NET Formation Drives Tophaceous Gout

Abstract

Gout is a chronic inflammatory disease characterized by the deposition of monosodium urate (MSU) crystals within joints, leading to recurrent acute flares and long-term tissue damage. While various hypotheses have been proposed to explain the self-limiting nature of acute gout attacks, we posit that aggregated neutrophil extracellular traps (aggNETs) play a central role in this process. This review focuses on the mechanisms underlying MSU crystal-induced formation of neutrophil extracellular traps (NETs) and explores their dual role in the clinical progression of gout. During the initial phase of acute flares, massive NET formation is accompanied by the release of preformed inflammatory mediators, which is a condition that amplifies inflammatory cascades. As neutrophil recruitment reaches a critical threshold, the NETs tend to form high-order aggregates (aggNETs). The latter encapsulate MSU crystals and further pro-inflammatory mediators within their three-dimensional scaffold. High concentrations of neutrophil serine proteases (NSPs) within the aggNETs facilitate the degradation of soluble inflammatory mediators and eventually promote the resolution of inflammation in a kind of negative inflammatory feedback loop. In advanced stages of gout, MSU crystal deposits are often visible via dual-energy computed tomography (DECT), and the formation of palpable tophi is frequently observed. Based on the mechanisms of resolution of inflammation and the clinical course of the disease, building on the traditional static model of “central crystal–peripheral fibrous encapsulation,” we have expanded the NETs component and refined the overall concept, proposing a more dynamic, multilayered, multicentric, and heterogeneous model of tophus maturation. Notably, in patients with late-stage gout, tophi exist in a stable state, referred to as “silent” tophi. However, during clinical tophus removal, the disruption of the structural or functional stability of “silent” tophi often leads to the explosive reactivation of inflammation. Considering these findings, we propose that future therapeutic strategies should focus on the precise modulation of NET dynamics, aiming to maintain immune equilibrium and prevent the recurrence of gout flares.

1. Introduction

Gout is an inflammatory disease caused by abnormal purine metabolism leading to deposition of monosodium urate (MSU) crystals, which manifests as acute arthritis, tophus formation and chronic joint damage [1]. Besides neutrophils, macrophages have been in the focus in gout research. In MSU crystal-induced inflammation, Besides in phagocytosis (including crystal phagocytosis), degranulation and superoxide generation neutrophils exert their effects through the formation of neutrophil extracellular traps (NETs) and aggregated neutrophil extracellular traps (aggNETs), particularly in the context of spontaneous resolution of gout and the development of tophi.

Pathogens and various stimuli, including MSU crystals, can activate neutrophils, leading to the formation of NETs. Generally, NETs are composed of a DNA network scaffold, histones, and various proteases [2]. Their primary roles include promoting inflammatory responses, mediating pathogen clearance, and confining stimuli. However, during gout flares, NETs demonstrate a dual-edged role. They can amplify the inflammatory response in the early stages, while also playing a critical role in facilitating the spontaneous resolution of inflammation and maintaining the silent state of tophi [3]. In this review, we will discuss the mechanisms underlying MSU crystal-induced NETs formation and highlight recent advances in understanding the role of NETs in both acute and chronic gout. By highlighting the importance of NETs in gout pathology, we aim to investigate whether disrupting NET formation or accelerating NET degradation represents therapeutic options for the development of new treatment strategies.

2. Formation of MSU-NETs

Since the discovery of NETs in 2004 [2], NETosis has been adopted to describe NET formation accompanied by neutrophil death [4]. It has long been known that neutrophils can survive enucleation (Roos, JCB, 1983) [5] and still active within inflamed tissues. Moreover, Pilsczeck and collegues (2010) later also demonstrated that NETs can be released from viable neutrophils in response to Staphylococcus aureus, independent of ROS generation [6]. This finding revealed a limitation in the definition of NETosis, prompting the adoption of a refined classification that distinguishes lytic from non-lytic NET release based on plasma membrane integrity [7,8]. Lytic NET release involves chromatin decondensation, nuclear envelope breakdown, and membrane permeabilization, leading to neutrophil death [7]. In contrast, non-lytic NET release involves vesicular packaging of chromatin, allowing neutrophils to remain viable and immunologically active [7,9].

