Bet on NETs! Or on How to Translate Basic Science into Clinical Practice

Neutrophil extracellular traps (NETs) comprise neutrophil DNA, citrullinated (deiminated) histones and proteases (Radic and Marion, 2013; Grayson and Kaplan, 2016). NET formation in vivo mostly occurs at sites of inflammation. Neutrophils, adhering to the endothelium or after extravasation, generate NETs upon interaction with microbes, activated platelets, cytokines, alarmins such as high mobility group box 1 (HMGB1) or uric acid. Environmental cues, such as hyperosmolarity or hyperglycemia, neurotransmitters and some autoantibodies also trigger NET generation (Kessenbrock et al., 2009; Mitroulis et al., 2011; Maugeri et al., 2014a; Grayson et al., 2016; Gupta and Kaplan, 2016). In vitro, two main pathways are activated: suicidal and vital NETosis (Yipp and Kubes, 2013). In the former, membrane integrity is disrupted and neutrophils die. In contrast after “vital” NETosis neutrophils still migrate, chase bacteria and extrude residual nuclear material through exocytosis (Pilsczek et al., 2010). Generation of reactive oxygen species (ROS) and fusion of neutrophil primary granules with the nuclear membrane that promote interactions between elastase, myeloperoxidase (MPO) and DNA are features of suicidal NETosis. Their role in vital NETosis is debated (Metzler et al., 2011; Yipp and Kubes, 2013; Zhao et al., 2015; Maugeri et al., 2016). Activation of the autophagic pathway is intermingled with NET generation. Autophagy sustains the metabolic requirements of the extensive intracellular vesicular formation, transport and fusion associated to NET generation. It also sustains neutrophil survival(Maugeri et al., 2014a).Neutrophils undergo extensive ex vivo manipulations and methodological approaches vary among research groups, possibly explaining some discrepancies and nurturing some healthy skepticism (Nauseef and Kubes, 2016). Some reports indicate a role of the mitochondrial DNA, which is devoid of histones, in NET generation. The relative contribution of nuclear vs mitochondrial DNA to extracellular traps generation remains a controversial issue (Yousefi et al., 2009).Under physiological conditions, NETs enhance the host response to microbes by: i) providing a template concentrating humoral innate immune molecules such as pentraxin 3 (PTX3) and complement, together with microbicidal molecules such as histones, MPO, proteinase 3 (PR3), or cathelicidin and ii) contrasting the hematogenic spread of pathogens through immunothrombosis (i.e. activation of platelets and of the coagulation cascade through NET components) (Engelmann and Massberg, 2013). The actions of NET-embedded von Willebrand factor, of citrullinated histones and of negative charges on platelet recruitment/activation and on the progression of the coagulation cascade contribute to thrombosis. Excellent reviews on this issue have been published (Gould et al., 2015; Kimball et al., 2016). The bioactive molecules integrated within the chromatin threads may vary according to the inciting stimuli or the environment. The characterization of the NETs proteome is a fascinating challenge for the near future. Neutrophils are abundant in the blood and their concentration and activation state increase following surges of systemic cytokines during acute phase responses. Thus NETs are readily available and easily renewable tools for the first-line response to infectious agents. However, there is a price to pay. During acute infections (e.g. during sepsis) NETs favor progression towards septic shock, disseminated intravascular coagulation and acute respiratory distress syndrome (ARDS), as well as tissue damage and organ failure, possibly because of the deleterious effects of immunothrombosis. On the other hand, the imbalance between the rate formation and degradation of NETs might favor the persistence and the presentation of autoantigens and facilitate autoimmunity (Radic and Marion, 2013). Systemic lupus erythematosus (SLE) is a paradigm of the pathogenic continuum that links nuclear antigens exposure to autoimmunity. Neutrophil from patients with SLE show prolonged survival (possibly through enhanced autophagy) and are thus endowed with additional chances to generate NETs. In addition the clearance of extracellular DNA in SLE is impaired either as a genetically determined trait influencing waste disposal or due to antibodies towards regulatory