Molecular Pathophysiology of Gout

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We also discuss new findings, such as how aggregat- ing neutrophil extracellular traps (NETs) might drive the resolution of arthritis and how these structures, ...

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Review

Molecular Pathophysiology of Gout Jyaysi Desai,1,y Stefanie Steiger,1,y and Hans-Joachim Anders1,*,@ Three contradictory clinical presentations of gout have puzzled clinicians and basic scientists for some time: first, the crescendo of sterile inflammation in acute gouty arthritis; second, its spontaneous resolution, despite monosodium urate (MSU) crystal persistence in the synovium; and third, immune anergy to MSU crystal masses observed in tophaceous or visceral gout. Here, we provide an update on the molecular pathophysiology of these gout manifestations, namely, how MSU crystals can trigger the auto-amplification loop of necroinflammation underlying the crescendo of acute gouty arthritis. We also discuss new findings, such as how aggregating neutrophil extracellular traps (NETs) might drive the resolution of arthritis and how these structures, together with granuloma formation, might support immune anergy, but yet promote tissue damage and remodeling during tophaceous gout.

Trends The early phase of acute gouty arthritis is characterized by an auto-amplification loop of regulated necrosis and inflammation; MSU crystals trigger both cell necrosis and inflammation in a direct as well as indirect manner. The later phase of gouty arthritis is characterized by the resolution of symptoms despite persistence of MSU crystals. Numerous immunoregulatory pathways as well as aggregated neutrophil extracellular traps contribute to this process. Chronic gout is characterized by tophus formation; that is, masses of MSU crystals and NETs encapsulated by a foreign body reaction involving giant cells, macrophages, and fibroblasts that contribute to the destruction of surrounding tissues.

Towards an Increased Understanding of Necroinflammation in Gout Gout presents most commonly as acute episodic arthritis in patients, while the symptoms and signs of visceral and chronic gout (see Glossary) are less well defined [1]. The symptoms of an acute attack of gouty arthritis include articular and/or periarticular swelling and inflammation in joint with rapid onset (12–24 h), whereas chronic gout develops after some years of recurrent acute gout attacks, leading to pain and stiffness due to progressive tissue destruction [2]. Guidelines based on current knowledge of how to manage gouty arthritis are available, but any future developments in gout management will derive from a deeper understanding of the molecular pathophysiology of gout. Gout is triggered by MSU crystals. Crystal formation itself results from local uric acid supersaturation as a consequence of systemic uric acid overload, either from massive cellular release, its increased production during metabolism, or from impaired renal clearance [2]. The genetic, metabolic, and environmental causes of hyperuricemia and the physicochemical processes that lead to uric acid crystallization have been reviewed in detail elsewhere [1]. Here, we focus on the molecular and cellular pathomechanisms underlying the various clinical presentations of gout. We provide an update on how MSU crystals induce tissue injury and why the response of the immune system can range from acute sterile inflammation to immune anergy. This update extends beyond the recently established concept of NLR Family Pyrin Domain-Containing 3 (NLRP3)/interleukin (IL)-1b inflammasome-mediated innate immunity in gout, and incorporates evolving data on the interaction of crystal-induced cell necrosis with crystal-induced inflammation, referred to as ‘necroinflammation’, and of NETs in the molecular pathogenesis of gout. In addition, we discuss recent discoveries of how the immune system counterbalances MSU crystal-mediated necroinflammation via the negative regulation of inflammatory mediators, cell clearance, aggregated NETs, and granuloma formation.

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Dissecting the molecular and cellular pathogenesis of gout can help to identify innovative drug targets.

1

Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany y These authors contributed equally. @ Twitter: @Crystallopathy *Correspondence: [email protected] (H.-J. Anders).

http://dx.doi.org/10.1016/j.molmed.2017.06.005 © 2017 Elsevier Ltd. All rights reserved.

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Clinical Presentations of Gout

Glossary

The most frequent clinical presentation of gout in humans is the presence of recurrent episodes of acute arthritis involving one joint at a time [3]. By contrast, tophaceous and visceral gout are characterized by the formation of tophi, which are abscess-like creamy masses comprising MSU crystals and dead immune cells. The clinical features of intermittent acute gout and chronic gout are described below.

Aggregated neutrophil extracellular traps (aggNETs): aggregated masses of MSU crystals and NETs that can trap and degrade proinflammatory cytokines and chemokines, thereby contributing to the resolution of inflammation. Complement factors: several small proteins that enhance the phagocytic function and induce inflammatory responses in innate immunity. Crystals: a crystal is a solid material whose constituents (such as atoms, molecules, or ions) are arranged in a specific microscopic structure. MSU crystals are an example of crystals formed inside the body during gout. Cystine: an oxidized dimer of the amino acid cysteine that is slightly soluble in water. Danger-associated molecular patterns (DAMPs): proteins released by necrotic cells (i.e., HMGB1 or histones) that can act as danger signals to promote inflammatory responses when released in the extracellular space. Ectosomes: small vesicles (0.1– 1 mm in diameter) that are released from the plasma membrane into the extracellular space. They comprise proteins, mRNA, and miRNA of the cell of origin. Components and nature of ectosomes vary for different stimuli, and for different states of the cell (resting versus activated). Gouty arthritis: form of arthritis associated with acute onset of joint pain and swelling due to the deposition of MSU crystals. Granuloma: a focal collection of inflammatory cells, such as activated mono- and multinucleated macrophages, neutrophils, cell debris, and lymphocytes, at the site of tissue injury or infection. High mobility box 1 (HMGB1): a co-factor protein that can bind to DNA and regulate gene transcription. It is a well-known DAMP that can induce an inflammatory response when released into the extracellular space. Immune anergy: defined as the incompetence of an immune cell to respond against its targets (i.e., foreign substrates, such as pathogens or crystals). Kinins: members of the kininkallikrein system mediating inflammation. M1-like macrophage: conventional designation of a subset of

