Introduction

Gouty arthritis (GA) is an inflammatory arthritis caused by abnormal monosodium urate (MSU) deposition that occurred in joints and peripheral tissues1 Ample evidence has suggested that the incidence and prevalence of GA are increasing around the world,2 bringing burdens on health systems.3 Patients with GA are characterized by high levels of serum uric acid (UA).4 experiencing bursts of symptoms and remission including intense pain, redness and swelling.5 Uric acid can accumulate in the bloodstream due to its excessive production or impaired renal excretion, ultimately resulting in the formation of needle-shaped crystals in the joints and adjacent tissues.6 Subsequently, the deposition of uric acid crystals in joints triggers an immune response as the body identifies the crystals as foreign substances, thereby initiating an inflammatory cascade that ultimately culminates in the distressing symptoms of gouty arthritis.7,8 Nevertheless, the underlying mechanisms behind GA onset and its interaction with the immune system are relatively vague.

Neutrophils are the most abundant immune cells in human and animal peripheral blood.9 As the first-line cells in immune system, neutrophils exhibit rapid responsiveness to both microbial and inflammatory cues emanating from damaged tissues. Neutrophils are swiftly mobilized to the site of injury to combat various pathogens, such as microcrystals.10 In homeostatic conditions, neutrophils are maintained at a low baseline density.11 However, when tissue damage or infection occurs, neutrophils assume critical roles as immune effectors, with their density increasing dramatically and their recruitment to the damaged site being promptly activated.12

Extracellular traps (ETs) are web-like structures containing DNA, histones, and various granule proteins like myeloperoxidase (MPO), released by activated neutrophils and other immune cells to capture and eliminate invading pathogens.13 Among them, neutrophils extracellular traps (NETs) have been implicated in different pathological conditions, such as GA.14 During GA development, NETs are released in response to MSU crystal stimulation, triggering an inflammatory cascade. It seems that inhibiting the formation of NETs, also called NETosis, appears as a feasible approach to alleviate GA.15 Several studies have also identified various factors that regulate the formation and degradation of NETs.16,17 However, evidence also showed that aggregated NETs (aggNETs), consisting of high-density of NETs, appeared to exert protecting role from inflammation via packing MSU crystals, and the mechanisms of which still remain elusive.18 Of note, targeting components from NETs offers new avenues for developing NET-based therapies for the GA treatment. In this review, we undertake a comprehensive review of the constituents of NETs and the diverse mechanisms underpinning NETosis, providing a detailed illustration. Additionally, we synthesize current knowledge on clinically available pharmacological interventions for GA and innovatively evaluate natural and synthetic product-based inhibitors that target NET formation as novel therapeutic options. This review aims to illuminate potential therapeutic strategies that leverage our understanding of NETs to combat GA effectively.

NET: The Special Network Structure Released by Neutrophil

NETs have gained a wide range of attention for their production, which is accompanied by the following inflammatory cascade. NETs are reticular skeletons composed of DNA released by neutrophils outside the cell, to which nuclear proteins (eg, histones), cytoplasmic proteins, granular proteins (eg, neutrophil elastase (NE), myeloperoxidase (MPO)), and antimicrobial peptides, etc., are attached (Figure 1). Most of these components exert different effects when they encounter different responses in the immune system.

Figure 1 Neutrophils extracellular trap formation. After being stimulated by various inducers including microbial products, cytokines, immune complexes and etc, neutrophils undergo chromatin decondensation and nuclear membrane breakdown, leading to the release of NETs. NETs are mainly DNA-based skeleton structures whose surfaces are inlaid with histones, NE, MPO PAD4. Moreover, NETs are characterized by antibacterial effect through capturing and killing pathogenic microorganisms.

The extracellular DNA in NETs is derived from the chromatin of the neutrophil nucleus, which is extruded from the cell and released into the extracellular space. The process of NETosis, characterized by the dissolution of the nuclear envelope, decondensation of chromatin and extrusion of DNA, is activated in response to various stimuli.19,20 As the major component of NETs, extracellular DNA is considered to be a damage-associated molecular patterns (DAMPs)21,22 that induces pro-inflammatory cascades.23 NETs have been implicated in the pathogenesis of various of inflammatory and autoimmune diseases, including sepsis, rheumatoid arthritis, and lupus.24,25 The DNA in NETs can activate different immune cells, such as macrophages and dendritic cells, leading to the release of pro-inflammatory cytokines and amplification of the inflammatory responses.26,27 A study28 has shown that the level of cell-free DNA in the plasma of patients with sepsis was positively correlated with mortality, meaning the higher the level of cell-free DNA, the higher the mortality rate of sepsis. Furthermore, DNA has been identified as a key factor in extending the lifespan of neutrophils. Notably, stimulation of neutrophils with mitochondrial DNA has been shown to enhance their activity.29 DNA’s significance as the fundamental framework of NETs is undeniable.

