Psoriasis is a chronic inflammatory disease that affects the skin and is characterized by a complex immune response, since cellular, molecular, and vascular components participate in the perpetuation of the inflammatory process. To date, T helper 1 (Th1) and Th17 lymphocytes are considered as the sole regulatory cells of the immune response in psoriatic lesions (121, 122). However, Lin et al. have shown that IL-17 is abundantly produced by neutrophils and mast cells, both of which are found in the cellular infiltrates of psoriatic plaques (123). Initially, interferon alpha (IFN-α)- and tumor necrosis factor alpha (TNF-α)-secreting plasmacytoid dendritic cells (pDCs) are activated through TLRs upon the recognition of LL37–nucleic acid complexes released by damaged keratinocytes to secrete their cytokines. Initially, pDCs are activated through TLRs upon the recognition of LL37–nucleic acid complexes released by damaged keratinocytes, and consequently, the pDCs release IFN-α and TNF-α (124). These cytokines might favor the activation of DCs and macrophages and their production of IL-23 and IL-1β, which in turn induce the activation and production of IL-17, TNF-α, CXCL2, chymase, and tryptase by mast cells through mast cell extracellular trap (MCET) formation (125). Additionally, these mediators promote neutrophil migration toward the epidermis, where they may become activated by IL-23 and IL-1β and produce NETs. Together, both neutrophils and mast cells secrete IL-17 and other proinflammatory mediators to amplify neutrophil migration, which will then contribute to the formation of Munro’s abscesses (126).

On the other hand, IL-17 in keratinocytes increases the expression of LL37 and defensins such as beta-defensin 2 (HBD-2), S100A7, S100A8, and S100A9 (127, 128), which mediate cellular infiltration and both MCET and NET formation. In this context, ET-derived DNA–LL37 complexes are usually generated. These complexes may lead to pDC activation and the consequent production of IFN-α, further promoting NETosis and inflammation in psoriatic lesions even in the absence of infection (124) (Figure 3).

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by immune complexes and high levels of IFN-α induced by the presence of autoantigens due to a failure to eliminate products derived from apoptotic or necrotic cells (129, 130). Loss of tolerance toward self-antigens leads to the activation of autoreactive B cells and the production of autoantibodies against nucleic acids and AMPs, which are released by infiltrating neutrophils that undergo NETosis in the skin and kidneys of patients with SLE. The generated immune complexes may deposit in different tissues, resulting in injury and inflammation, provoking mainly cutaneous lesions, nephritis, and cardiovascular disease (131, 132). Furthermore, opsonized autoantigens induce pDCs to secrete IFN-α, also known as an “IFN-α signature,” and induce neutrophils to form NETs (133, 134).

In patients with SLE, nucleic material derived from dead cells accumulates due to the failure of its elimination. These self-antigens are presented to autoreactive B cells in germinal centers in secondary lymphoid organs by follicular dendritic cells, thus generating autoantibodies against cellular components derived from NETosis and apoptosis (134, 135). The production of immune complexes activates the complement system and induces inflammation, vascular injury, thrombosis, and brain damage. Immune complexes are internalized by pDCs through type II Fcγ receptor-mediated endocytosis; afterward, they associate with TLR7 and TLR9 on endosomes, which leads to the activation of IFN-α-secreting pDCs and additional formation of NETs (136–138).

Patients with SLE have been found to possess elevated numbers of a subpopulation of neutrophils in the blood known as low-density granulocytes (LDGs). These are immature neutrophils that quickly undergo apoptosis and release ROS in vitro, thus acting as potent NET inducers in SLE patients (132). Through NET formation, LDG intracellular contents are released into the microenvironment and include several molecules such as LL37, α- and β-defensins and HMGB1; these molecules associate with nucleic acids and induce pDC activation though TLR9 stimulation, which subsequently induces IFN-α synthesis. It has been observed that, in SLE patients, IFN-α is a potent NETosis inducer (139) and, along with activated pDC-derived IL-6, promotes the differentiation of autoantibody-secreting autoreactive B cells (140). Another possible mechanism for the release of autoantigens such as HMGB1 and nucleic acids from apoptotic cells is through secondary necrosis, a phenomenon observed when apoptotic bodies are not properly removed by phagocytes. HMGB1 then associates with DNA and activates pDCs due to its recognition by TLR9 and receptor for advanced glycation end products (RAGE) in pDCs, thus inducing pDC activation (141). DNA–HMGB1 complexes may also be recognized by autoreactive B cells through B cell receptor–TLR7/9–RAGE; this results in the production of autoantibodies. Likewise, DNA–HMGB1–immunoglobulin may activate pDCs by interacting with RAGE–FcR–TLR9, which will lead to IFN-α synthesis and a positive feedback cycle (Figure 4) (142).

