Editorial: NETosis 2: The Excitement Continues

1Department of Internal Medicine 3 – Rheumatology and Immunology, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Universitätsklinikum Erlangen, Erlangen, Germany, 2 Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, United States, 3 Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN, United States

iNtrodUCtioN Chromatin externalization to form extracellular traps has first been described in neutrophils (1) but can also be observed in mast cells (Mollerherm et al.) as well as in other myeloid cells like eosinophils and basophils (2). DNA externalization-based defense emerged more than 1 billion years ago. Sentinel cells of the social ameba Dictyostelium discoideum release redundant mitochondrial DNA and in an altruistic manner chromatin that sequester colony invading bacteria (Zhang and Soldati). Leukotoxic hypercitrullination and deficient mitophagy can initiate mitochondrial DNA expulsion (Konig and Andrade). These processes can be discriminated from canonical NET formation (1). However, since they cause DNA externalization they could be considered non-canonical forms of NET formation. Although the generation of reactive oxygen species is reportedly involved in several pathways of NET formation it is not strictly required for others (3). Furthermore, under hypoxic conditions, the PMA-induced NET formation is reported to be reduced, though not abrogated (Branitzki-Heinemann et al.). This editorial summarizes the collection of articles of the research topic "NETosis 2, the excitement continues. " to diE or Not to diE There is ongoing discussion whether the neutrophils are still viable when they release their chromatin and if they release chromatin at all. There are reports that claim the cells mainly externalize mitochondrial DNA (Yousefi and Simon). Some investigators question the notion that NETs form by cytolysis. They argue that it seems inconsistent with neutrophil "containment" processes (Malachowa et al.). Furthermore, the term "NETosis" has been criticized by some researchers who prefer the term "NET formation. " Surveying the published data, including those published in this special issue, we want to summarize the current knowledge (I) Depending on the stimulus, NET formation can be a lytic death process eventually leading to disintegration of the neutrophil or a process where neutrophils remain migratory and impermeable to ionic dyes; (II) these processes have been observed in vitro as well as in vivo; (III) the inducer and its concentration determine, at least in part, these processes as well as the content of the NETs. For purposes of this review, we will use the term NET formation rather than NETosis to include the various forms of this cellular processes as described by various research groups.

iNHiBitorS oF NEt ForMatioN
The increased capacity to form NETs during gestation is controlled by the inhibitory activity of progesterone. This hormone reduces the nuclear translocation of the neutrophil elastase leading to decreased NET formation (Giaglis et al.

NEt CoMPoNENtS
The NET backbones mainly consist of chromatin with modified, often citrullinated, histones and is overall similar in most NETs reported. However, there are considerable differences in their protein cargo (Mitsios et al.). These proteins may either be derived from the neutrophils' granules, from the vicinity of the NETs or from bystander cells. Neutrophils are endowed with a "tool kit" for alternative complement activation. They secrete properdin and deposit C3-derived complement fragments on NETs and on NET-bound bacteria (Yuen et al.

aNtiBodiES BiNdiNG NEts aNd tHEir CoMPoNENtS
Deimination is a physiological process that is amplified at sites of inflammation. However, only individuals with genetic predispositions for rheumatoid arthritis develop antibod-

MEtHodS For tHE aNalYSiS oF NEts
In general, NETs can be detected by staining of DNA and its co-localization with granular and modified nuclear proteins in web-like, spiky or cloudy structures, exceeding the size of a neutrophil. In neutrophil-rich areas like inflamed synovium or densely infiltrated tissues, these NETs tend to aggregate (4, 5) and form extended chromatin clumps decorated with an effective pathogenocidal armament (Brinkmann et al. In an animal model of paw edema by injection of nanodiamonds into wild-type mice and in those with deficient capacity for oxidative burst and NET formation (Ncf1** mice), the inflammatory response resolves in the former and becomes chronic in the latter as a result of the failure to dampen the neutrophildriven inflammation (Biermann et al.). Importantly, ANCA in general are reported to drive immune complex-mediated pathologies but they may also aid in the clearance of circulating NETs or NET remnants (Soderberg and Segelmark).

NEt ForMatioN aNd EXtraVaSCUlar diSEaSE
Though NETs have initially been described as mechanism of bacterial defense, they must be considered as double-edged

tHEraPEUtiC iNtErVENtioNS
NETs are discussed as a source of the citrullinated autoantigens pathognomonic for patients with rheumatoid arthritis and for the DNA observed in tissues and in the circulation of patients with SLE. Consequently, PAD4 is proposed to be a potential therapeutic target for these chronic inflammatory rheumatic diseases (Konig and Andrade). The synthetic peptide P140/Lupuzor™ selectively modulates chaperonemediated autophagy but not NET formation in sensu stricto (Ramirez et al.). As high fat diet reportedly increases the formation of NETs dietary intervention and reduction of fat intake may be beneficial in NET-associated disorders (Moorthy et al.).
Targeting NET formation as well as NET-associated chromatin decondensation may delay the pathogenesis of Alzheimer's disease (Pietronigro et al.), the early inflammatory responses to Sendai virus infection (Akk et al.), and the disseminated intravascular coagulation observed in severe forms of malaria (Boeltz et al.). As already established for patients with cystic fibrosis, the clearance with recombinant human DNaseI of the NET-associated DNA, the neutralization of NETborne proteins using anti-histone antibodies, as well as inhibitors for NET-bound proteases are discussed as therapeutic options for pulmonary diseases involving alveolar NETs in Porto and Stein.
From the papers included in this Research Topic, it is evident that the field of NET research is now more mature and sophisticated than even just a few years ago. Physiological and disease conditions that induce abundant NET formation are now firmly established and experimental methods for detection of NETs in vivo and in vitro have been carefully defined. It is the hope of the authors that the combined efforts presented here will contribute to further shape the consensus in the field, energize efforts to understand NET biology, and lead to novel therapies for major human disorders that present with abnormal NET release or impairments in NET degradation.

aUtHor CoNtriBUtioNS
All authors wrote and revised the manuscript.