Histones are a group of highly basic and conserved proteins located in the nucleus and are a key component of the web-like structures that are released by activated neutrophils. Several histone subtypes, including histones H2, H3, and H4 constitute a complex with DNA called nucleosome.30 Histones, a key component of NETs, contribute significantly to the antimicrobial activity of NETs. Specifically, the positively charged amino acids in histones negatively interact with the charged cell membranes of microorganisms, promoting their adherence to the NETs and leading to subsequent elimination.31 Not only do histones possess antimicrobial properties, but they also exhibit toxic effects on the host, triggering pro-inflammatory responses upon their release from the nucleus into the extracellular space.32,33 The release of histones from dying cells, including from NETs, has been implicated in the pathogenesis of a range of inflammatory and autoimmune diseases, including sepsis, rheumatoid arthritis, and lupus.34 On the one hand, histones can be passively released through the necrotic program to exacerbate inflammatory responses. Moreover, histones can also be actively released during NETosis to exert an antibacterial effect.35 The role of histones as damage-associated molecular patterns (DAMPs) can trigger toll-like receptors (TLRs), activate the NLRP3 inflammasome, and induce calcium influx, leading to the initiation of inflammation in conditions, such as acute pancreatitis.36 In septic mice, histones can be expected to result in endothelial dysfunction, organ failure and even death.37 It must be mentioned that high mobility group box 1 (HMGB1) is a key component of NETs, where it plays an important role in immune response like histones. HMGB1 is a highly conserved nuclear protein present in most mammals. In its capacity as DAMPs, HMGB1 can promote the aggregation of neutrophils at sites of tissue damage and enhance inflammatory responses via interaction with other DAMPs (such as DNA) or pathogen-associated molecular patterns (PAMPs) (such as lipopolysaccharide (LPS)).38,39 HMGB1 containing disulfide bonds binds to TLR4, inducing cytokine production by macrophages and also promotes NETs formation by neutrophils.40 The relationship between histones and HMGB1 in the context of NETs is complex and involves both synergistic and antagonistic interactions.41 HMGB1 is known to promote the release of histones from neutrophils and to enhance their ability to bind to bacterial membranes, thereby increasing the efficacy of NETs in killing invading microorganisms.42 However, HMGB1 is able to exert both pro-inflammatory and anti-inflammatory effects, depending on the cellular and molecular context when histones begin to activate immune cells and promote inflammation.43,44

Peptidyl arginine deiminase 4 (PAD4) is a calcium-dependent enzyme that plays a critical role in NETs formation. During the process of NETosis, activated neutrophils undergo chromatin decondensation and nuclear membrane breakdown, allowing for the release of NETs composed of DNA, histones, and other proteins.45 The citrullination of histones by PAD4 is a crucial step in this process, as it alters their charge and structure, facilitating their release from chromatin and promoting the formation and stability of NETs.46,47 PAD4 has been implicated in the pathogenesis of a range of autoimmune and inflammatory diseases.48–50 Wesley et al demonstrated that mice lacking PAD4, a key enzyme involved in NETosis, exhibited a significant reduction in NET formation and pro-inflammatory cytokine production, leading to protection against acute kidney injury induced by renal ischemia/reperfusion.51 Renal function was restored to 48 hours after ischemia/reperfusion, whereas renal function in wild-type mice gradually deteriorated. The PAD-specific inhibitor YW3-56 was used for validation, which indicates that PAD4 plays a key role in ischemia/reperfusion-induced acute kidney injury. It is also a strategic point to differentiate apoptosis52 and necrosis.53,54

MPO is present in specific tissues within neutrophils, monocytes, and macrophages. The majority (95%) of MPO found in the bloodstream originates from neutrophils, making changes in its levels a reflection of alterations in neutrophil functionality. Some studies55,56 have found that MPO in NETs was biologically active and exhibited bactericidal ability after binding to DNA in NETs, where it generated hypochlorous acid and other reactive oxygen species (ROS) that contributed to the microbicidal activity of NETs.57 MPO is also involved in the modification of histones, which helps increase the antimicrobial activity of NETs.58 Simultaneously, by utilizing hydrogen peroxide (H2O2) and chloride ions, MPO has the ability to generate hypochlorite, which effectively contributes to the eradication of bacteria. This evidence demonstrates that MPO can enhance the bactericidal efficacy of NETs, while also causing tissue damage. Therefore, MPO may act as an antigen in the pathogenesis of anti-neutrophil cytoplasmic antibody-associated vasculitis.59 Meanwhile, MPO has emerged as a key player in the pathogenesis of numerous inflammatory conditions, such as vasculitis, systemic lupus erythematosus, and acute pancreatitis.60–62 By producing ROS and other substances, MPO can inflict tissue damage and exacerbate inflammation, thereby contributing to the pathophysiology of these diseases.63 Recent studies have shown that inhibiting MPO activity with a specific inhibitor is capable of suppressing the formation of NETs.57,64 Collectively, these findings underscore the importance of MPO as a critical component of NETs in mediating their antimicrobial activity, but also emphasize the need to regulate its activity to prevent its contribution to inflammatory disease pathogenesis.

Neutrophil elastase (NE) represents a serine protease variant prominently housed within the azurophilic granules of neutrophils, exerting a pivotal role as a fundamental constituent within NETs. The presence of both NE, as well as MPO, within neutrophils is notable, as these enzymes are extensively associated with the fiber network of NETs.65 During NETosis, translocation of NE to the nuclear membrane is required for chromatin decondensation, whereas MPO can bind to chromatin and enhance chromatin dedensification.66,67 In addition to degrade extracellular matrix proteins and inflammatory mediators such as cohesin, various membrane proteins and IL-8, etc., NE is capable of inhibiting tissue factors and promoting the formation of vascular fibrin.66,68,69 Studies have shown that the formation of NETs involved with MPO and NE depends on the different types of stimuli. Parker59 et al found that the generation of phorbol 12-myristate 13-acetate (PMA)-stimulated NETs required the involvement of MPO without the need for Staphylococcus aureus or E. coli. Conversely, Leishmania parasites70 induced the generation of NETs that required NE without the need for MPO and ROS. As a potent inducer of NETs, PMA is widely used in the research about NETs based on the different kinds of signaling pathways, enzymes, and ROS requirements.71 Another inducer of NETs is the MSU crystal. In gout, MSU crystal is the main precipitated form of urate in the blood, at the same time, is also an important participant in activating the neutrophils, which facilitates the release of NETs.72 It is worth noting that, unlike PMA, the release of NETs induced by MSU crystal is uncertain if the ROS involvement is needed. According to the in vivo research from Davidson and her companions,73 NET formation induced by MSU crystals was independent of ROS production. The same findings were also found in other studies. Tatsiy et al found that under the activation of MSU crystals, NETosis was found to be independent of endogenous ROS, but under the control of PAD4.74 Conversely, NETosis caused by MSU crystals in vitro was found to be in an ROS-dependent manner. What’s more, inhibiting ROS through different anti-oxidants restrained NETosis caused by MSU crystals.1 This controversial view deserves a deeper exploration, which may provide insights into the progression of NETosis in diseases.