Finally, the release and persistent presence of these products represents a source of self-antigens that enhance the autoimmune and inflammatory process, leading to tissue injury in SLE.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by persistent synovial inflammation that leads to cartilage and bone injury in the joints (143). The synovial fluid at the synovial cavity of RA patients becomes infiltrated with neutrophils that readily form NETs; furthermore, even the circulating neutrophils of RA patients are more easily stimulated to NETosis than those from healthy subjects (144, 145). As occurs in other autoimmune diseases, NETs may act as a source of extracellular autoantigens; for instance, citrullinated peptides generated from histone citrullination via PAD2 and PAD4 activity are overexpressed in neutrophils and can be detected even in the synovia of RA patients (144, 146, 147). Such citrullinated peptides are recognized by α-citrullinated peptide antibodies (ACPAs), which form immune complexes that induce NET formation, resulting in the release of neutrophil granular contents as well as cytoplasmic self-antigens in the joints. They may also release receptor activator of nuclear factor kappa-β ligand and B-cell activating factor, which activate osteoclasts and B cells, respectively (148, 149), leading to excessive innate and adaptive immune responses in the joints and tissue injury. ACPAs are detected in the serum of RA patients at early stages of the disease and even before clinical symptoms appear, and they thus represent an early biomarker of RA (150). Khandpur et al. have found that in addition to autoantibody NET induction, IL-17 and TNF-α may also have this ability, and they found these cytokines to be elevated in RA patient serum (144).

High-mobility group box 1 is another autoantigen related to RA pathogenesis. It can be found elevated in the pannus of RA patients, particularly in the bone-cartilage interphase and areas with tissue hypoxia (151). Under hypoxic stress, cells release HMBG1 and induce production of the proinflammatory molecules TNF-α and IL-1, suggesting that HMGB1 is closely associated with hypoxia and inflammation in RA (152). Additionally, this protein binds IL-1α and IL-1β to form complexes that enhance the immune response in joints, thus provoking inflammation (141).

Some patients may develop Felty syndrome, a severe presentation of RA that manifests in patients as neutropenia and splenomegaly. The latter seems to be related to the oligoclonal expansion of T cells and autoantibodies against PAD4 (153).

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the destruction of β pancreatic cells in genetically predisposed individuals, leading to hyperglycemia. The destruction of β pancreatic cells also permits the presentation of autoantigens that are recognized by autoreactive T cells, followed by the production of specific autoantibodies for β cell antigens, including glutamic acid decarboxylase autoantibody, insulinoma-associated protein 2 autoantibody, and zinc transporter-8 autoantibody, which are used clinically as predictors of and diagnostic for T1DM, although they are not considered pathogenic (154–157).

In individuals with T1DM, infiltrates of predominantly CD8 T cells along with CD4 T cells and B cells participate in the destruction of β pancreatic cells through the release of granzymes and perforins, activation of the FasL pathway, and production of proinflammatory cytokines, namely, IFN-γ and TNF-α (158). Innate immune response cells also play an important role in the pathogenesis of T1DM, since macrophages, monocytes, DCs, and neutrophils can be found within infiltrates in pancreatic islets, wherein they synthetize IFN-α and ROS, thus promoting the synthesis of proinflammatory cytokines (154, 158, 159).

Several studies have shown that T1DM patients and individuals at risk of developing the disease suffer neutropenia (160), which may partially be attributed to increased NETosis and neutrophil infiltrate in pancreatic tissue (160–162).

Neutrophils produce superoxide and cytokines when exposed to hyperglycemic conditions. In diabetic individuals, TNF-α is elevated and activates neutrophils to form NETs and, subsequently, to release their intracellular contents, which include neutrophil serine proteases, such as neutrophil elastase (NE), PR3, and CG (161–163). T1DM patients show elevated concentrations of serum NE and PR3, as well as elevated levels of activity for these enzymes (162). These proteins are important in T1DM pathogenesis given their implication in the maturation and release of the cytokines IFN-α, IL-1β, and IL-18, as well as in inducing the expression and activation of TLRs, which are important mediators of insulitis and the destruction of pancreatic β cells (158, 164). They also favor neutrophil recruitment to sites of inflammation, providing negative feedback and contributing to pathogenesis in autoimmune diabetes (162).

