"Neutrophil extracellular traps" (NETs) are released by neutrophils. Neutrophils act as the first line of defense during infection and inflammation. Once activated, they are able to fulfil numerous tasks to fight inflammatory insults while keeping a balanced immune response. Besides well-known functions, such as phagocytosis and degranulation, neutrophils are also able to release “neutrophil extracellular traps” (NETs).. In response to most stimuli, the neutrophils release decondensed chromatin in a NADPH oxidase-dependent manner decorated with histones and granule proteins, such as neutrophil elastase, myeloperoxidase, and cathelicidins. Although primarily supposed to prevent microbial dissemination and fight infections, there is increasing evidence that an overwhelming NET response correlates with poor outcome in many diseases. Lung-related diseases especially, such as bacterial pneumonia, cystic fibrosis, chronic obstructive pulmonary disease, aspergillosis, influenza, and COVID-19, are often affected by massive NET formation. Highly vascularized areas as in the lung are susceptible to immunothrombotic events promoted by chromatin fibers. Keeping this fragile equilibrium seems to be the key for an appropriate immune response. Therapies targeting dysregulated NET formation might positively influence many disease progressions.
1. Mechanisms of NET formation
The underlying mechanisms leading to NET formation show variable characteristics, and studies demonstrated that the signaling pathway vary depending on the respective stimulus. NET formation can be stimulated via G protein-coupled receptors (GPCRs), chemokine and cytokine receptors, Toll-like receptors (TLR), and Fc receptors (FcR). The subsequent downstream signaling comprises mostly the activation of the NADPH oxidase (NOX) complex, but exceptions were also described . Upstream of oxidant production, the molecules Raf-MEK-ERK have been shown to be involved . Cytoskeletal rearrangement  and glycolytic ATP production  are both required for NET formation and are dependent on ROS produced in the context of mitochondrial dysfunction and NOX activation . ROS initiates the dissociation of NE from a membrane-associated complex into the cytosol and activates its proteolytic activity in an MPO-dependent manner. Subsequently, NE degrades F-actin to arrest actin dynamics followed by translocation into the nucleus, where NE and MPO drive chromatin decondensation and histone cleavage , which can be supported by PAD4-dependent histone citrullination  ( Figure 1 ).
Nevertheless, NE- and PAD4-independent pathways have been described, too . Van Avondt and colleagues demonstrated that the inhibition of the signal inhibitory receptor on leukocytes-1 (SIRL-1) could prevent NET production without affecting oxidant production . Cell cycle proteins  support nuclear envelope breakdown followed by the release of chromatin into the cytosol, where nuclear and cytosolic proteins are mixed . The final cell lysis and NET release involves Gasdermin D (GSDMD), which is able to form pores in granule and plasma membranes . This kind of NET formation ends up with cell death and is often described as lytic NET release or NETosis and occurs within a rather long time frame of three to eight hours. In contrast, the non-lytic NET release can be observed rapidly after 5–60 min of stimulation. Here, neutrophils do not undergo cell death, which was observed for neutrophils in close contact with activated platelets  or in response to Staphylococcus aureus infections . Similar to NETosis, the non-lytic NET formation also involves the translocation of NE to the nucleus, histone citrullination, and chromatin decondensation . Conversely, the membrane does not disintegrate, and the protein-decorated chromatin is released via vesicles  ( Figure 1 ). Even the remnants of non-lytic NET formation, cytoplasts, are able to keep their mobility and fulfill important functions, such as phagocytosis, the activation of DCs, and the release of cytotoxic molecules .
Figure 1. Schematic illustration of the essential steps of NET formation. Several pathogens are capable of inducing NET formation directly or via release of peptides or damage associated molecular patterns (DAMPs). Receptors like TLR, FcR or GPCR transmit signals into the cell and activate predominantly the NADPH-oxidase complex (NOX), which subsequently catalyzes production of reactive oxygen species (ROS). In azurophilic granules, NE gets released from the membranes in a ROS-dependent manner and translocates into the nucleus and in parallel degrades actin. NE activity results in the decondensation of chromatin, which is further supported by the PAD4-dependent citrullination of histones. The chromatin, decorated with microbicidal molecules like histones, MPO and NE, is released in the environment. This occurs either in a non-lytic procedure, where DNA fibers are suggested to be released via vesicles, or in a lytic process, followed by the breakdown of the nuclear envelope and the cell membrane, ending with cell death.
2. NET-Targeting Therapies
As described above in detail, the abundance of NETs is a fragile balance, which tends to tilt over to a rather deleterious influence during several infectious diseases. To date, several attempts were taken to keep NETs in the right frame. Nevertheless, the combination of DNA with potentially damaging molecules makes it difficult to find the perfect treatment. Here, we summarize the most relevant and promising treatment strategies.