The Formation of NETs: Vital NETosis and Suicidal NETosis

The formation process of NETs is called NETosis which is a novel type of programmed cell death distinguished from neutrophil apoptosis, necrosis and pyroptosis. The concept of NETosis was firstly purposed by Brinkman and his colleagues in 2004.75 They offered the opinion that neutrophils were activated and released extracellular meshwork, called NETs, that was decorated with granule proteins and chromatin, leading to degrade virulence factors and kill bacteria. Following an extensive span of nearly two decades devoted to research, there has been a discernible augmentation in the comprehension of NETosis, leading to a more comprehensive and profound knowledge of this biological process. The process of NETosis can be triggered by various stimuli, such as microbial products, cytokines, and immune complexes, and can occur via distinct mechanisms, mainly involving vital and suicidal NETosis that depend on the viability of neutrophils.19,45,76

The most striking difference between NETosis and other modes of death is the changes in the nucleus and the release of NETs. Upon stimulation, the nuclear membrane of neutrophils disintegrates into vesicles, while chromatin begins to deconcentrate. Then, antimicrobial peptides are released from intracellular particles to adhere to lost chromatin. Simultaneously, upon the disruption of the plasma membrane, a diverse array of intracellular components, including proteins, DNA, and other entities, are extruded into the extracellular milieu. Notably, this process does not entail the condensation of chromatin, commonly known as chromatin pyknosis. Although researches have proved that the formation of NETs improved the body’s defense mechanism, NETs are believed to be capable of causing the body to break the pro- and anti-inflammation balance.77,78 Apoptosis is cell shrinkage accompanied by DNA rupture and nucleus condensation. The whole cell forms apoptotic bodies through blebbing and other methods. Of note, the process of apoptosis does not cause a rupture of the plasma membrane and the release of NETs, which is the biggest difference between apoptosis and NETosis.79 Additionally, neither inflammatory mediators nor inflammatory responses occur in this process. Cell necrosis leads to the swelling and rupture of cells, including the processes like the swelling of various intracellular organelles such as the nucleus, the rupture of the plasma membrane, the release of cellular contents, and the triggering of inflammation.79 What distinguishes with NETosis is that in the process of cell necrosis, DNA is barely released. Fuchs et al80 performed live cell imaging of NETs to reveal the difference between NETosis, apoptosis and pyroptosis. Pyroptosis is typified by cellular shrinkage, accompanied by DNA fragmentation and degradation, which is similar with the apoptotic process.81 Pyroptosis is distinguished by cell necrosis accompanied by the process of cell swelling, rupturing and releasing intracellular inflammatory substances, which induces inflammation in the body without production and release of NETs structure.82,83

Vital NETosis, also known as non-lytic NETosis or “vivacious NETosis”, is a relatively new type of NETosis that was first described in 2011.84 Unlike suicidal NETosis, vital NETosis does not result in the death of the neutrophil, but rather, the release of NETs while the neutrophil remains alive and functional.85 During vital NETosis, the neutrophil undergoes significant morphological changes, including the formation of nuclear lobes and the extrusion of chromatin into the extracellular space, which is accompanied by the release of antimicrobial molecules such as myeloperoxidase and neutrophil elastase. Vital NETosis has been suggested to play a role in promoting wound healing and preventing excessive inflammation, as it can limit the spread of bacterial infections and promote tissue regeneration.86

On the other hand, suicidal NETosis, also known as lytic NETosis or “suicidal NETosis”, is a more well-known and characterized type of NETosis that results in the death of the neutrophil.87 Suicidal NETosis is distinguished by the rupture of the nuclear envelope of neutrophils, subsequently leading to the extracellular release of chromatin and granular proteins. This process is mediated by the activation of the NADPH oxidase complex, which produces ROS that lead to DNA damage and histone citrullination, as well as the activation of proteases and endonucleases that degrade the neutrophil’s nuclear and cellular components.88 Suicidal NETosis is important for the host defense against various pathogens, including bacteria, fungi, and viruses, but it can also contribute to tissue damage and the development of autoimmune and inflammatory diseases.89

In summary, vital and suicidal NETosis are two distinct forms of neutrophil death that contain the process of releasing extracellular traps to fight against pathogens. Vital NETosis is currently considered as a relatively new and less characterized process that enables neutrophils to remain viable and functional. In contrast, suicidal NETosis is a well-established mechanism that results in the death of neutrophils and is critical for host defense against infections.

The Underlying Mechanism of NETs Formation

According to the formation mechanism of NETs, it is mainly divided into three pathways: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NADPH oxidase 2, NOX)-dependent, -independent and others (Figure 2).