Small vessel vasculitis (SVV) is a systemic disease of unknown etiology. SVV patients exhibit blood vessel inflammation affecting arterioles, venules, and capillaries, which may also involve arteries during disease exacerbation. In these cases, necrotizing inflammation occurs in small blood vessels and may potentially damage any organ, the primary ones being the kidneys, lungs, skin, and peripheral nerves (165). Antineutrophil cytoplasmic antibodies (ANCAs) can be detected in most SVV patients (166). Since prolonged exposure to proteins released during NETosis, such as myeloperoxidase (MPO), PR3, histones, HMGB1, and NE, is the main cause of ANCA production, these proteins are considered proinflammatory mediators that activate the complement system and lead to endothelial damage. MPO and PR3 are the principal targets of ANCAs, and it has been shown that α-PR3 and α-MPO ANCAs induce NETosis during active disease; additionally, high levels of DNA-MPO complexes are associated with disease activity (167–170). The presence of ANDA-PR3 and ANCA-MPO may activate neutrophils and perpetuate an inflammatory state through complement system activation and neutrophil chemotaxis toward the site of injury (171).

Infections may also induce the production of ANCAs through molecular mimicry. Additionally, microorganisms may induce NET formation, leading to autoantigen release (172, 173). However, since NETs have been found in SVV patients in remission, it is also important to consider that the presence of ANCAs may also help to remove NET-derived products and contribute to host homeostasis (174, 175).

Thrombosis is caused by the release of TF, cytokines, and other inflammatory mediators during NETosis occurring as a result of infection, autoimmune disease, and cancer (75, 176).

Small vessel vasculitis patients show elevated levels of NETs in the bloodstream. LDGs have been proposed as the main source of NETs due to their capacity to spontaneously generate NETs (177). These NETs may potentially amplify the inflammatory process, causing endothelial injury and activating the alternative complement pathway (170, 178). Additionally, histone presence in NETs has been demonstrated, which contributes to thrombus formation and promotes TF production, which in turn induces thrombin (165, 179). In mouse models, activated platelets have been observed to stimulate NET formation and provoke thrombosis in deep veins (180). Finally, an increased presence of neutrophil-platelet aggregates in SVV patients’ circulation has been shown to correlate to disease activity, which suggests a relationship between NETs and thrombin formation in SVV (181).

Gout is an autoinflammatory disease characterized by the deposition of monosodium urate (MSU) crystals in the joints, which attracts leukocytes and forms structures known as tophi that mediate tissue damage. After uptake by phagocytes, MSU crystals are major stimulants of the immune response through NLRP3 inflammasome-mediated IL-1β production due to the osmotic disequilibrium caused by a sudden increase in the intracellular sodium concentration coupled with water influx and subsequent potassium dilution. Additionally, MSU-activated neutrophils secrete IL-8, TNF-α, and IL-6. These cytokines not only promote neutrophil recruitment but also induce NET formation. In particular, NETs could participate in tophi formation, since their components are closely related (Figure 5) (182, 183).

As in other diseases, NETs have been reported to promote inflammation in gout (184). However, unlike other diseases, NETs also seem to play an important role in regulating the inflammatory process and stopping gout episodes (185). Initially, NETosis reduces neutrophil density, as they indicate neutrophil death. Second, DNA nets encapsulate MSU crystals and protect them from further phagocytosis. Finally, NET-derived proteases inactivate cytokines and abrogate their proinflammatory effects (186).

Crohn’s disease is a complex systemic disease that clinically manifests as gastrointestinal disorders and inflammation of the ileum and colon (187). Though inflammation is a major component of CD, the cellular components involved in its pathology remain somewhat unclear. Regarding neutrophils, their activity becomes altered. While their chemokine-mediated migration is reduced, ROS production is enhanced; furthermore, bacterial uptake seems altogether unaltered (188).

