls of blood vessels throughout the body, leading to pathological changes such as obstruction of capillaries and inflammation. Our group made the surprising discovery that NETs cause blood vessels to become sticky by expressing cytoadhesive proteins, which facilitate binding by Plasmodium-infected cells. In the absence of NETs, infected mice tolerated the presence of parasites and showed no signs of disease.
Similarly harmful effects of NETs have been described in noninfectious diseases such as lupus, an autoimmune condition. In these instances, NET release is dysregulated, triggering chronic pathological inflammation, with NETs potentially acting as a source of autoantigens and immunostimulatory molecules. (See “Commensal Menace,” The Scientist, June 2019.)
The process of cells releasing their DNA as a protective mechanism might be ancient.
As large, fibrous extracellular structures, NETs are also excellent scaffolds for thrombosis, the formation of blood clots that are the main cause of heart attack and stroke. Denisa Wagner’s group at Harvard Medical School has shown that NETs are found in both arterial and deep vein thrombosis and that their degradation by DNase 1 is protective in animal models of the disease. Before being linked to NETs, histone-packaged bits of DNA called nucleosomes were known to have procoagulant properties.
NETs can also be subverted by malignant cells to facilitate metastasis. The link between inflammation and cancer progression has long been established, but it was a seminal study by the laboratory of Mikala Egeblad at Cold Spring Harbor Laboratory that demonstrated NETs can reawaken dormant tumor cells. Working in mouse models of breast and prostate cancer, the Egeblad lab showed that bacterial antigen–triggered NETs remodel the microenvironment to encourage activation and proliferation of tumor cells. Importantly, researchers were able to block metastasis by either interfering with NET production or by blocking downstream signaling effects. Inhibition of NETs is thus a potential strategy for developing novel cancer therapies, and is likely to be the focus of intense research in coming years.
. . . AND DISEASE
NETs have a dark side that makes them dangerous when inappropriately deployed. The structures have been implicated as contributors to a range of conditions.
Despite very active research, there are still a number of unknowns concerning NETs. Mechanistically, we do not fully understand how many pathways exist to induce neutrophils to make NETs, or how these pathways might interact. And the pathological effects of NETs are only beginning to be detailed, while their potential healing properties are currently understudied.
Another important question is what determines whether a neutrophil undergoes lytic or non-lytic NET formation. The concept of neutrophils releasing NETs while remaining motile and capable of engulfing bacteria is intriguing, but more studies are required to elucidate when and where this occurs.
Finally, an interesting and unsolved question is how evolutionarily conserved the process of NET formation is. Plant root cells release chromatin to defend against pathogens, and a recent study described NET-like structures released from phagocytes of invertebrates, such as crabs, mussels, and anemones. The process of cells releasing their DNA as a protective mechanism might therefore be ancient. Further studies will hopefully identify the origin of NETs, and thereby shed light on this fascinating and important process.
Borko Amulic is an assistant professor at the University of Bristol in the UK and a Medical Research Council Fellow. Gabriel Sollberger is a postdoctoral researcher at the Max Planck Institute for Infection Biology in Berlin.