Distinct forms of NET formation can be elicited by different types of stimuli, each engaging specific intracellular pathways [10]. Among them, MSU crystals are widely recognized to induce lytic NET release. Not all neutrophils, however, are equally responsive-evidence suggests that only certain subsets possess the capacity to form NETs under stimulation with MSU [11]. After contact with MSU crystals, neutrophils activate signaling cascades that rapidly elevate cytosolic calcium levels. This increase, in turn, facilitates the activation of NADPH oxidases (NOX), which catalyze the conversion of molecular oxygen into reactive oxygen species (ROS) [9,12]. Acting as central signaling hub, calcium and ROS subsequently trigger cascades of downstream events that culminate in NET release: (1) activation of PAD4 catalyzes the citrullination of histone arginine residues, thereby promoting chromatin decondensation and rendering the DNA scaffold more accessible for extrusion [9]. (2) neutrophil elastase (NE) and myeloperoxidase (MPO) are activated, together with antimicrobial effectors such as defensin-1, into a multi-component complex known as the azurophilic granules [13,14]. During the early phase of NET formation, azurophilic granules undergo membrane fusion and release their contents into the cytoplasm. Subsequently, NE is translocated into the nucleus along the microtubule network, where it facilitates chromatin fiber unraveling [15]. (3) activation of calpain and consequent cleavage of nesprins promote the breakdown of the nuclear envelope [16]. (4) upregulation of the palmitoyltransferases ZDHHC5, ZDHHC7, and ZDHHC9 causes the palmitoylation of gasdermin D (GSDMD). This lipid modification facilitates the translocation of the N-terminal domain of GSDMD (GSDMD-NT) to the plasma membrane, where it binds phosphatidylinositol phosphates and assembles into stable β-barrel pores [17,18]. The resulting membrane perforation contributes to cellular lysis and promotes the extracellular release of NETs [7,19,20]. (5) activation of the RIPK1–RIPK3–MLKL necroptosis pathway contributes to NET formation. Pharmacological inhibition of this axis with necrostatin-1 (Nec-1) or necrosulfonamide (NSA), markedly suppresses NET release [21,22].

Nevertheless, emerging evidence suggests that MSU crystals may also initiate NET formation through alternative routes that bypass ROS-dependent signaling [23,24]. Following MSU crystals stimulation, one arm of this pathway involves the nuclear translocation of autophagy-related protein 7 (ATG7) in concert with p53. Together, these molecules upregulate the expression of PAD4, thereby enhancing histone modification and facilitating chromatin decondensation-a prerequisite for NET release [25]. In parallel, lysosomal membrane permeabilization (LMP) has been proposed as a trigger for noncanonical NET formation, independent of classical ROS-mediated pathways. Earlier work showed that phagocytosed MSU crystals can disrupt phagosomes through interaction with granule membrane cholesterol, and that this disruption is essential for crystal-induced neutrophil death (Rich et al., Inflammation 1985) [26]. The present study extends these findings by directly linking LMP to noncanonical NET formation.

In vigorous NET formation many different pathways and amplification loops work simultaneously. These include ROS production, calcium influx, and mediator release. The role of mitochondrial DNA is still elusive.

3. NETs Drive a Self-Amplifying Inflammatory Loop During Acute Gout Attacks

Elevated serum urate levels, driven by increased purine intake, enhanced xanthine oxidase activity [27] or impaired urate excretion [28], surpass the solubility threshold, leading to the formation of MSU crystals that accumulate in joint-associated structures. As potent pro-inflammatory signals, MSU crystals trigger the release of a cascade of inflammatory cytokines and chemokines, including IL-1β, IL-6 and TNF [29], within the deposition microenvironment. This initiates a robust inflammatory response.