Onset and Peak of Acute Gouty Arthritis Gouty arthritis is characterized by sudden onset, often beginning during the night so that the patient wakes up with a painful joint early in the morning. This classical feature of gouty arthritis is associated with articular and periarticular swelling and heat. When smaller joints are affected, redness may also be present. The peak of pain and swelling is reached approximately 6–12 h later. Periarthritis, tendinitis, or bursitis may develop accordingly. The clinical features of gouty arthritis can be indistinguishable from those of septic arthritis, especially because an acute gout attack can present with symptoms and signs of systemic inflammation, such as fatigue and fever [4] (Table 1). Spontaneous Resolution of Gouty Arthritis A characteristic feature of acute gouty arthritis is its self-resolving nature after a few days [2]. Given that MSU crystals persist in the joint, this spontaneous resolution may imply the subsequent activation of mechanisms that suppress crystal-induced inflammation, such as negative regulators of sterile inflammation, and clearance of dead cells and aggregated NETs, as discussed below. Chronic and Visceral Gout In contrast to acute gouty arthritis, chronic tophaceous gouty arthropathy involves persistent crystal masses deposited subcutaneously that cause a smoldering local or systemic inflammation via granuloma-like foreign body reactions [5].

The Crescendo of Acute Gouty Arthritis: The Necroinflammation Loop Acute gouty arthritis is triggered by MSU crystals inside joints, a process promoted by a local imbalance of uric acid supersaturation as well as potential pre[457_TD$IF]-existing structural joint damage, such as osteoarthritis [1] (Figure 1, Key Figure). MSU Crystal-Induced Necroptosis MSU crystal deposition in the synovium triggers the release of reactive oxygen species (ROS) and reactive nitrogen species in human fibroblasts, (e.g., synoviocytes), leading to cell death [6]. However, MSU crystals can also induce direct cytotoxicity, inflammation, and inflammationdriven cell necrosis in the synovium [7]. Recently, MSU crystals as well as calcium oxalate, calcium pyrophosphate, or cystine crystals were reported to induce cell necrosis in a variety of human and murine epithelial and mesenchymal cell types in vitro [8]. Of note, necrosis may involve different biochemical signaling pathways, referred to as regulated necrosis [9]. Using a range of murine and human epithelial and mesenchymal cell types in vitro as well as in vivo mouse models of different crystal-related diseases, it was recently reported that crystal-induced cell necrosis can be mediated by the receptor-interacting protein (RIP) kinase-1, RIP kinase-3 and the pseudokinase mixed-lineage kinase domain-like (MLKL)-driven necroptosis pathways [8]. Once RIPK3 and MLKL form the necrosome complex, the latter can integrate within, and disrupt, the plasma and mitochondrial membranes, leading to cell death [10]. These necrotic cells further induce an inflammatory response by releasing immunostimulatory molecules, such as [458_TD$IF]danger-associated molecular patterns (DAMPs), which include histones, High mobility box 1 (HMGB1), DNA, as well as alarmins, such as IL-1a [11] (Figure 1). Thus, necrotic cells within the synovium further activate the immune