molecules, such as DNases (Hakkim et al., 2010; Leffler et al., 2013). Anti-neutrophil cytoplasmic antibodies (ANCA) represents a variant on the theme of autoantibodies elicited following enhanced generation and/or persistence of NETs. The formation of NETs at sites of vascular inflammation in ANCA-associated vasculitides (AAV) and the ability of ANCA to perpetuate the generation of NETs were described in 2009 (Kessenbrock et al., 2009). Later on, dendritic cells have been found to capture ANCA targets (i.e. MPO and PR3) from NETs and to present them to T lymphocytes while the susceptibility to ANCA generation was found to be deeply rooted in genetics. Neutrophil priming with HMGB1, a well-established inducer of NETs and of autophagy, contributes to the action of ANCA(Ma et al., 2016). In rheumatoid arthritis, NETting neutrophils infiltrate the synovium and rheumatoid nodules while anti-citrullinated peptides antibodies apparently boost the generation of NETs (Khandpur et al., 2013). B cells that expand and differentiate within ectopic synovial germinal centers frequently target deiminated antigens generated during NETosis (Pratesi et al., 2014; Corsiero et al., 2015) and/or as a consequence of the release of bioactive peptidyl arginine deiminases in the synovial fluid. Anti-phospholipid antibodies in combination with ROS and platelet-assisted TLR4 stimulation induce neutrophils to form NETs, which in turn precipitate intravascular thrombosis (Engelmann and Massberg, 2013). Conversely, NET degradation appears to be impaired in a fraction of patients with anti-phospholipid syndrome(Leffler et al., 2013). Neutrophils infiltrate the pancreas of patients with type 1 diabetes mellitus (T1DM) (Valle et al., 2013) and, according to experimental models and indirect clinical evidence (systemic levels of NETs byproducts in serum), cause an IFNα response, autoantibody generation and β-cells destruction through in situ NETosis (Diana et al., 2013; Wang et al., 2014).Indicators and markers of NETosis. The notion that NETs contribute to a wide range of autoimmune diseases so far has had little impact on the clinical practice. Reasons include: i) the low threshold of activation of neutrophils, that limits the development of robust, easy-to-perform and cheap diagnostic assays;ii) high costs of clinical trials;iii) the lack of insight on appropriate targets to safely target NETosis.Three main approaches are currently used in research laboratories:• analytical assays based on fluorimetry for cell-free DNA or on ELISAs for soluble NET byproducts (DNA-MPO or -neutrophil elastase complexes, citrullinated histones) (Kessenbrock et al., 2009; Maugeri et al., 2014a)• confocal microscopy for neutrophil enzymes along extracellular DNA lattices (Kessenbrock et al., 2009; Maugeri et al., 2014a)• flow cytometry, based on nuclear morphology and variations in MPO distribution (Zhao et al., 2015) or staining of citrullinated histones or DNA (Gavillet et al., 2015).Fluorimetry is not time-consuming and generate semi-quantitative information that can be associated to clinical variables. However it does not unambiguously identify the source of DNA (neutrophils or other cells) or the process by which DNA was released (NETosis, necrosis or necroptosis etc.). The association of DNA fragments to neutrophil enzymes and the citrullination of histones are relatively specific for NETosis. Thus determination of MPO-DNA complexes and/or of citrullinated histones selectively reveals the amount of NET by-products in biological fluids. Human studies monitoring in vivo NETs formation (Kessenbrock et al., 2009; Maugeri et al., 2014a), also revealed a concordant rise in NETs by-products in plasma, suggesting that ELISA is sufficient per se to assess the degree of NET formation in human inflammatory diseases (Figure A-F). Furthermore, parallel quantitation of in vitro-generated NETs by confocal microscopy and concomitant measurement of DNA, DNA-MPO complexes and citrullinated histones in cell-free supernatants drives to concordant results (Figure G-I). Thus, we believe that at present only ELISA assays on plasma samples meet the requirements of robustness, reproducibility and easiness for widespread clinical use.However, it does not discriminate between vital or suicidal NETosis nor assess the relative contribution of NET generation and catabolism. Standardized protocols will be necessary for transfer these