Concurrently, the formation of NETs during inflammation is orchestrated by a multitude of synergistic factors, giving rise to a striking self-amplifying loop. Specifically: (1) MSU crystals directly stimulate neutrophils to release NETs enriched with damage-associated molecular patterns (DAMPs), including histones and high-mobility group box 1 (HMGB1) [30,31,32]. (2) Within the joint cavity, the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and IL-1β enhance NET formation through activation of the NADPH oxidase-dependent pathway [14,33,34]. (3) Following phagocytosis of NETs by macrophages, their DNA components are specifically sensed by cyclic GMP-AMP synthase (cGAS) and triggers a type I interferon response. The latter further promotes NET formation via a positive feedback loop [35]. (4) MSU-induced activation of macrophages leads to the secretion of platelet-activating factor (PAF), which in concert with activated platelets, robustly augments NET release [36]. The multi-tiered signaling events converge to drive an explosive generation of NETs. Meanwhile, the core components of NETs DNA, histones, and HMGB1 play a central role in the initiation and amplification of inflammation [37]. These DAMPs drive inflammation through multiple mechanisms. On one hand, they activate Toll-like receptors (TLR), triggering MyD88-dependent signaling pathways, which in turn induce the production of key pro-inflammatory cytokine [38]. On the other hand, they activate the NLRP3 inflammasome [39,40,41], promoting the polarization of macrophages toward the pro-inflammatory M1 phenotype [24,42]. This shift initiates a cascade of cytokine release, including TNF-α and IL-1β [43], which synergizes with TLR signaling outputs, ultimately establishing a self-sustaining positive feedback loop of inflammation. DAMPs embedded within tophi including urate can also stimulate pattern recognition receptors, promoting the secretion of CXCL1 and CXCL2 [44]. These chemokines act via G-protein-coupled receptors (GPCR), and perpetuate the recruitment of neutrophils to the inflamed tissue [45]. MSU crystals, neutrophil extracellular trap (NET)-derived components such as myeloperoxidase (MPO) and neutrophil elastase (NE), as well as Granzyme K—primarily secreted by CD8⁺ T cells—can activate the complement cascade [46,47]. In turn, the key complement fragment C5a reciprocally enhances NET formation, fueling a vicious cycle of “complement–NET–inflammation” [48].

This pathological feedback loop not only perpetuates neutrophil activation and complement amplification but also escalates inflammatory responses and drives tissue injury. Notably, complement activation itself is known to have pro-fibrotic consequences, especially in chronic inflammatory contexts [49]. Collectively, these interactions define an amplified “inflammation–NET–inflammation” axis, in which the complement system plays a pivotal and amplifying role.

This self-reinforcing circuit culminates in a cytokine storm and extensive NET formation, both of which contribute to profound joint damage. Clinically, it manifests as the hallmark features of acute gout: intense swelling, erythema, and excruciating pain. At its core, this pathological process reflects a dynamically sustained inflammatory network orchestrated by NLRP3 inflammasome activation, NET release, complement cascade, and coordinated immune cell modulation.

Given the synergistic role of NETs and complement in early inflammation, a combined strategy using C5 inhibitors (e.g., eculizumab, ravulizumab) and DNase I has been proposed to concurrently suppress complement activation and degrade NETs. This approach shows therapeutic potential in SLE and APS [50]. Notably, while MSU crystals form the structural and immunological foundation of canonical tophi in gout, this phenomenon is not universally observed across all crystal-induced arthropathies. The crystal-bound proteome includes IgG, C5, and LDL [46] and proteins of the complement cascade are found in tophi [51]. CPPD (Calcium pyrophosphate deposition disease), while capable of inducing pseudogout, does not form canonical tophi [52], and no studies to date have demonstrated that it binds complement similarly to MSU crystals.