On the other hand, NET formation in CD has not been studied. Arguably, since ROS production is enhanced, neutrophils may be more prone to NET formation. Accordingly, L. rhamnosus, an important probiotic with both protective and corrective activity against CD, effectively inhibits NETosis (43).

Similar to CD, UC is characterized by inflammation of the gastrointestinal tract. Together, CD and UC form a clinical entity known as inflammatory bowel disease (IBD). However, unlike CD, UC is mostly restricted to colon inflammation (189, 190). Also similar to CD, the cellular components of clinical inflammation in UC are mostly unknown. Unsurprisingly, NETs have been observed in UC and correlated with inflammation by proteomic studies (191), though clearly more cellular and biochemical research is required for a clear understanding of NET involvement in IBD.

Diabetes mellitus (DM) is characterized by chronic inflammation that involves humoral factors and different types of white blood cells, including mononuclear and polymorphonuclear leukocytes. It is known that in diabetic individuals, there is an increased neutrophil count and dysfunction of phagocytic activity (195).

It has been observed that the diabetic microenvironment can favor NETosis, as in diabetic conditions (hyperglycemia), neutrophils generate oxidative stress and produce cytokines such as IL-6 and TNF-α, which predispose neutrophils to produce ETs (Figure 6) (196, 197). However, hypotheses linking NETosis deregulation and hyperglycemia, oxidative stress, inflammation, and further complications of the disease remain to be confirmed (198).

Studies in humans and murine models have shown that hyperglycemia predisposes neutrophils to release NETs regardless of diabetes type when stimulated with ionomycin, PMA, or LPS. In addition, protein expression of PAD4 was found to be increased fourfold in neutrophils from individuals with diabetes when compared to those from healthy controls, suggesting that increased PAD4 may favor chromatin decondensation. However, it is still unclear whether high glucose concentrations upregulate the protein expression of PAD4 at the transcriptional or post-translational level (197).

In the sera of type 2 diabetes patients, NET-related biomarkers (elastase, mono- and oligonucleosomes, and dsDNA) are increased when compared to non-diabetic subjects; additionally, these biomarkers positively correlate with glycated hemoglobin (HbA1c) levels. In these patients, dsDNA has also been correlated with the IL-6 concentration, which may suggest a role for NETosis in the interactions between hyperglycemia and inflammation as well as in the consequences of inflammation (198).

Joshi et al. investigated whether hyperglycemic conditions could modulate NET release. They found that neutrophils exposed to high glucose concentrations and neutrophils isolated from diabetic patients had altered potential for NET release when exposed to LPS stimuli (196). Thus, they hypothesized that the chronic proinflammatory conditions present during hyperglycemia promote the constitutive formation of NETs, yet a weak response to stimuli. Fadini et al. found similar results when they analyzed NET-generation pathways in neutrophils isolated from patients with diabetic ulcers, i.e., NOX-dependent and NOX-independent pathways. In this model, neutrophils show enhanced spontaneous NETosis but a compromised capacity for induced NETosis (199).

On the other hand, neutrophils from non-diabetic individuals exposed to a high glucose concentration (25 mM) have been shown to be more susceptible to spontaneous and PMA-induced NETosis when compared to those exposed to a low glucose concentration (5 mM) and mannitol (25 mM). Since non-energetic sugars did not affect NETosis, this phenomenon could be explained by enhanced ROS production through an increase in glycolysis (198).

In summary, these data suggest that NET formation is enhanced in hyperglycemic conditions independent of diabetes type and origin.

It is important to note that in DM patients, slower wound healing represents one of the main complications. Cicatrization is a process that involves endothelial cells, fibroblasts, leukocytes, platelets, and keratinocytes. Since inflammation is a typical characteristic of the cicatrization process, neutrophils are one of the first cells recruited to the injury site, where they also act as antimicrobial cells. However, some studies report that excessive activation of NETosis-inducing neutrophils may contribute to poor wound healing (199–202).

It has been shown that PAD4-deficient mice (both diabetic and non-diabetic) possess faster wound healing and re-epithelization processes than their wild-type (WT) counterparts, independent of wound infection. This suggests that NETosis could hinder wound healing by limiting keratinocyte migration and, consequently, adequate re-epithelization. As such, NETs are a putative therapeutic target for one of the main disabling complications of diabetes (197).