assays to the clinics (Masuda et al., 2016). For example, DNases for sample enrichment influence the sensitivity of MPO/DNA-based analytical assays (Figure G-H). Microscopy allows a direct visualization of NETs. In expert hands, it remains a powerful and informative tool, even more when combined with the determination of NET by-products (Figure A-F)(Kessenbrock et al., 2009; Maugeri et al., 2014a) and with high throughput proteomic assays. However, due to the high inter-observer variability, it is not routinely used in clinical practice. Novel semi-automated image analysis techniques might circumvent this limit. Flow cytometry yields accurate and reproducible data. Large trials are needed to pave the way to their widespread use (Zhao et al., 2015; Masuda et al., 2016). Besides improvements in NET-detection, caution in the pre-analytical sample handling is mandatory, since neutrophils and platelets respond to physical and chemical stimuli during blood sampling and transportation. Activated platelets in particular release various signals – both soluble and associated to microparticles - that impact on neutrophil functions, including NET generation (Manfredi et al., 2016; Maugeri et al., 2016)NETs, drug development and repositioning. Citrullination of histone residues is a key step in NETosis. Pharmacological or genetic tools to inhibit deiminating enzymes reduce the formation of NETs and their detrimental consequences in preclinical models (Knight et al., 2013; Wei et al., 2013; Kusunoki et al., 2016). The actual selectivity and potential safety risks of the available pharmacological inhibitors are not yet established.Mitochondrial generation of ROS triggers NET formation. In addition, oxidized mitochondrial DNA within NETs could contribute to its immunogenic potential. Interference with the respiratory chain and/or ROS scavenging exerts antinflammatory effects and clinical benefit in mice models of sepsis and SLE (Tsoyi et al., 2011). Targeting platelet/neutrophil reciprocal activation and platelet microparticle-associated moieties, HMGB1 in particular (Maugeri et al., 2014a; Maugeri et al., 2014b; Maugeri et al., 2016), and finding strategies aimed at restoring the phagocytosis of apoptotic substrates by neutrophils can exert a calming influence over NET generation (Manfredi et al., 2015) and thus appear promising. Other anti-NET treatments could aim at restoring and/or potentiating the NET clearance. NET-mediated lung injury in cystic fibrosis abates in response to nebulized DNase supplementation. Studies improving the drug delivery could pave the way for the application of this anti-NETs treatment to inflamed joints, kidney, lung or skin in the setting of autoimmune diseases.The repositioning of known drugs and agents has advantages over the development of new drugs, since toxicity/safety profiles are usually known and cost and time to bring agents to market abate. A proof-of-concept trial links reduced SLE flares to the metformin inhibition of mtDNA-enriched NETs. An action of metformin on HMGB1 release (Tsoyi et al., 2011; Maugeri et al., 2014a) could also be involved. Heparins are being used in a wide range of diseases for effects independent of their anticoagulant properties (Mulloy et al., 2016), including conditions in which NETs generation possibly plays a role. Heparins might interfere with the metabolic needs for NET generation since they restrict the activation of the autophagic flux (Manfredi et al., 2016). Prophylactic doses of low molecular weight heparins, that are routinely used for thrombo-prophylaxis and for prevention of pregnancy complications, indeed interfere with autophagy induction of neutrophils of healthy subjects and virtually abrogate the ability to generate NETs in response to various stimuli (Manfredi et al., 2016). Thus interference with NETosis might be involved in the benefit of treatments selected mainly on empirical basis. Unfractionated heparin has also been shown to dismantle NETs after they have been generated, by liberating histones from the DNA backbone and destabilizing the chromatin threads(Fuchs et al., 2010).In conclusion, accumulating data on the role of NETs in autoimmune diseases and in highly prevalent inflammatory conditions such as sepsis demonstrate that novel bioindicators and treatments might readily become available and improve the quality of patients care from tomorrow. It’s time to take the next step.