4. Formation of aggNETs: A Key Mechanism for Spontaneous Resolution of Inflammation During Acute Gout Flares

Acute gout flares exhibit a self-limiting clinical course and often resolve spontaneously within days to weeks. The underlying molecular mechanisms orchestrating this resolution remain incompletely defined. Current mechanistic studies suggest that this process involves multiple levels of regulation: (1) The anti-inflammatory cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) are upregulated during the resolution phase and suppress pro-inflammatory amplification cascades [53]. (2) Macrophages undergo phenotypic polarization toward an anti-inflammatory M2 state, a shift accompanied by enhanced efferocytosis-the efficient clearance of dying cells and cellular debris [54]. (3) Neutrophil-derived ectosomes (PMN-ectosomes), a class of microvesicles, have been shown to impede NLRP3 inflammasome activation by blocking C5a receptor signaling [55]. (4) Apoptotic neutrophils, rather than contributing to further tissue damage, are actively removed through efferocytosis. This promotes resolution of inflammation and restores tissue homeostasis [56]. However, mice depleted of neutrophils, as well as those carrying mutations in Ncf1 (a key gene required for ROS-dependent NET formation) exhibited markedly prolonged and even chronic inflammatory responses [3,57]. This breakthrough redirected attention toward neutrophils and, more specifically, their capacity to form aggregated neutrophil extracellular traps (aggNETs) as central orchestrators of spontaneous resolution of inflammation. During the acute phase of inflammation, MSU crystals provoke the rapid formation of NETs, characterized by decondensed chromatin DNA scaffolds measuring 15–17 nm in diameter and densely decorated with histones H1 through H4 [58]. These NET structures perpetuate neutrophil activation via a feed-forward loop, continuously recruiting and priming incoming neutrophils. When cell densities exceed a critical threshold (≥5 × 10⁶/mL), this dynamic culminates in the formation of aggNETs [3], with MSU crystals at the core and successive layers of NETs concentrically surrounding them. Ultrastructural analyses via electron microscopy reveal that in vitro formed NET-associated neutrophil aggregates display a distinct fibrillar architecture. NET strands connect activated neutrophils; strikingly different from the uniform monolayer of unstimulated cells [57]. AggNETs orchestrate the inflammatory response through a unique spatial structure-function coupling mechanism. The chromatin-based meshwork physically entraps damage-associated molecular patterns (DAMPs), such as extracellular histones, thereby limiting their diffusion. Simultaneously, the porous nature of the matrix permits the entry of the small soluble inflammatory mediators, which are funneled into the proteolytically active interior compartments enriched with active NE and proteinase 3 (PR3). The neutrophil serine proteases (NSPs) proteolytically degrade most of the inflammatory mediators [3,58,59]. Moreover, the complex three-dimensional topology of aggNETs exerts a steric shielding effect that partially impedes access of endogenous macromolecular protease inhibitors such as the serpin α1-antitrypsin (AAT) to the intraluminal NSPs, thereby preserving enzymatic activity within the core microenvironment [60]. At the heart of this dynamic equilibrium lies an intrinsic “self-braking” mechanism. In the early phase of inflammation, MSU crystals act in concert with nascent NETs to amplify pro-inflammatory signaling cascades. At this stage, the enzymatic activity of NSPs remains effectively restrained by rapid neutralization through endogenous protease inhibitors. As inflammation progresses and neutrophil accumulation gives rise to the formation of aggNETs, a fundamental shift occurs. These NET-based aggregates encapsulate MSU crystals along with a multitude of inflammatory mediators, intercept with the pro-inflammatory loop, and dampen the pro-inflammatory signals. Notably, the NET-MSU complexes actively modulate the phenotype of newly recruited neutrophils, favoring their polarization toward the anti-inflammatory phenotype. This reprogramming enhances the secretion of interleukin-1 receptor antagonist (IL-1RA) and simultaneously suppress the release of canonical pro-inflammatory cytokines [61].