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response and the influx of innate immune cells, such as neutrophils and macrophages, into the synovium [9]. MSU-Induced Macrophage and/or Dendritic Cell Activation In addition to direct MSU crystal uptake by resident mononuclear phagocytes, DAMPs released from cells killed by MSU crystals activate pattern recognition receptors on the surface of, or within, these cells, a process that can amplify the local inflammatory response [12] (Figure 1). In particular, MSU crystals activate the NLRP3 inflammasome, a protein complex inside the intracellular cytosol that integrates numerous danger signals leading to caspase-1-dependent cleavage of pro- IL-1b, thereby triggering the release of mature IL-1b from the cell [13]. The NLRP3 inflammasome is a multiprotein oligomer complex formed by the cytosolic proteins NLRP3, ASC, and caspase-1 [14]. The NLRP3 molecule incorporates danger signals (i.e., mitochondrial ROS release, potassium efflux, or protease leakage from lysosomal compartments) into the inflammasome complex and activates caspase-1 [14]. Recently, NLRP3 interaction with NET7, a member of the NIMA-related kinases, was described to mediate MSU crystal-induced IL-1b secretion in vivo [15,16]. Caspase-1 subsequently cleaves pro-IL1b into its bioactive and secreted form, and promotes macrophage polarization into a proinflammatory M1-like phenotype [17]. The release of IL-1b from macrophages and dendritic cells can activate other cells via the IL-1 receptor (IL-1R) to induced proinflammatory cytokines and chemokines, leading to local inflammation and, sometimes, systemic effects, for example, clinically presenting as chills and fever [18]. However, how MSU crystals trigger inflammasome activation is not fully understood. MSU crystals can initiate NLRP3 activation simply by binding to the outer plasma membrane of bone marrow macrophages, which involves Syk signaling [19,20]. Phagocytosis of MSU crystals by immune cells, such as macrophages, can then lead to the massive release of sodium inside the cell, increasing tonicity and resulting in water influx; this process dilutes the intracellular potassium concentration, which constitutes a classical intracellular danger signal and activates the NLRP3 inflammasome [21]. Additionally, MSU crystals have also been shown to induce IL-1b release from mast cells in a [459_TD$IF]mouse model of acute gouty arthritis generated by intra-articular injections of MSU crystals in mice with constitutive or inducible mast cell deficiency [22]. In this model, MSU crystals have been reported to activate resident mononuclear phagocytes to release of IL-1b as a trigger for joint inflammation [22]. MSU Crystals Can Induce Neutrophil Necroptosis At the onset of acute gouty arthritis, accumulated MSU crystals can induce massive infiltration of inflammatory cells, such as neutrophils and monocytes, into the site of MSU crystal deposits in patients [23]. IL-1 that is released from these immune cells further triggers the release of various proinflammatory cytokines and chemokines, such as IL-8, IL-6, and CXCL8, upregulating selectins and integrins on the luminal surface of endothelial cells, which can further enhance neutrophil recruitment. MSU crystals have been found to activate such infiltrating neutrophils, not only by triggering cytokine secretion, but also by inducing neutrophils to form NETs [24,25]. In vitro evidence suggests that human as well as murine neutrophils release their chromatin decorated with granular enzymes, such as neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G, and others, forming extracellular net-like structures in response to bacterial infection, proinflammatory cytokines (e.g., TNF-a and IL-8) [24], DAMPs, MSU crystals [25], and other triggers. Peptidylarginine deiminase-4-mediated histone citrullination has been noted to enforce chromatin decondensation and chromatin release during NET formation [26]. The process of NET formation can be different for different stimuli (i.e., bacteria, PMA, MSU crystals, etc.), but usually involves NADPH oxidase-mediated ROS production in human as well as murine neutrophils [27]. Crystal-induced NET formation in human neutrophils has been found to lead to neutrophil death, previously referred to as ‘NETosis’ [28]. The RIPK3-MLKLdependent signaling pathway can mediate MSU crystal-induced neutrophil death; thus,

macrophages associated with the production of high amounts of proinflammatory cytokines, such as IL1-b, TNF, IL-12, and so on. They are usually associated with host– pathogen responses and the promotion of TH1 responses. Phenotypically, M1 macrophages express major histocompatibility complex class II (MHC II), the CD68 marker, and co-stimulatory molecules CD80 and CD86. M2-like macrophages: conventional designation of a subset of resident macrophages associated with the production of high amounts of anti-inflammatory cytokines, such as IL-10 and TGF-b, and low levels of proinflammatory cytokine IL-12. Tumor-associated macrophages usually display this phenotype. Macrophages: large white blood cells that can recognize, engulf, and digest cellular debris, microbes, or foreign substances as a consequence of injury or infection. Necroinflammation: a process whereby cell necrosis and inflammation enhance each other in an auto-amplification loop, such as in the early crescendo phase of gouty arthritis. Necroptosis: form of regulated necrosis induced by ligands binding to TNF death domain receptors and followed by phosphorylation of receptor interaction protein kinase 1 (RIPK1) and RIPK3 to form a necrosome complex. The necrosome further activates mixed-lineage kinase-like (MLKL), executing cell death by migrating to the plasma membrane, where it is associated with pore formation and plasma membrane rupture. Necrosome complex: formed by receptor-interacting protein (RIP) kinase-3 and the pseudokinase mixed-lineage kinase domain-like (MLKL) executing regulated necrosis downstream of RIP kinase-1. Neutrophil extracellular traps (NETs): upon activation by different stimuli (i.e., bacteria, crystals, LPS, pr PMA), neutrophils can explode and release their chromatin, bearing granular serine proteases to form net-like structures known as NETs. NLR Family Pyrin DomainContaining 3 (NLRP3)/interleukin (IL)-1b inflammasome: danger signaling platform in the cytosol, comprising mainly macrophages and dendritic cells. It integrates several danger signals into the secretion of

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crystal-induced NETosis might be classified as necroptosis [24]. Specifically, Ripk3 deficiency, as well as using small-molecule modulators of RIPK1, such as necrostatins (inhibitors of the necroptosis pathway), inhibited MSU crystal-induced NET formation and gout-like tophus formation in mouse models in vivo [24]. Moreover, a study recently showed that purinergic P2Y6 receptor, which is involved in neutrophil migration [29], was required for MSU crystalinduced NET formation in human neutrophils in vitro, because the P2Y6 receptor antagonist MRS2578 inhibited MSU crystal-induced NET formation [30]. However, MRS2578 also blocked crystal-NET aggregate formation indirectly by repressing neutrophil migration and recruitment, as evidenced from live cell imaging of PMNs [30]. In another in vitro model system, human macrophage-derived IL-1b enhanced MSU crystal-induced NET formation, suggesting that the interplay between macrophages and neutrophils during gout attack accelerates MSU crystal-induced NET release in the synovium [31]. Overall, NET release appears to be one of the events that serves to amplify necroinflammation in acute gout. NETs release various DAMPs, such as histones or DNA-activating Toll-like receptors (TLR2, TLR4, TLR9) or NLRP3 inflammasomes [11], leading to the release of proinflammatory cytokines (IL-8), which can further activate the immune system. Moreover, several studies have shown that histones can have direct cytotoxic effects when they are released in extracellular spaces [32]. Extracellular histones can induce necrosis of endothelial cells in vitro as well as in mouse models of inflammation in sepsis [33]. Since extracellular histones are a major component of NETs, histone release during NET release may have an important role in progressing inflammatory response and cell death. Thus, histones that are released during NET formation may constitute an important trigger of necroinflammation during acute gout episodes. However, this hypothesis remains to be fully tested. In summary, MSU crystals in the synovial fluid trigger a series of events, some of which might involve necroptosis and inflammation, which might in turn enhance each other [34,35], thus contributing to the crescendo of clinical symptoms, such as local inflammation in the joint, as well as redness and intense pain that are typically observed during the early phase of acute gouty arthritis.