Figure 2 The underlying mechanism of NETs formation. The mechanism of NETs formation can be classified into NOX-dependent pathway, NOX-independent pathway and others. The NOX-dependent pathway to induce NETs formation contains RAF-MEK-ERK pathway, DAG-PKC pathway and iICs-FcγRIIIB Pathway. On the other hand, NETs formation is caused by NOX-independent pathway including the mitochondrial DNA pathway, Calcium pathway and histone acetylation pathway. In some special cases, NETs formation can be performed through neither NOX-dependent pathway nor NOX-independent pathway, as referred to rapid release of NETs formation. On the contrary, pathway like the mTOR pathway is involved in NETs formation by either NOX-dependent pathway or NOX-independent pathway.

NOX-Dependent Formation of NETs

The NADPH oxidase complex is a critical enzyme involved in the generation of ROS that is necessary for the formation of NETs. It has been reported that NOX-dependent formation of NETs is a critical mechanism in the pathogenesis of various inflammatory and autoimmune diseases.90 The NOX-dependent pathway is the predominant mechanism involved in the production of NETs.80 Plenty of pro-inflammatory mediators can stimulate neutrophils to generate NETs, such as PMA, LPS, interlukin-6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-ɑ), as well as bacteria, fungi, and chemicals.91 The activation of NOX by stimuli causes the generation of ROS, including O2, H2O2, and HOCl. In addition to killing microorganisms,92 ROS is capable of activating NE and MPO in neutrophils93 and interacting with the nucleus without restraint, in which serine proteases and NE cleave histones to promote chromatin decondensation.94,95 Soon afterwards, the nuclear membrane loses integrity, leading to the release of chromatin into the cytosol and subsequently the final form NETs.65,69 Here, the pathways involved in the NOX-dependent formation of NETs are illustrated in the following.

RAF-MEK-ERK Pathway

The rapidly accelerated fibrosarcoma (RAF)-mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway (RAF-MEK-ERK pathway) is one of the mitogen-activated protein kinase pathways. This signaling pathway exhibits the capacity to convey extracellular signals to the nucleus, facilitating interactions with specific transcription factors and subsequently eliciting context-dependent cellular responses.96 The RAF-MEK-ERK pathway can be activated by various stimuli, for example PMA and Fcγ receptors (FcγRIIIb), to induce NETosis.97 Some studies98,99 have shown that the RAF-MEK-ERK pathway is crucial in the process of PMA-induced NETs generation, while downregulating the expression of apoptotic protein Mcl-1 to prevent cell apoptosis. This downregulation can be blocked with protein kinase C (PKC), cRaf and MEK inhibitors.100 In addition, this pathway also acts as a vital role in NETs induced by unconventional stimuli. During the process of amoebiasis, neutrophils form the host are stimulated and NETs are induced by amoeba. Study has proved that selective inhibition on RAF and ERK exhibited prevention of E. histolytica induced NETs.101 The evidence strongly implies that RAF-MEK-ERK pathway serves as an upstream modulator of NADPH oxidase, thereby implicating its involvement in the formation of NETs through the activation of NADPH oxidase and up-regulation of anti-apoptotic proteins. While the study also showed that the inhibitor targeted NADPH could not block the E. histolytica-induced NETs formation,101 which might be explained by that ROS generated by trophozoites and processed by the extracellular MPO during the contact with neutrophils were of relatively importance for E. histolytica induced NETosis.102 These finding sheds light on the intricate regulatory mechanisms underlying NETosis and further underscores the interconnectedness of signaling pathways in orchestrating neutrophil functions.

DAG-PKC Pathway

Protein kinase C (PKC) is mainly distributed in the cytoplasm in an inactive state and is activated by diacylglycerol (DAG) and then transferred to the cell membrane to participate in the formation of NOX complex, leading to the increased generation of ROS and the sequent formation of NETs.103–105 The DAG-PKC pathway can be considered as an upstream signaling pathway for NOX-dependent formation of NETs.105 DAG mimicking PMA can stimulate neutrophils to form NETs,106 therefore, leading to the association between PKC signaling and NETs formation via NADPH oxidase-ROS pathway and PKC. Gray et al106 used LY333531 to inhibit PKCβ (LY333531 is a specific potent inhibitor of PKCβ that is a subtype of the PKC family), and the results showed the respiratory burst activated by NADPH oxidase was inhibited, and the formation of NETs was also suppressed. The findings of this study provide corroborative evidence for the involvement of the DAG-PKC pathway in the formation of NETs. Notably, among the PKC isoforms, PKCβ emerges as the primary regulatory isoform governing the process of NET generation.

iICs-FcγRIIIB Pathway

Immobilized immune complexes (iICs) are immune complexes that consist of autoantibodies and self-antigens, and Fc gamma receptor IIIb (FcγRIIIB) is a receptor expressed on neutrophils that binds to the Fc portion of IgG antibodies.107 Upon binding of iICs to FcγRIIIB receptors which presents on the surface of neutrophils, a signaling cascade is initiated, enhancing the induction of NETosis and subsequent formation of NETs in an ROS release-dependent manner.107 Alemán et al108 demonstrated that activation of FcγRIIIB receptors using specific antibodies effectively induces the generation of NETs, akin to the stimulatory effect observed with PMA. These findings highlight the iICs-FcγRIIIB pathway as a significant contributor to the mechanistic landscape of NET formation. Upon binding of iICs to FcγRIIIB, a plethora of downstream signaling cascades is triggered. Among these, the activation of protein kinases, particularly the Src family kinases, plays a pivotal role in phosphorylating and activating subsequent downstream effectors. Notably, this includes the guanine nucleotide exchange factor Vav1, which, upon activation, serves as a crucial mediator engaging the small GTPase Rac, thereby facilitating its activation. Consequently, Rac activation leads to the initiation of the NOX complex, ultimately enhancing the production of ROS. This robust ROS generation contributes significantly to the formation of NETs, thus substantiating the integral involvement of this signaling pathway in the NETosis process.107 Alongside the involvement of the Src family kinases and Vav1, a constellation of additional downstream signaling pathways is likely implicated in the activation of the NOX complex and subsequent ROS production. These pathways encompass the RAF-MEK-ERK pathway and the DAG-PKC pathway.100,109 These pathways can be activated by various signals, including cytokines, growth factors, and G protein-coupled receptors, and can activate the NOX complex and the subsequent formation of NETs.