Meanwhile, the high local concentration of NSPs within the aggNET microenvironment facilitates the breach of protease inhibitor barriers (such as AAT). Once this inhibitory threshold is overcome, NSPs preferentially degrade key cytokines such as IL-1β and IL-6. This proteolytic predominance progressively outpaces the rate of pro-inflammatory mediator production and release, tipping the balance toward spontaneous resolution of inflammation [59,60].

Lei Liu and colleagues provided direct experimental support for this mechanism [62]. In a murine model of spontaneously resolving gout, exogenous administration of AAT during the resolution phase led to a counterintuitive local rebound of the pro-inflammatory cytokines IL-1β and IL-6 and caused a relapse of the inflammation. This striking observation highlights two key insights: (I) it confirms that the proteolytic degradation of inflammatory mediators by NSPs embedded within aggNETs constitutes a central driver of spontaneous resolution of inflammation. (II) it reveals an important limitation of the spatial resistance conferred by the three-dimensional topology of the aggNETs.

Although the chromatin meshwork of aggNETs sterically hinders access of endogenous inhibitors such as AAT to the active sites of NSPs, this barrier is not absolute. Under certain pathological conditions, such as during urate-lowering therapy, which has been shown to markedly elevate systemic AAT levels [63], the inhibitory threshold may be surpassed. Once breached, AAT penetrates the aggNET architecture, neutralizes NSPs activity, and disrupts the finely tuned protease-antiprotease balance that governs inflammatory resolution. The consequence is a functional collapse of the enzymatic containment system, culminating in renewed cytokine accumulation and reversal of inflammation regression.

5. Formation and Maturation of Tophi

In clinical practice, dual-energy computed tomography (DECT) has emerged as a highly sensitive imaging modality for detecting intra-articular MSU crystals deposits in patients with gout [64]. Remarkably, MSU crystals persist within the joint cavity throughout the disease course, from acute flares to clinically quiescent stages of chronic gout [65]. This persistence, however, does not reflect a diminished inflammatory potential of the crystals themselves. Rather, it underscores a critical immunological observation: during these phases, the pro-inflammatory capacity of MSU crystals appears to be tightly regulated. Both the self-limiting nature of acute attacks and the clinical stability observed in late-stage gout suggest that intra-articular tophi exist in a state of dynamic equilibrium, in which specific regulatory mechanisms actively constrain MSU-induced inflammation and prevent its uncontrolled propagation.

The structural organization of gouty tophi has been described as tripartite: a central core mainly composed of MSU crystals; an intermediate layer enriched with macrophages (CD68⁺) and plasma cells; and an outer rim consisting of fibrous vascular tissue interspersed with small numbers of B and T lymphocytes [66]. The fibrotic capsule, in particular, has been proposed to serve as a physical barrier that confines the inflammatory process and limits its peripheral spread [67]. Another prevailing hypothesis suggests that neutrophils within the tophus undergo apoptosis or necrosis, followed by macrophage-mediated clearance. In this process, transglutaminase 2 (TG2) expressed by M2 macrophages at the efferocytosis synapse enhances the clearance of apoptotic cells and MSU crystals, promotes the release of TGF-β1, and thereby sustains the anti-inflammatory phenotype while driving the self-limitation of MSU crystal-induced inflammation [68]. Tophi isolated from gout patients displays hallmark features of NETs [3,59] including active NE. Building on this evidence, we propose that aggNETs represent a structural and functional core component of tophi. These NET-based aggregates encapsulate MSU crystals, thereby contributing fundamentally to the structural integrity and immunological stability of the tophi.