The Decrescendo of Acute Gouty Arthritis: Counter-Regulation of Sterile Inflammation If not counter-regulated, the crescendo of acute inflammation has the potential to end in a cytokine storm, systemic clotting, organ failure, and death. As described below, the crescendo of crystal-induced necroinflammation involves the subsequent activation of numerous immunoregulatory elements that limit necroinflammation and promote the resolution of the acute inflammatory response (Figure 2A). Negative Regulators of Inflammasome and TLR Signaling in Sterile Inflammation Negative feedback loops are crucial for reducing DAMP-induced sterile inflammation. Two negative inflammasome regulators are the pyrin domain (PYD) and the caspase-containing domain (CARD). PYD and CARD domains are short proteins that modulate inflammasome activation by acting as decoys and disrupting ASC recruitment to the inflammasome in response to LPS [36,37]. Pyrin deficiency in mice has been shown to increase endotoxin sensitivity and enhanced caspase-1 activation in macrophages [38], which has also been implicated as a central signaling element in gout [13]. Furthermore, negative regulators of DAMP-induced TLR signaling have been identified to shut down parameters of sterile inflammation in animal models in vivo, as well as in human and murine in vitro experiments [39]. Such regulators can be broadly divided into three categories: (i) extracellular modulators, such as the soluble decoy TLR-4 [40–42] or globotetraosylceramide [43]; (ii) transmembrane proteins, such as the suppressor of tumorigenicity 2 [39,42,44,45] or C-type lectin receptors (CLRs); and (iii)

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IL-1b and IL-18 via activating caspase-1. Pattern recognition receptors (PRRs): proteins expressed by innate immune cells that can recognize various pathogens as well as DAMPs. Based on their function and ligand specificity, PRRs can be divided into signaling PRRs (e.g., Toll-like receptors, NOD-like receptors) and endocytic PRRs (i.e., glucan receptors). Phagocytosis: process of engulfing certain particles, debris, or microorganisms by specialized immune cells, such as neutrophils or macrophages. Receptor activator of nuclear factor-kB ligand (RANKL): membrane protein that is part of the TNF cytokine superfamily. It has a key role in the regulation of osteoclast formation. Regulated necrosis: class of a ‘programmed’ form of necrosis that includes multiple cell death pathways with distinct biochemical signaling cascade, such as necroptosis, parthanatos, ferroptosis, pyroptosis, and mitochondrial permeability transition pore (MPTP)-mediated necrosis. Resolution of inflammation: a coordinated and active process to restore homeostasis. Septic arthritis: a condition of joint inflammation caused by bacterial or fungal infection. Toll-like receptors (TLR): class of membrane-bound PRRs present on innate immune cells, such as macrophages and dendritic cells, that can recognize pathogens as well as DAMPs and further activate NFkB signaling and the MAP kinase pathway to generate an inflammatory response. Tophus: masses of monosodium urate crystals, NETs, and proteins surrounded by foreign-body granuloma-like encapsulating immune cells and fibroblasts. Visceral and chronic gout: visceral gout is a disease occurring in birds and humans due to impaired kidney function followed by accumulation of urate crystals in different organs. Chronic gout is a more severe condition that occurs in humans after repeated attacks of acute gout. The urate crystals accumulated in both of these conditions lead to the formation of white ‘tophi’ in joints or skin.

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[450_TD$IF]Table 1. Clinical Presentations of Gout and Known Related Molecular Pathomechanismsa Clinical presentation

Pathomechanisms

Refs

Acute gouty arthritis

MSU crystals directly and indirectly activate RIPK1-RIPK3-MLKL-mediated necroptosis of epithelial cells

[8]

Spontaneous resolution of gouty arthritis

Chronic gout/anergic tophus a

MSU crystals directly activate RIPK1-RIPK3-MLKL-mediated suicidal NETosis of neutrophils

[24]

NLRP3 inflammasome-mediated secretion of IL-1b and IL-1b-mediated induction of innate immunity

[13]

Induction of negative regulators of innate immunity

[36–45]

NET-related proteases digest inflammatory mediators

[81]

Macrophages clear NETs and crystals

[56]

NET-related proteases digest inflammatory mediators

[81]

Abbreviations: MLKL, mixed lineage kinase domain-like protein; RIPK1, receptor interacting serine/threonine kinase 1; RIPK3, receptor interacting serine/threonine kinase 3.