NOX-Independent Formation of NETs

While the canonical pathway of NET formation involves the production of ROS by the NOX complex, NETs can also be formed through NOX-independent mechanisms. The exact signaling pathways for NOX-independent formation of NETs are still not fully understood, but several mechanisms have been proposed.110,111 Here are some of the proposed signaling pathways for NOX-independent formation of NETs.

Mitochondrial DNA (mtDNA) Pathway

The release of mtDNA can activate the immune system and contributes to the pathogenesis of several diseases, including autoimmune diseases, cancer, and cardiovascular diseases.112–114 It should be noted that mtDNA can serve as a DAMP to promote the formation of NETs.115 The pathway for mtDNA release involves several steps. Initially, a critical event in the cellular milieu involves the occurrence of mitochondrial permeability transition, culminating in the release of mitochondrial constituents, such as mtDNA, into the cytoplasmic compartment. This process significantly contributes to the intricate dynamics of cellular homeostasis and underscores the pivotal role played by mitochondria in orchestrating essential cellular functions. Second, the mtDNA is recognized by the cGAS-STING pathway, which triggers the production of type I interferons and other inflammatory cytokines. Finally, the mtDNA can directly activate the NLRP3 inflammasome, which leads to the production of IL-1β and other pro-inflammatory cytokines.116 According to Yousefi et al, their understanding suggests that the release of mtDNA is a regulated process that enables neutrophils to use their DNA as a weapon against microbes. In addition, it has been proposed that mitochondrial DNA may exert immunomodulatory effects on other immune cells.113,117 Furthermore, one of the responsible underlying mechanisms entails the belief that SIRT1 possesses the capacity to stimulate the initiation of mitochondrial permeability transition pore channels, facilitating the liberation of mitochondrial DNA and consequently giving rise to the formation of mitochondria-dependent vital NETs, as opposed to the conventional citrullinated histone H3-dependent NETs.118 It is implied that neutrophil-SIRT1-vital NET pathway may be a potential strategy to prevent tumor metastasis.

Calcium Pathway

The calcium pathway refers to the signaling cascade triggered by the release of intracellular calcium ions in response to stimuli such as LPS.119 In the context of NET formation, calcium signaling has been shown to activate the NOX-independent pathway. This pathway is triggered by the calcium-activated potassium channel of small conductance (SK channel), which is the major calcium activated potassium channel known to be present on neutrophils. Among the SK channel members (SK1, SK2 and SK3), SK3 is expressed predominantly on neutrophils and the knockdown of SK3 in differentiated HL-60 (dHL-60) cells reduces the NETs formation induced by ionomycin-mediated NOX-independent NETosis. Ionomycin is a natural calcium ionophore produced by a different gram-positive bacteria Streptomyces conglobatus and has proved the ability to induce NOX-independent NETosis.110 On the other hand, David et al raised the conclusion that NOX-independent NETosis caused by calcium ionophores required the mitochondrial ROS, which was regulated by the NOX2 enzyme.110 However, evidence also present the correlation between calcium pathway and NOX-dependent NETosis. Calcium is a vital regulator of NETosis, as it activated enzymes and signaling pathways that involve in the production of ROS, which are essential for NOX-dependent NETosis.19 One of the calcium dependent-enzymes is PAD4, which modified histones and chromatin decondensation.120 Also, calcium is deemed as a regulator of NADPH oxidase, the main source of ROS in neutrophils.121

Histone Acetylation Pathway

Histone acetylation is a post-translational modification that loosens the chromatin structure and affects gene expression.122 Histone deacetylases (HDACs) are enzymes that removes acetyl groups from histones and modulates chromatin condensation. According to the study,123 histone acetylation (particularly H4K8) and spontaneous NETosis at baseline were increased by agents that block HDAC activity and enhance histone acetylation, known as HDAC inhibitors. Also, the same situation happened to the NETosis induced by PMA, A23187, or LPS in an additive manner. In the further investigation, inhibition or knockdown of HDAC1, HDAC2, HDAC3, or HDAC6 increased histone acetylation and spontaneous NETosis at baseline. And the inhibition of HDAC was beneficial for the elevation of chromatin decondensation during NETosis induced by PMA. Interestingly, there was a performance that increased in mitochondrial ROS production and caspase-3 activation, both of which were associated with NOX-independent NETs formation, displayed by the inhibition of HDAC. What’s more, it is showed that the neutrophil death form was available to be switched from NETosis to apoptosis with HDAC inhibitors in dose-dependent manner.124 The evidence mentioned above implied that histone acetylation may be involved in NOX-independent NETs formation by multiple pathways and deserves attention of researchers in the field of NETs. Despite the researchers’ partial comprehension of the histone acetylation process, there is currently a limited repertoire of drugs targeting NETosis through modulation of histone acetylation. Consequently, there is still great potential for drug development in this specific domain.