Tophus formation originates from the deposition of MSU crystals and proceeds through a three-stage process. Initially, urate ions undergo heterogeneous nucleation on exposed collagen fibers, giving rise to an amorphous precursor phase—amorphous MSU (AMSU). This intermediate subsequently undergoes gradual structural reorganization along the collagen scaffold, forming needle-shaped MSU crystals. Under sustained hyperuricemia, the crystals accumulate and thicken, eventually consolidating into clinically detectable tophi [29].

In healthy joints, collagen fibrils are enveloped by a protective layer of proteoglycans and lubricin (proteoglycan 4, PRG4), which together form a barrier that prevents urate adhesion and crystal nucleation. PRG4, in particular, has been shown to inhibit MSU crystal deposition in vitro [69], thereby acting as a natural suppressor of pathological crystallization. However, cartilage subjected to mechanical wear, aging, or inflammation becomes structurally compromised. Under hyperuricemic stress, chondrocytes upregulate matrix metalloproteinase-3 (MMP-3), accelerating proteoglycan degradation [70]. Together, these factors lead to collagen exposure, providing a high-affinity template for urate nucleation. Also, Type II collagen (CII) modulates the morphology of monosodium urate (MSU) crystals and enhances their uptake by macrophages, which may contribute to the onset of gout and the progression to erosive joint damage. Additionally, tissue factors released from damaged cartilage further promote MSU crystallization [71].

Concurrently, the physicochemical environment of the joint cavity facilitates crystal deposition. Subphysiological temperatures favor urate precipitation [72]; impaired microcirculation reduces local urate clearance; and continuous mechanical loading promotes crystal retention on articular surfaces. These factors converge to establish a permissive niche for MSU nucleation, growth, and anchorage—ultimately driving tophus development [71,73,74].

The absence of uricase in humans, a key evolutionary loss [75,76], eliminates an enzymatic mechanism that lowers soluble urate levels and thereby shifts the dissolution equilibrium of MSU crystals, and impairs the timely clearance of MSU crystals from the body. Consequently, these crystals persist in tissues and chronically stimulate innate immune cells. Macrophages respond with sustained activation, while neutrophils, upon encountering MSU crystals, release NETs that ensnare the crystals. We hypothesize that this entrapment marks the initiating step of tophus formation. This process does not occur in isolation. Rather, it triggers a self-amplifying feedback loop of neutrophil recruitment and activation. Over time, the accumulation of neutrophils and the successive release of NETs give rise to densely packed aggNETs, which not only capture and immobilize MSU crystals but also actively contribute to the resolution of acute inflammation by proteolytic clearance of inflammatory mediators.

Of particular interest, DECT imaging has revealed that intra-articular MSU deposits are frequently observed in patients who are not only asymptomatic but also clinically stable and without palpable tophi [77,78]. This finding suggests a critical immunological state: MSU crystals, while present, may be functionally neutralized by their sequestration within aggNETs. We refer to these latent, NET-encapsulated deposits as “tophus precursors”. They represent a transitional form with potentially anti-inflammatory properties that maintain joint homeostasis and prevent exacerbation of the flare.

The maturation of gouty tophi is inherently cumulative. Recurrent flares, often triggered by high-purine diets or alcohol intake [79,80], not only reignite acute inflammation but also drive episodic deposition of newly formed MSU crystals. Each inflammatory peak is accompanied by the local formation of fresh aggNETs, which serve to entrap and compartmentalize the expanding crystal load. Building on the widely accepted concept of a tophus as a central crystalline core surrounded by a fibrotic capsule, we refine this model by proposing a dynamic multilayered structure hypothesis: an initial episode of inflammation establishes a foundational aggNET-MSU complex. Subsequent flares progressively add concentric layers of newly deposited crystals and aggNETs, each cycle reinforcing the inflammatory scaffold. These layers actively recruit various immune cell populations, including monocytes/macrophages, T lymphocytes, and fibroblasts, which eventually become embedded within the surrounding fibrous tissue. Such an assembly mechanism implies that clinically palpable tophi are not the product of a uniform, radially symmetric process. Rather, they emerge from spatially heterogeneous and multicentric growth patterns. Consistent with long-standing clinical observations, the tophus is not viewed as a static anatomical entity but rather as a dynamic structure influenced by episodic inflammation, layered crystal accrual, and ongoing immune cell integration.