Extracellular chromatin and NETs can be digested by naturally occurring DNase1. It dismantles the DNA structure and liberates entangled components, which has to be calculated as a significant risk factor since, e.g., NE or MPO are capable of perpetuating inflammation. However, DNase is the only NET-targeting therapy already in clinical use. It is used for the treatment of virus-associated bronchiolitis 
, as well as cystic fibrosis, in order to improve lung function and reduce the occurrence of infectious exacerbations 
. Similarly, NET DNA in COVID-19 contributes to mucus accumulation, rigidity, and airway occlusion, indicating that severe cases of COVID-19 might also benefit from DNase treatment. A single-center case study was published recently, suggesting that nebulized dornase (recombinant human DNase) reduced supplemental oxygen requirements 
. Further clinical trials are currently underway investigating the effect of nebulized dornase-α during COVID-19 
One possible drawback of disentangling DNA fibers could be the subsequent release of histones or proteases, potentially causing cytotoxicity. Recently, a study suggested that the synergy between histone and DNA is critical for sub-lethal signaling 
. Accordingly, another study proposes a mechanism whereby aggNETs contribute to the detoxification of histones. Neutralization of histones might be a promising target in future, as demonstrated in different murine disease models 
. The C1 esterase inhibitor (C1INH), a serine protease inhibitor, is capable of targeting multiple pathways 
and can bind and neutralize histones due to its glycosylation-dependent overall negative charge. Additional studies revealed that C1INH treatment reduced neutrophil activation and improved inflammation and survival in sepsis patients 
. However, additional preclinical testing and investigation of different disease models is needed to further validate this promising inhibitor as a therapeutic agent during inflammation. Furthermore, a recent study suggests a promising role of an anti-citrullinated protein antibody (tACPA), which prevented NET-associated disease symptoms in different inflammatory pathologies in mice by inhibiting NET formation and increasing NET degradation through macrophages 
. Accordingly, another study demonstrated that neutralizing citH3 attenuates endothelial damage in vitro and has the capability to improve survival rates and inflammatory responses during LPS-induced sepsis in mice 
2.3. Neutrophil Elastase
Similar to the abovementioned release of histones, the liberation of NE might also contribute to tissue damage and inflammation since it is capable of disturbing the lung barrier, inducing the release of inflammatory cytokines, thereby fostering a cytokine storm which is often a life-threatening event during ARDS. Small-molecule inhibitors, such as sivelestat 
, alvelestat and Bay-8550, are possible therapeutics directed against NE and currently under investigation. Different studies and clinical trials with ARDS/SIRS patients indicate that sivelestat improves pulmonary function, and shortens the duration of mechanical ventilation and the length of ICU care 
, most likely through inhibition of the exaggerated signaling pathways and neutrophil chemotaxis 
2.4. Other Treatments
There are several molecules that are able to influence NET formation. Aspirin treatment decreases NET formation in the lung microcirculation and plasma 
and also decreases the deposition of platelets with neutrophils on the lungs’ vascular walls 
. TLR-mediated NET formation can be inhibited by the use of blocking antibodies, such as anti-CLEC or the bispecific anti-CLEC5A/TLR2 
. Additionally, hydroxychloroquine, also known as an anti-malarial and anti-inflammatory drug, inhibits the stimulation of pDCs by NETs via TLR9 
. The antidiabetic drug metformin directly binds the alarmin HMGB1, resulting in increased NET clearance, and attenuates the pro-inflammatory activity of NETs 
. Glucocorticoids, such as dexamethasone, belong to a class of drugs with anti-NET formation activity 
. Additionally, NET-inhibitory factors have been identified. They specifically inhibit NET formation in vitro and in vivo, thereby suggesting them to be a potential therapeutic agent 
. Further treatment options exist that do not directly target NET formation but rather neutrophil recruitment. For example, a CXCR2 antagonist reduced neutrophil influx into the airways following an LPS challenge in humans 
. Nonetheless, blocking neutrophil recruitment always harbors the risk of impairing the innate immune response. In regard to this, a promising therapy might be the use of the CD40 antibody M7, which was shown to limit inflammation without affecting the protective host defense in mice 
. A summary of possible interventions that are targeted against NETs or their components is listed in Table 1
Table 1. Summary of NET-targeting therapeutics or compounds in preclinical and clinical applications.
|Clinical Phase 2 study: COVID19
|C1 esterase inhibitor
|Clinical phase 4 study: ARDS
|Inhibition of NET formation
||Critically ill patients
NETs seem to play a fundamental role in the pathogenesis of several respiratory diseases often attributed to worse outcomes, but the treatment options are scarce. However, in the last years, the knowledge about NET formation increased, and it became certainly clear that there has to be a consensus about the definition of NETs, their stimulation, and their components 
. Results from experiments with non-physiologic triggers, such as PMA, are most likely not able to represent the in vivo situation. In addition, the detection of NETs should follow certain rules, since only staining of extracellular DNA does not necessarily detect NETs but also other necrotic cell remnants. Inconsistent methods of published studies complicate interpretation of their data. However, much more information about the underlying signaling pathways is required to establish potential therapies. With regard to respiratory diseases, it appears that NETs have a beneficial role in the early phase of disease. They often participate in capturing pathogens and prevent ongoing infection, secondary infections, and dissemination. A number of evasion mechanisms evolved by different bacteria support the importance of this preventive role. Nonetheless, in a later phase, where the disease has manifested, the anti-microbial components of NETs are no longer able to fight infection, but rather contribute to pathology due to their cytotoxic properties: this fragile balance complicates an effective treatment. Studies investigating NETs during different phases of disease are rare. The differentiation of the impact of NETs during onset, progression, and resolution of disease is of great interest and will provide essential contributions to the development of possible therapeutic interventions.