Others

Apart from NOX-independent and NOX-dependent way for the formation of NETs, some methods are found to be classified into neither of them, as referred to rapid release of NETs formation. With the in-depth study of NETs, it has been found that neutrophils can continue to remain active after the clearance of NETs and come into play with antibacterial effects. This process only takes 5–60 minutes, which is different from the release of suicidal NOX-dependent NETosis or NOX-independent NETosis lasting more than 3 hours.125 At the end of this process, neutrophils eventually rupture and die. Yipp et al45,126 proposed that the formation of nuclear DNA in rapid NETosis was dependent on the binding of LPS. Under the action of gram-negative bacteria, generally, LPS binds to TLR4 on the platelet surface to generate NETs in a NOX-independent manner. For gram-positive bacteria, TLR2 and complement receptor 3 are required. After a few minutes of S. aureus stimulation, neutrophils were observed under a microscope to depolymerize their chromosomes, forming beads of DNA strands and nucleosomes. Late chromosomes are excreted in the form of nuclear vesicles, and neutrophils appear to be anucleate. Parts of chromatin and dense granules are excreted from cells by exocytosis to form NETs. The process mentioned above only takes 10 minutes. Anucleate neutrophils are still chemotactic, and this is manifested by hyperpolarization and poly pseudopodia crawling. They still have the function of degranulation and engulfing NETs released by bacteria. It is worth mentioning that according to the research, process of rapid release of NETs did not require the participation of NOX.127–129

On the other hand, mechanistic target of rapamycin (mTOR) signaling pathway is not solely included in the NOX-independent and NOX-dependent pathway, as it can potentially play a role in both pathways. mTOR is a serine/threonine protein kinase that regulates cellular growth, proliferation, and survival.130,131 mTOR signaling plays an important role in the regulation of autophagy, which is a conserved catabolic process that degrades cellular components and organelles, leading to NETs formation.132 Neutrophils are highly metabolically active cells that require significant amounts of energy to perform their functions.133

It has been reported that inhibiting the mTOR signaling pathway with rapamycin enhanced both autophagy and NETs formation, while activating the mTOR signaling pathway with insulin suppressed both processes in neutrophils isolated from health donors. And blocking autophagy with 3-MA or chloroquine showed a decrease in spontaneous NETs formation.134 It is suggested that spontaneous NETs formation is negatively regulated by the mTOR signaling pathway. However, a different trend in the relationship among mTOR, autophagy and NETs formation was found. Two β-lactam antibiotics, meropenem and ceftazidime/tazobactam, induced the activation of mTOR signaling pathway and inhibited autophagy in neutrophils and HL-60 cells. Moreover, they showed that blocking the mTOR signaling pathway with rapamycin or inhibiting β-lactamase with clavulanic acid attenuated the NETs formation induced by the antibiotics.135 Another study displayed that inhibiting mTOR pathway with rapamycin or Torin 1 enhanced autophagy and reduced NETs formation in PMA-stimulated neutrophils, while activating the mTOR pathway with insulin-suppressed autophagy and increased NETs formation in LPS-stimulated neutrophils. Also, blocking autophagy with 3-MA or bafilomycin A1 increased NETs formation in PMA-stimulated neutrophils, while enhancing autophagy with trehalose decreased NETs formation in LPS-stimulated neutrophils.136 Based on the aforementioned evidence, mTOR signaling may regulate NET formation by modulating autophagy, although the underlying mechanism appears to depend on the specific stimuli. Nonetheless, the exact mechanisms and implications of this regulation remain elusive and warrant further investigation.

In summary, various mechanisms do exist to achieve the purpose of NETs formation through specifical stimuli. It is supposed to be note that some pathways mentioned above possess the interaction with each other, rather than being independent, suggesting an objective and noticeable view that the classification used to distinguish either NOX-dependent pathway or NOX-independent pathway is relative.

Regulatory Role of NETs in GA Inflammation

GA is a common disease associated with inflammation. The main cause of GA can be attributable to a disorder of purine metabolism leading to excessive production or insufficient excretion of UA. GA attack usually occurs late at night accompanied by joint pain, swelling, redness and fever. Symptoms can cause discomfort or tingling in the joints and can get worse within 24 hours.5,137 In most cases, the acute manifestations of inflammation tend to subside spontaneously within a few days to weeks, without any notable residual effects. This observation suggests the existence of efficacious mechanisms that effectively mitigate acute inflammation.138

According to the pathogenesis of gout, there are 4 stages of gout, including asymptomatic hyperuricemia, acute gouty arthritis, intercritical gout and chronic tophaceous gout.139 GA is typically associated with the acute stage of gout. Nonetheless, there is a significant information that not all patients with gout will experience GA, and GA attacks will not only occur in the acute gouty arthritis stage but also in the chronic tophaceous gout stage.137,140 As reported that GA was the most commonly associated with the acute stage of gout and was the most common presenting symptom of gout causing inflammatory symptoms.141,142 Also, the chronic tophaceous gout stage is the most advanced stage of the disease and is characterized by the presence of tophi, which are deposits of urate crystals in the soft tissues with the less severe inflammation compared to the early stage.139 It is implied that there must be some physiological and pathological activity that acts as a regulator of inflammation.

Among different targets, NET is a crucial role in regulating inflammation.92,143 As is mentioned above, neutrophils can be activated through various mechanisms and pathways leading to the releasing of NETs. During the process of GA, stimuli like MSU crystals are beneficial for the recruitment of neutrophils and the generation of NETs, which is accompanied by the release of DAMPs such as histones, activate the immune system to release pro-inflammatory cytokines around joints resulting in an inflammatory environment.74,144,145 Herein, it is necessary to figure out the regulatory role of NETs in GA inflammation.