6. “Stable” Tophi: A Pseudo-Stable “Time Bomb”

Advanced gout represents the terminal stage of disease progression, and a proportion of these patients eventually develop tophi. At this stage, patients have typically experienced multiple bouts of acute inflammation, after which the synovial microenvironment enters a phase of chronic reorganization. This transition culminates in the formation of structurally mature, immunologically contained tophaceous lesions referred to as stable tophi. They are composed of aggNETs, MSU crystals, various immune cell subsets, enclosed in a fibrous matrix.

Crucial to this structure is the accumulation of aggNETs generated across successive inflammatory episodes. These chromatin-based networks perform two major functions: (I) physically isolating pro-inflammatory mediators such as MSU crystals and histones within a dense three-dimensional scaffold and (II) simultaneously providing a proteolytic microenvironment rich in NSPs capable of degrading inflammatory mediators. In vitro, we observed that co-incubation of aggNETs with recombinant IL-1β and TNF-α, led to a time-dependent reduction in their concentrations, highlighting the degradative capacity of aggNETs [3].

Histones, as a core component of NETs, are potent DAMPs. Once released into the extracellular space, they can induce pyroptosis and activate Toll-like receptor (TLR) signaling cascades, thereby amplifying the secretion of pro-inflammatory cytokines and chemokines. This initiates a vicious cycle of tissue damage and propagation of inflammation [81]. However, pre-treatment with aggNETs markedly attenuates histone-induced cytotoxicity [58]. These findings suggest that stable tophi do not merely serve as passive containers for inflammatory debris but are functionally equipped to proteolytically neutralize such stimuli.

Taken together, this evidence supports the notion that aggNETs-rich stable tophi possess intrinsic anti-inflammatory potential. Their capacity to simultaneously sequester and degrade pro-inflammatory mediators provides a mechanistic basis for clinical quiescence, even in the ongoing presence of immunogenic stimuli such as MSU crystals, extracellular histones, and cytokines within the joint cavity.

Although stable tophi rarely precipitate sudden acute flares, their long-term persistence poses a significant cumulative burden on the host. At the local level, progressive MSU crystals deposition directly erodes osseous and periarticular structures, resulting in irreversible bone destruction and loss of joint function. At the same time, low-grade crystal-driven inflammation, maintained by persistent release of pro-inflammatory mediators such as IL-1β, contributes to progressive tissue fibrosis and may give rise to compression-related complications, including neurovascular entrapment syndromes [82,83,84]. Therefore, removal of tophi is essential for the treatment of gout. However, the clearance process is often accompanied by episodes of inflammatory flares.

Based on these observations and current mechanistic understanding, we propose the following hypothesis: Stable tophi, though clinically quiescent, may in fact represent a latent immunological “bomb”-encapsulating a dense reservoir of pro-inflammatory signals. This immunegenic burden is maintained through two principal mechanisms: (I) NETs act as both producers and carriers of inflammatory mediators. They entrap MSU crystals and enshroud danger-associated molecular patterns (DAMPs) within their chromatin meshwork. (II) NETs are enriched in filamentous actin (F-actin), which integrates into the NET scaffold to form a robust fibrous matrix. These F-actin networks confer resistance to enzymatic degradation, particularly by DNase I, thereby enhancing the structural persistence of NET-DNA complexes. The DNase resistance allows NETs to serve as long-term depots for pro-inflammatory mediators [85], including IL-1β, effectively transforming stable tophi into reservoirs of latent inflammation primed for reactivation. We hypothesize that any perturbation of the structural integrity or proteolytic balance within these NET-rich tophi, be it mechanical, enzymatic, or immunological, may act as a “switch,” unleashing the stored inflammatory content and precipitating acute flares. Thus, stable tophi are not merely inert remnants of past inflammation but immunologically reactive structures with the potential to reignite the pathology.