intracellular negative regulators, such as MyD88 short [39,46] (Table 2). The seesaw of inflammation in gouty arthritis is conceptually similar to the phenomenon of endotoxin tolerance: endotoxin exposure to bone marrow-derived macrophages first induces proinflammatory and, subsequently, anti-inflammatory mediators [47,48], as identified by transcriptome analysis [49–51]. Given that MSU microcrystals can be considered to be important DAMPs during cell necrosis [52], immune cells, such as murine neutrophils, monocytes, dendritic cells, and macrophages, are also equipped with surface receptors that recognize MSU crystals and can directly turn down inflammatory signaling pathways, for example via the inhibitory C-type lectin receptor (CLR) Clec12a [53] (Table 2). Indeed, the Clec12a receptor, when engaged by MSU crystals, can downmodulate human and murine neutrophil activation by inhibiting ROS production [53]. Moreover, DAMPs released from necrotic cells, as shown for thymocytes, splenocytes, or HEK293 cells, have also been found to trigger the expression of two CLRs, Mincle and Clec9a [54,55], on murine myeloid cells, thus inhibiting neutrophil function via Clec12a ligation [53]. Interestingly, Clec12a deletion does not appear to affect MSU crystalstimulated macrophages, indicating that the NLRP3 inflammasome pathway may not be directly involved by Clec12a signaling [53]. Collectively, these findings suggest a role for Clec12a in modulating neutrophil function, leading to indirect regulation of sterile inflammation, when MSU crystals are taken up by neutrophils in the context of cell necrosis. Regulation of Proinflammatory Cytokines in MSU Crystal-Activated Macrophages The intrinsic IL-1R antagonist (IL-1Ra) has the ability to block the proinflammatory activities of IL-1 cytokines released by MSU crystal-stimulated immune cells, such as macrophages, indicating a regulatory role for IL-1Ra as a competitive inhibitor of inflammasome-related IL-1R signaling in patients with rheumatoid arthritis [56] as well as in acute septic and aseptic inflammation in mice [57–59] (Table 2). IL-1Ra release can be induced by the anti-inflammatory cytokine TGFb1 in human circulating monocytes [60,61] as well as neutrophils [62]. Of relevance, the levels of IL-1Ra are elevated in synovial fluid during the phase of resolving acute gouty arthritis [63]. Furthermore, the recombinant IL-1Ra anakinra has been found to be effective and well tolerated in the short-term control of acute gouty arthritis in several clinical trials by rapidly relieving inflammatory symptoms within 5 days [64–66] (Figure 2A). Therefore, anakinra represents a safe alternative treatment for acute gouty arthritis in medically complex hospitalized patients when standard therapy has failed [67–69]. The synovial fluid of patients with gouty arthritis also contains high levels of the soluble TNF receptors I and II (sTNFR-I/II), as well as of IL-10 [63]. Data from a murine air pouch model and MSU crystal-stimulated peritoneal macrophages in vitro demonstrated that soluble sTNFR-I/II

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Key Figure

Necroinflammation in Acute Gouty Arthritis

Figure 1. The molecular mechanisms underlying intra- and periarticular monosodium urate (MSU) crystal formation remain poorly understood but hyperuricemia and precedent abnormalities in joint structure are known to have an important role (1). MSU crystals elicit direct cytotoxic effects on epithelial cells by activating necroptosis via receptor-interacting protein kinase (RIPK)-1 and 3 and mixed-lineage kinase domain-like protein (MLKL), which form the necroptosome and disrupt cell membranes (2). Epithelial cell death implies the release of damage-associated molecular patterns (DAMPs) and alarmins (3). Resident mononuclear phagocytes take up MSU crystals, a process activating the NLR Family Pyrin Domain-Containing 3 (NLRP3)/interleukin (IL)-1b inflammasome and inducing local secretion of mature IL-1b as well as IL-1a (4). Both forms of IL-1 activate the IL-1R on parenchymal and immune cells to secrete numerous proinflammatory mediators (5). Among these mediators, several promote the rapid recruitment of neutrophils (6) that, once they encounter MSU crystals, undergo necroptosis and neutrophil extracellular trap (NET) formation (7). Necrotic neutrophils and NETs release various proinflammatory mediators, such as lytic proteases and cytotoxic histones, that further contribute to the crescendo of the auto-amplification loop of necroinflammation.

and IL-10 can inhibit proinflammatory mediators, such as TNFa and the macrophage inhibitory protein 1a and -b [70] (Table 2). Also, it is well known that certain miRNAs can regulate the gene expression of proinflammatory cytokines. In addition, screening miRNA expression in human macrophages identified miR-146a as a possible therapeutic candidate for mediating and limiting the inflammatory response of these cells to MSU crystals [71]. TGFb1 can induce endogenous IL-37, an anti-inflammatory cytokine with intracellular and extracellular functions [72]. Blood monocytes from patients with asymptomatic gout express higher levels of IL-37 compared with those from patients with a gout flare [73]. Furthermore, in vitro silencing of IL-37 in MSU crystal-stimulated human peripheral blood mononuclear cells (PBMCs) was reported to increase the production of IL-1b, IL-6, and TNFa, indicating that IL-37 could suppress proinflammatory cytokine production [73] (Table 2). The suppressive effect of rhIL-37 has been confirmed in in vivo mouse models of gouty arthritis [74]. Moreover, several intracellular