Inflammatory Initiation

GA is caused by the deposition of monosodium urate (MSU) crystals in joints, which triggers a strong inflammatory response. The MSU crystals are recognized by the innate immune system, which activates resident macrophages and recruits neutrophils to the joint resulting in the accompanying inflammation.146 When the crystals accumulate in the joint, they trigger an inflammatory response by activating the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, a large protein complex that regulates the immune response. The activation of the NLRP3 inflammasome leads to the secretion of pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and IL-18, which causes the characteristic pain, redness, and swelling associated with gout. The activation of the NLRP3 inflammasome and subsequent release of IL-1β and IL-18 are triggered by innate immune cells like macrophages and dendritic cells, which recognize danger signals and respond by initiating the inflammatory response.147,148 During the process of GA, it is widely regarded that MSU stimulate macrophages to produce IL-1β and IL-18 via the NLRP3 inflammasome, which in turn leads to the recruitment of neutrophils and subsequent release of NETs. Meanwhile, neutrophils then undergo a process of activation and degranulation, leading to the release of their intracellular contents, including ROS, citrullinated histones, and granule enzymes, which together promote the formation of NETs.149 Also, neutrophils can release NLRP3 inflammasomes through a process called phagocytosis, which is the ingestion of foreign particles such as urate crystals.95,150 The urate crystals are recognized by the neutrophils and internalized through the formation of phagosomes. The phagosomes then fuse with lysosomes to form phagolysosomes, which contain enzymes and ROS that can degrade the urate crystals.151–154 This process results in the release of DAMPs, such as mitochondrial DNA and ATP, which can activate the NLRP3 inflammasome and bring about the generation of IL-1β and IL-18, ultimately promoting NET formation.18,155,156

NETs are deemed to be a vital role in initiating GA inflammation. Excessive formation of NETs is a sign of increased GA flare and a critical factor that contributes to GA pathology.157 NETs can cause tissue damage and trigger inflammation in GA in multiple ways. First, the histones and granular proteins from NETs are capable of activating the complement system and promoting the recruitment of other immune cells, such as monocytes and macrophages, to the site of inflammation.34,158,159 The recruited immune cells further propagate the inflammation by secreting pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6.15,137,160 What’s more, NETs can directly foster inflammation by various pro-inflammatory molecules such as histones, DNA, and granule proteins that can activate the NLRP3 inflammasome.84,161 (Figure 3) For example, histones from NETs directly activate the NLRP3 inflammasome by disrupting lysosomal membranes and releasing cathepsins into the cytosol, which in turn activates the NLRP3 inflammasome that assembles with ASC and pro-caspase-1 followed by the step to cleave the pro-caspase-1 into caspase-1 and finally the release of IL-1β (Figure 3).33 Last but not least, NETs can cause mitochondrial damage, resulting in the release of mtDNA, which can activate the NLRP3 inflammasome in the diabetic wound.162,163 In the recent years, NETs have been treated as a potential target for the treatment of gout. Compelling evidence suggests that loganin exerts its inhibitory effects on NLRP3 inflammasomes by repressing mitochondrial stress in macrophages.164 However, similar finding has not been seen in studies of MSU crystals induced NETosis, leading to a valuable direction that needed explored. An interaction between macrophages and neutrophils has been used as a method for the treatment of gout. Ji Hye Jeong and his colleges proposed that synovial fluid macrophages were capable of clearing NETs by means of enhancing engulfment of MSU crystals without inducing any immunological response.15 Taken together, it is believed that NETs function as an intrinsic alarming that mainly initiates the inflammasome activation and elicits the inflammatory response in GA.

Figure 3 Regulatory role of neutrophil extracellular traps in gouty arthritis. MSU crystal deposition is formed at the joints due to high serum uric acid concentration, leading to the appearance of inflammatory response of neutrophils. During the early period of gouty arthritis, the NLRP3 inflammasome, which is activated by MSU crystals directly or indirectly, is responsible for turning pro-caspase-1 into the mature form of caspase-1 after the assembly of NLRP3. Pro-IL-1β and pro-IL-18 are transformed into IL-1β and IL-18 with the help of caspase-1, exhibiting pro-inflammatory effects and promoting the generation of NETs, which are accompanied by adherence to neutrophil elastases, myeloperoxidases and citrullinated histone 3. Additionally, macrophages and monocytes recruited by MSU crystals contribute to the generation of IL-1β and IL-18. With the recruitment of neutrophils, increasing numbers of neutrophils cause the aggregation of NETs, as referred to aggNETs. aggNETs manifest anti-inflammatory function by neutrophils serine proteases including NE, proteinase 3, cathepsin G and neutrophil serine proteases. At the same time, anti-inflammation is associated with the process of aggNETs engulfing and degrading MSU crystals.

Inflammatory Resolution

In the chronic stage of gout, the symptoms that occurring at the beginning of gout are relieved and the inflammation around the joints is reduced.165 Inflammation during GA can expand without limitation, leading to joint necrosis and even death if there is no regulatory mechanism.166,167 There is no doubt that multiple mechanisms are utilized to prevent endless inflammation from happening. Evidence has showed that several key mechanisms are involved in inflammatory regression during gouty arthritis.157,168–170 Among them, NETs serve as a vital role in inflammation resolution in the form of aggregation, referred to as aggNETs. Here, we describe an inflammatory resolution that is regulated by NET.