Recently, it has been shown that an excess of NET-related proteins is associated with wound healing alterations and poor resolution. Furthermore, NE, oligo- and mononucleosomes, neutrophil gelatinase-associated lipocalin, and PR3 are increased in biopsies with poor healing prognosis compared to those in remission or completely healed (199).

These studies show a link between neutrophils, inflammation and tissue injury in diabetes. However, more investigation is required to fully understand the mechanisms behind NETosis and glucose metabolism.

Obesity is characterized by an excess of adipose tissue produced as a consequence of a loss of equilibrium between energy intake and expenditure (203). The development of obesity implies a complex interaction of genetic and environmental factors that are also frequently associated with other chronic complications (hyperglycemia, dyslipidemia, hypertriglyceridemia, and high blood pressure). Individuals with three of these criteria may be clinically diagnosed as presenting MS according to the World Health Organization. MS increases the risk of developing metabolic diseases such as type 2 DM and cardiovascular diseases (194).

Obesity has been associated with low-grade chronic inflammation in white adipose tissue (192). Adipocytes are able to secrete adipokines such as TNF-α, IL-6, and IL-8, which due to their proinflammatory properties, have been associated with enhanced activity of peripheral neutrophils, such as production of superoxide radicals and NET formation (Figure 7A). However, the effects of inflammation on adiposity and its association with NETosis are not yet clear (204–206). Since adipose tissue promotes a potentially neutrophil-activating proinflammatory environment, there exists a need to study whether the increase in adiposity may contribute to NETosis. Additionally, enhancement of glucose metabolism may lead to increments of mitochondrial-derived ROS. This phenomenon is also observed in obesity, which provokes activation of inflammatory pathways (192).

Feeding mice a high-fat diet (HFD) induces neutrophil recruitment to adipose tissue (Figure 7B). As a consequence, it is possible that neutrophils could play a role in triggering the inflammatory cascade in response to obesity (207). Accordingly, when mice fed an HFD are infected with influenza virus, viral titers are increased threefold when compared to infected mice fed a low-fat diet (LFD); the H₂O₂ concentration is also relatively higher in HFD mice, suggesting increased oxidative stress in their lungs. The neutrophils of these mice are more prone to spontaneous NET formation compared to neutrophils derived from LFD mice. Finally, the authors conclude that because of the elevated cytokine levels and the proinflammatory oxidative stress caused by these cytokines, adiposity associated with higher lung viral titers could represent stronger stimuli for NET formation, suggesting that in individuals with morbid obesity, lung NETs could be formed at significant levels as a response to influenza infection, thus aggravating pulmonary injury and resulting in further complications of influenza-provoked pneumonia (208).

Talukdar et al. have reported that neutrophils derived from mice fed an HFD possess higher granular content and that their NE activity is significantly higher than that of mice fed an LFD, which could promote insulin resistance through degradation of insulin receptor substrate-1 (Figure 7B). They also observed decreased insulin signaling and higher glucose production in human hepatocytes and murine adipocytes. Additionally, they showed that NE-knockout mice are more sensitive to insulin than WT mice, suggesting that the ablation of NE leads to higher hepatic insulin sensitivity and decreased expression of proinflammatory genes (207).

Oxidized low-density lipoprotein (oxLDL) is a complex mixture of LDL composed of oxidized bioactive elements with intrinsic proinflammatory activity capable of stimulating ROS production and improving the degranulation capacities of human neutrophils. Additionally, oxLDL is able to induce ROS-dependent NETosis in human neutrophils in a dose- and time-dependent manner. Interestingly, TLR2/TLR6 heterodimers seem to be necessary for oxLDL-induced NETosis as well as the PKC–IRAK–MAPK pathway, which indicated that NETosis is a multifactorial process that requires not only respiratory burst but also sequential activation of several signaling events that depend on the nature of the NET inductor. In conclusion, the inflammatory environment characterized by oxLDL and proinflammatory cytokines (IL-1β, TNF, IL-8) may be considered a potential inductor of NETs in the absence of microbial stimuli, further aggravating systemic inflammatory response syndrome, atherosclerosis and other sterile inflammatory conditions (209).

The accumulating data on the role of neutrophils in infectious, autoimmune, autoinflammatory and metabolic diseases via NET structures demonstrate that they constitute novel bioindicators of prognosis and represent candidates for therapeutic targets by blocking the formation of or locally neutralizing NET signaling.