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[49_TD$IF]Figure 2. Tophus Formation in Chronic Gout. (A) Persistence of larger crystal masses trigger massive neutrophil extracellular trap (NET) formation or aggregated NETs (aggNETs). These NETs, as well as apoptotic neutrophils, are cleared by macrophages in a non-inflammatory manner (1). AggNETs release various proteases that cleave proinflammatory mediators, a mechanism contributing to immune anergy in tophaceous gout (2). In an attempt to clear crystals and aggregated NETs, macrophages localize around the tophus formed by MSU crystal masses and NETs (3). (B) This can lead to foreign-body granuloma formation with tophus masses at the center, surrounded by giant cells and epitheloid cell layers (5), a process contributing to bony lesions, soft tissue damage, and tissue remodeling. Negative regulators of surface receptors, (Toll-like receptors; TLRs), negative regulators of inflammasomes in neutrophils, as well as macrophages, have a key role in mediating the resolution of a gout attack (4).

mediators limit inflammation, such as signal transducers and activators of transcription (STATs), cytokine-inducible SH2-containing protein (CIS), and suppressors of cytokine signaling (SOCS) [63,75]. Of note, overexpression of CIS and SOCS3 has also been observed in in vitro MSU crystal-activated human monocyte-derived macrophages and synovial fluid mononuclear cells from patients with gouty arthritis [63]. In addition, intracellular CIS can decrease MSU crystal-induced IL-1b and TNFa production and simultaneously enhance STAT3-mediated transcription of TGFb1 [63] in mouse macrophages and human monocyte-derived macrophages from healthy donors (Table 2). This implies that increased production of antiinflammatory mediators and intracellular CIS and SOCS3 expression may be associated with the spontaneous resolution of acute gouty arthritis. Neutrophils, NETs, and Clearance of Dying Cells Data now indicate that in vitro MSU crystal-stimulated human blood neutrophils and neutrophils isolated from a murine model of MSU crystal-induced peritonitis release microvesicles (ectosomes) from their surface; a process initiated by macrophage-derived complement C5a in gout [76] (Table 2). Such ectosomes express phosphatidylserine (PS) on their surface, and can suppress NLRP3 inflammasome-mediated IL-1b release from mouse peritoneal macrophages

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Table 2. Known Mechanisms Associated with the Resolution of Gouty Arthritisa[451_TD$IF] [452_TD$IF]Pathomechanism

Mechanism of [453_TD$IF]action

Refs

Negative regulators of inflammasome and TLR signaling

Inflammasome regulators: PYD and CARD

[36,37]

Regulation of pro-inflammatory cytokines

Neutrophils, NETs, and cell clearance

Pre-resolving mediators

a

TLR regulators: sTLR-4, Clec12a, CLRs, and MyD88 short

[39–42,46,48,53]

Downregulation of IL-1R by TGFb1

[60–62]

Endogenous receptors: IL-1Ra and sTNFR-I/II

[56,63]

miRNA miR-146a

[71]

Intracellular CIS and SOCS3

[63,75]

Cytokine IL-37

[73]

C5a-mediated release of ectosomes by neutrophils

[76]

AggNETs to degrade and digest cytokines

[81]

Phagocytosis of dying neutrophils and release of TGFb

[82–85]

Resolvins, lipoxins, maresins, and prostaglandins

[89–91]

Short-chain fatty acids and high-fiber diet

[97]

Abbreviations: C5a, complement 5a; CIS, cytokine-inducible SH2-containing protein; IL-Ra, IL-1R antagonist; SOCS3, suppressors of cytokine signaling 3; ST2, suppressor of tumorigenicity 2; sTLR4, soluble decoy toll-like receptor-4; sTNFR-I/II, soluble tumor necrosis factor receptors I and II; TGFb, transforming growth factor beta; TRAILR, TNF-related apoptosis-inducing ligand receptor.

as well as from human monocyte-derived macrophages and dendritic cells [76–78]. This process in turn induces TGFb1 release from macrophages, implying that ectosomes might have an anti-inflammatory effect [79] in gout, which warrants further investigation. Furthermore, large DNA/MSU crystal structures are formed when neutrophils and, therefore, NETs are present in high numbers [aggregated NETs (aggNETs)], as shown in a murine air pouch and MSU crystal-induced paw models [80,81]. Human aggNETs can trap and degrade proinflammatory chemokines, such as chemokine ligand CCL2, and cytokines, such as IL-1b and IL-6 via neutrophil serine proteases in response to MSU crystals in vitro [81] (Figure 2A). Absorbance and trapping of inflammatory cytokines as well as of needle-shaped MSU crystals further limit inflammatory response by MSU crystals. Thus, neutrophils have a crucial role in initiating the resolution process of gout [81]. Another mechanism that has been implicated in the resolution of acute gouty inflammation is the non-inflammatory phagocytosis of dying neutrophils by macrophages [82,83]. Monocytes infiltrating sites of MSU crystal-induced necroinflammation in vivo have been reported to exhibit a higher phagocytic capacity as they differentiate into a M2-like macrophage phenotype in a murine MSU crystal-induced peritonitis model [17]. Human M2-like macrophages are able to phagocytose human apoptotic neutrophils [82,83] as well a small number of human NETs [84] (Figure 2A), and this mechanism has been associated with the production of TGF-b1 [17,82,85]. Neutrophils might also clear dead cells [85]. In addition, in patients with gout, elevated TGF-b1 levels have been found in their synovial fluid during the resolution phase of acute inflammation [86–88]. This suggests that the phagocytosis of dying cells and TGF-b1 production contribute to the resolution of MSU crystal-induced necroinflammation. Anti-Inflammatory and Proresolving Circuits Lipid mediators, such as resolvins, lipoxins, maresins, and prostaglandins, have been reported to contribute to the resolution of inflammation in a mouse model of zymosan-induced peritonitis [89–91] and elevated levels of pro-resolving mediators have been found in the plasma and, to a