Considering the emerging evidence regarding the role of aggNETs throughout the course of gout, it becomes evident that the existing therapeutic paradigm, while foundational, may not fully address all immunological aspects of disease progression. On one hand, urate-lowering therapy, primarily through xanthine oxidase inhibitors, can effectively reduce serum urate concentrations to below the theoretical solubility threshold of 360 μmol/L [86,87]. However, this pharmacological intervention is frequently accompanied by a marked elevation in circulating AAT, which in turn suppresses NSPs activity. As a result, the functional stability of aggNETs is compromised, weakening their ability to sequester and neutralize inflammatory mediators and thus eroding their role as immunological buffers. On the other hand, surgical debridement, though efficiently and rapidly removing tophaceous deposits [88], introduces another form of disruption. The mechanical disassembly of aggNET architectures leads to the abrupt release of sequestered pro-inflammatory cytokines and cytotoxic components, triggering a secondary inflammatory burst able to exacerbate tissue damage.

These considerations do not negate the central role of urate-lowering therapy; rather, they highlight the potential benefit of concurrently addressing NET-associated dynamics as a complementary strategy within the existing therapeutic framework. They suggest that future interventions need not transcend the traditional focus on urate solubility but rather extend it to include efforts to preserve, or even recalibrate, the delicate equilibrium of NET homeostasis. To this end, the development of precision-targeted molecular tools capable of modulating NET dynamics and balancing inflammatory clearance with immunological containment may offer a more sophisticated and sustainable strategy that complements existing approaches inmanaging advanced gout.

7. Conclusions

The formation of NETs generally proceeds through two mechanistic routes: lytic NET release and non-lytic NET release. Notably, NETs induced by MSU crystals, referred to as MSU-NETs predominantly employ the former.

Canonically, this involves ROS-dependent activation of PAD4, However, recent studies have revealed alternative, ROS-independent mechanisms, whereby MSU crystals can elicit NET release through autophagy-related pathways or lysosomal membrane permeabilization [25,89].

Functionally, NETs play a dichotomous role in immune regulation. On one hand, their release is accompanied by a spectrum of DAMPs that activate the NLRP3 inflammasome and promote M1 polarization of macrophages and establish a feed-forward inflammatory loop, the “inflammation–NETs–inflammation” axis. On the other hand, when neutrophil density surpasses a critical threshold, NETs coalesce into aggNETs, initiating a self-regulatory phase. These three-dimensional chromatin networks physically sequester pro-inflammatory triggers such as cytokines, chemokines, histones and MSU crystals, while the high intranetwork concentrations of NSPs overcome endogenous protease inhibitors. This shifts the balance from the release to the degradation of inflammatory mediators. The net result is spontaneous resolution of inflammation.

Yet during repeated inflammatory flares, NET-MSU complexes accumulate like onion skins and ultimately lead to clinically palpable, stable tophi. The latter are spatially heterogeneous structures with multicentric architectural features. While they may sustain local immune quiescence through persistent proteolytic activity, they also harbor substantial pro-inflammatory cargo, including IL-1β and HMGB1. They are stabilized within a DNase-resistant, F-actin–reinforced scaffold. In this context, stable tophi function as “molecular time bombs”: any disruption of aggNET integrity, whether mechanical trauma from surgical debridement or AAT elevation during urate-lowering therapy, can trigger an explosive release of the sequestered cytokines, thus inducing secondary inflammatory flares.

This pathophysiological insight underscores a critical shift in therapeutic logic: rather than solely targeting MSU crystallization, future strategies must be designed to precisely modulate NET homeostasis. Such approaches could be key to maintaining immunological balance in gout and preventing recurrent flare-ups.