The process of aggNETs formation begins when neutrophils are exposed to uric acid crystals. The crystals stimulate neutrophils to release NETs, which then aggregate and entrap the crystals within the web-like structure of the NETs.149 The aggregation of NETs and uric acid crystals leads to the formation of aggNETs that is capable of entrapping and degrading MSU crystals.149,171 In the research from Christine et al, neutrophils recruited to the site of inflammation and underwent oxidative burst caused by MSU crystals, which led to the formation of NETs.171 As neutrophils recruited to the site of inflammation, aggregation of NETs occurred under a high density of neutrophils and then resulted in the degradation of cytokines and chemokines. In another study, aggNETs displayed it anti-inflammation via the protection from antiproteases in vivo and in vitro.172 Actually, the process of manifesting anti-inflammatory function by aggNETs needs the involvement of neutrophil serine proteases (NSPs). As a kind of neutrophils granule, NSPs contain NE, proteinase 3 (PR3) and cathepsin G. In the context of gouty arthritis, NSPs are believed to contribute to the resolution of inflammation. It has been shown that neutrophils release elastase and PR3 together with aggNETs, which can then engulf and degrade MSU crystals, thus preventing further activation of the NLRP3 inflammasome and subsequent release of pro-inflammatory cytokines like IL-1β and IL-18 (Figure 3).173–175 Moreover, NSPs have been found to directly degrade and inactivate a range of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and chemokines such as IL-8.171 Other than that, a report from Jasmin et al showed that the cytotoxic effect of histones on epithelial cells could be weakened by aggNETs’s function of sequestering and degrading histones, and the process of which required the participation of serine proteases, NE and PR3.176 The research suggested that the resolution of inflammation induced by histones resulted from degradation and detoxification of aggNETs. Collectively, aggNETs with the effector NSPs can exert anti-inflammatory effects through various mechanisms, including the degradation and inactivation of pro-inflammatory cytokines and chemokines. In recent times, the concept of NSP has received limited attention in the context of gout treatment. Researchers have instead directed their focus towards investigating distinct components of NSP for in-depth study and exploration. This shift in research emphasis signifies a renewed interest in understanding the intricate roles played by individual NSP constituents and their potential implications for therapeutic interventions in the management of gout and related inflammatory disorders.

Subsequently, the focus of attention falls on the formation of aggNETs, as its formation is currently viewed differently. It is believed that the release and degradation of inflammatory mediators is a dynamic balance.177 NSPs bonding to aggNETs cause the resolution of inflammation, which implies that the ability to degrade inflammatory mediators is greater than the ability to release them. According to the research by Jonas et al,172 peak generation and maximum supernatant concentrations of cytokines and chemokines appeared at a neutrophil density of 20–40×106/mL during the stimulation of MSU crystals. The study also found that the contents of inflammatory cytokines and chemokines began decreasing at higher densities of neutrophils with the help of NSPs. Moreover, maximum release of inflammatory mediators and chemokines occurred at a neutrophil density of 20–40×107/cm3 in arthritis mice model induced by MSU crystals, whereas the contents of the mediator reduced at higher density of neutrophils.172 The evidence mentioned above suggests the neutrophil induced inflammation is a self-limiting condition containing a dynamic process of releasing and degrading inflammatory mediator and chemokines, and more importantly, the formation of aggNETs seems to depend on the density of neutrophils. While in another study, the authors held the opinion that aggNETs formation depended on ROS in vivo and in vitro. In this study, it turned out that neutrophils had a weaker ability to degrade inflammatory mediators and generate aggNETs when compared to normal.171 In line with this, ROS-deficient Ncf1**mice which was characterized by the inability to produce ROS and represent mice model of CGD, showed a reduction in NETs aggregation and an increase in inflammatory mediators during the stimulation by MSU crystals when compared to air pouches of wild-type mice. It is assumed that the number of neutrophils only determines the size of the aggregates and then the aggNETs formation starts at 50 μg/mL of MSU crystals in a dose-dependent manner. Moreover, the level of neutrophils does not appear to be a critical factor, as NETs aggregation can still be induced by other stimuli such as ATP or lactoferrin, even at low neutrophil concentrations (5×106/mL).171 There exists uncertainty regarding the underlying mechanism for the formation of aggNETs that requires clarification.

NETs as Therapeutic Target for GA Treatment

The evidence we mentioned above strongly illustrates the close connection between GA and NETs. With the increasing research on GA in recent years, well-established anti-GA drugs and decoction have been widely used in the gout population.178–181 In addition to this, raising number of pharmaceutical agents are presently undergoing development to counteract GA in accordance with its pathogenesis. NETs have emerged as a pivotal target in the context of GA, and their inhibition has garnered considerable interest as an attractive therapeutic strategy for managing this condition. Consequently, an increasing array of drugs aimed at modulating NETs formation are currently under exploration and development, signifying the potential for novel and promising therapeutic avenues in GA treatment.

Clinical Approaches for GA Prevalent Drugs

According to the American College of Rheumatology Guideline for the Management of Gout182 published in 2020, first-line drugs commonly used in clinical practice for GA attack involve colchicine, non-steroidal anti-inflammatory drugs (NSAID) and glucocorticoid. Colchicine has been a medicine widely used in GA for a long history.183,184 The incidence of adverse reactions is high since the therapeutic dose of colchicine is close to the toxic dose.185 For patients with acute gout attacks, it is recommended to accept colchicine treatment in the first dose of 1 mg, 0.5 mg after 1 h, and 0.5 mg after 12 h, with a frequency of once a day or twice a day.186 Small doses of colchicine combined with NSAID also are strategies recommended to cure acute GA attacks.187,188 Glucocorticoids are appropriate for the patients suffering GA attacks while having trouble with drug administration orally.182 In addition, lowering uric acid drugs, including allopurinol, benzbromarone and febuxostat are also vital treatments for gout. However, both advantages and disadvantages exist in drug administration. Studies have shown that side effects, including liver functional disorder and leukopenia, occur after taking allopurinol for a long time.189 Seriously, allop