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lower extent, the synovial fluid of patients with arthritis [92]. Generated from essential fatty acids, such as omega-3 polyunsaturated acids, eicosapentaenoic acid, and g-linolenic acid, these bioactive molecules limited neutrophil chemotaxis, enhanced apoptotic cell clearance by macrophages, and prevented inflammation by inhibiting NLRP3 inflammasome activation in vitro and in an in vivo mouse air pouch model [93–96] (Table 2). Recently, the short-chain fatty acid acetate was shown to regulate MSU crystal-induced inflammation in mice when given either orally or in drinking water [97]. Acetate treatment promoted caspase-dependent neutrophil apoptosis and decreased IL-1b release by macrophages, triggering the secretion of anti-inflammatory cytokines, including IL-10 [97] (Table 2). Similar results have been observed by treating animals on a high fiber diet in a peritonitis model of acute gouty arthritis [97]; the increased consumption of fiber also alleviated certain clinical symptoms in patients with gout [98], indicating that food sources rich in dietary fiber may limit gout occurrence. Thus, acute necroinflammation appears to trigger several counter-regulatory mechanisms that limit MSU crystal-induced inflammation, promoting its resolution. These implications may be relevant because they might explain why the signs and symptoms of acute gouty arthritis can resolve to a certain degree, despite the persistence of MSU crystals. However, notwithstanding the cessation of symptoms, MSU crystals usually persist and can trigger subsequent gout attacks. Thus, questions that remain include why MSU crystals often remain asymptomatic and why or how flares occur.

Anergic Tophi: Crystal Masses Devoid of Inflammation In contrast to acute gout, the mechanisms of chronic tophaceous gout are less well understood. Chronic gout is characterized by the formation of tophi in joints, skin, or bursae that resemble chronic granulomatous lesions containing MSU crystal-NET masses at the center surrounded by mono- and multinucleated phagocytes (‘giant cells’) and fibroblasts (Figure 2B) [99]. The tophus comprises three central compartments: (i) a central crystalline core formed of MSU crystals; (ii) a surrounding area of dense populations of innate immune cells, such as CD68+[45_TD$IF] macrophages, plasma cells, and NETs; and (iii) the fibrovascular outer compartment containing T and B lymphocytes in smaller numbers [5]. The presence of cytokines, such as IL1b, IL-6, TNF-a, and TGF-b1, within tophaceous lesions of patients with gout has been demonstrated, suggesting that these cytokines have a role in granuloma formation in such patients [99]. However, tophi can present without apparent manifestations of acute gouty inflammation (e.g., heat, swelling, pain, or erythema), despite the presence of persistent MSU crystal deposits at the center of the lesions. Recent data suggest that, under high neutrophil densities at the site of inflammation, NETs aggregate to form aggNETs and degrade proinflammatory cytokines and chemokines via serine proteases [81]. This study highlights a novel role for aggNETs in effectively shutting down MSU crystal-induced inflammation [80,81] (Figure 2B). Proteomic analysis of a tophus from a patient with chronic gout identified a variety of proteins that are present within the tophus [100]. These proteins included immunoglobulins and complement factors; pro- and anti-inflammatory proteins, such as myeloperoxidase and TGF-b1; connective tissue; matrix proteins; apolipoproteins; fatty acid-binding proteins; and histones [99,100]. Co-expression of inflammatory (e.g., IL-1b) and inflammatory proteins (e.g., TGF-b1) suggests the occurrence of an inflammatory response upon MSU crystal accumulation, followed by resolution via anti-inflammatory proteins [99]. Moreover, it is possible that the formation of a large granuloma around the tophus might also limit exposure of inflammatory factors to neighboring cells, thereby limiting MSU crystal-induced inflammation. Granuloma formation is a typical feature of certain infectious and non-infectious diseases, when the host is unable to clear pathogens or DAMPs, respectively (Figure 2B). In chronic gout, accumulated MSU crystal-NET masses and microenvironmental factors, such as cytokines, can serve as triggers to induce the palisading of activated macrophages, which, together with

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Box 1. Clinician’s Corner [45_TD$IF] The fundamental concept of managing gout involves urate-lowering therapeutics (ULT), such as xanthine oxidase inhibitors or uricosuric drugs, to prevent uric acid supersaturation and crystal formation [104].  In acute gouty arthritis, colchicine, nonsteroidal anti-inflammatory drugs (NSAIDs), or steroids can control the signs and symptoms of acute necroinflammation [105] (see Figure 1 in the main text).  IL-1 inhibitors, such as anakinra, canakinumab, and rilonacept, can suppress the clinical manifestations of acute gouty arthritis or prevent gout flares [64,106] (see Figure 1 in the main text). Currently, only canakinumab is approved as a third-line therapy for frequently relapsing gout. However, the high costs of this biological drug remain a concern.  Short-chain and long-chain fatty acids, such as acetate and butyrate, have been reported to exert anti-inflammatory effects by suppressing inflammatory cytokine production in animal models of acute gout as well as in PBMCs isolated from healthy volunteers and patients with gout [97,107] (see Figure 1 in the main text).  Treatment of chronic tophaceous gout is difficult and based on ULT to substantially reduce serum urate levels (to below 5 mg/dl) [105]. Recombinant uricase has been used as an off-label option to resolve persistent MSU crystal masses in tophaceous gout (see Figure 2 in the main text), but hypersensitivity and loss of efficacy after repetitive dosing remain problematic.

other immune cells, such as lymphocytes (e.g., CD4+ T cells), form granulomas around tophi [101]. These large-body giant cell granulomas comprising mono- and multinucleated macrophages, in addition to osteoclast-like cells around tophi, can be responsible for extensive bone resorption [5]. In vitro, MSU crystals can inhibit osteoclast viability, differentiation, and mineralization, leading to a reduced capacity for new bone formation around tophi. These in vitro data are supported by human bone samples that are affected by tophi because of the absence of osteoclasts at sites of erosion (reference PMID: 21622970). In addition, human chondrocytes, when stimulated with MSU crystals in vitro, have shown reduced viability and function, suggesting that the interaction of MSU crystals and chondrocytes contributes to the loss of cartilage in joints [102] (Figure 2B). This appears to be consistent with observational data from joints of patients with gout that reveal that the normal cartilage architecture is lost, with empty chondrocyte lacunae [102]. Thus, it is surprising that tophi might be strongly associated with joint and bone damage in chronic tophaceous gout. For example, a recent study reported increased protein expression of receptor activator of nuclear factor-kB ligand (RANKL) in tophus samples from patients with chronic gout [103], and IL-1b and TNFa expression within the tophus might also enhance bone resorption by osteoclasts [99,103]. Presently, however, information on the role of multinucleated giant cells in tophaceous gout is limited. An appropriate animal model for chronic tophaceous gout and robust studies would be needed to unravel the functional contribution of these giant cells to granuloma formation and bone resorption in gout.

Concluding Remarks Acute gouty arthritis is a paradigmatic example of the crescendo and decrescendo of crystalinduced necroinflammation. Understanding the role of IL-1 in the molecular pathogenesis of gout has raised the possibility of using IL-1-targeting drugs as a novel treatment option for patients with recurrent gout. The recent discovery of crystal-induced cytotoxicity offers additional drug targets. Chronic gout is possibly an underdiagnosed disease entity, which can now be spotted with dual-energy computed tomography. Its pathogenesis is exemplified by granuloma formation around crystal masses with regional differences in immune cell phenotypes. However, important questions remain to be addressed by further research efforts (Outstanding Questions and Box 1). Among those, developing better animal models will undoubtedly help the translational research process. There is increasing interest in gout as a potentially treatable, noncommunicable disease. Moreover, in recent years, a novel xanthine oxidase inhibitor, febuxostat, and a new URAT-1 inhibitor, lesinurad, have become available. Nevertheless, there is great hope that a better understanding of the molecular pathophysiology of acute and chronic gout will lead to further drug options for treatment, particularly since gout represents the most prevalent destructive inflammatory joint disease.

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Outstanding Questions Could small-molecule inhibitors of the NLRP3 inflammasome or IL-1R offer less costly ways to target IL-1-mediated pathogenesis (and clinical manifestations) in gout? Such agents have become available but have not yet been tested in the context of gout. Can small-molecule inhibitors of necroptosis attenuate the symptoms of acute gouty arthritis? Such inhibitors could block MSU crystal-induced necrosis of epithelial cells, and neutrophils, including the formation of neutrophil extracellular traps. Does the dual inhibition of cell necrosis and inflammation elicit additional effects on the symptoms of acute gouty arthritis? It remains unclear whether interrupting MSU crystalinduced necroinflammation at both ends would be more efficient. Can immunoregulatory mediators enforcing the resolution of gouty arthritis serve as therapeutic targets to control the symptoms of acute gouty arthritis? The hierarchical importance of single immunoregulatory elements versus redundancy remains to be elucidated. Are there convenient ways to inhibit MSU crystal formation despite local supersaturation to prevent future gout attacks? Controlling hyperuricemia is not always possible. Preventing MSU crystal formation in such settings could be useful. How can we overcome the limitations of current recombinant uricase preparations to solubilize MSU crystals and improve outcomes of recurrent and chronic gout? Hypersensitivity reactions and tachyphylaxis have been reported with current products. What are the molecular and cellular pathomechanisms of the foreign-body reaction around tophi? Modulating these pathomechanisms could help limit tissue destruction in chronic gout.

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Acknowledgments We apologize to all colleagues whose work could not be cited or discussed in detail due to space and format restrictions. This work was supported by grants from the [460_TD$IF]Deutsche Forschungsgemeinschaft [461_TD$IF]to H.J.A. (AN372/14-3, 16-1, 20-1, and 24-1) and to S.S. (STE2437/2-1).

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