Epigenomic Reprogramming in Gout

Abstract

Gout is a crystal-induced arthropathy in which monosodium urate (MSU) crystals precipitate within joints as a result of persistent hyperuricemia and elicit an inflammatory response. An intriguing aspect is the occurrence of gout in only 10–15% of hyperuricemic individuals, suggesting the presence of additional risk factors. Although MSU crystal deposition is widely recognized as the cause of gout flares, the variability in initiating the inflammatory response to hyperuricemia and MSU deposition is not well understood. Several studies bring up-to-date information about the environmental and genetic influences on the progression towards clinical gout. Elevated urate concentrations and exposure to different external factors precipitate gout flares, highlighting the potential involvement of epigenetic mechanisms in gouty inflammation. A better understanding of the alteration of the epigenetic landscape in gout may provide new perspectives on the dysregulated inflammatory response. In this review, we focus on understanding the current view of the role of epigenomic reprogramming in gout and the mechanistic pathways of action.

1. Introduction

General Introduction About Gout (Incidence, Clinical Manifestation, Associated Comorbidities, Pathophysiology)

Gout is a common crystal-induced arthropathy with a clinical description that dates back to ancient times, often referred to as the disease of the kings, with elevated urate concentrations being correlated to increased intake of purines [1,2,3]. Nowadays, gout is an impairing pathology with an increasing prevalence worldwide, with differences observed among ethnicities, age groups, and sexes [4]. The well-known risk factor for gout is hyperuricemia; however, not all patients with elevated serum urate concentrations develop this disease [5]. Over time, extensive literature has been generated on understanding how the immune system mediates the inflammatory response to urate and urate crystals [6,7].

Hyperuricemia represents the elevation of urate concentrations above the threshold of solubility. Multiple factors increase the risk of gout due to a predisposition to hyperuricemia [8]. The imbalance between overproduction and underexcretion of urate leads to supersaturation of the serum. This leads to urate precipitation in the form of monosodium urate (MSU) crystals which accumulate in the joints and trigger an inflammatory response [9]. Hyperuricemia is a necessary but not a standalone condition to develop gout; only 10–15% of hyperuricemic individuals will progress to clinical manifestations of this disease [10,11].

To attain a comprehensive understanding of gout, it is essential to elucidate the interconnection among urate, hyperuricemia, and the manifestation of gout [12].

In humans and other primates (hominoid primates), urate is the end product of purine catabolism due to successive mutations during evolution that lead to an inactivated gene encoding urate oxidase (uricase) [13]. Uricase is an enzyme synthesized in the liver that oxidizes urate to its more soluble form, called allantoin. The loss of uricase functionality leads to higher serum urate concentrations in humans than in uricase-conservative species, thereby increasing the risk of developing gout [14].

Gout is a chronic pathology that develops in four stages [1]: asymptomatic hyperuricemia, acute gouty attack, intercritical period, and chronic tophaceous gout.

The initial clinical manifestation of gout is the occurrence of an acute attack (flare) caused by the deposition of MSU crystals within the joints, which sets off an immediate inflammatory response mediated by synovial macrophages [8]. Most typically, inflammation is monoarticular and affects the metatarsophalangeal joint by abrupt onset characterized by intense pain, tenderness, and swelling [15]. The flare is preceded by asymptomatic hyperuricemia, which, after a clinical gout diagnosis, requires urate-lowering therapy (ULT) [16]. The complete resolution of flares might require 2–3 weeks but is not correlated with the resolution of urate crystals, and subclinical inflammation may persist [17,18]. Untreated, it can progress to the chronic stage, marked by polyarticular involvement, the formation of gouty tophi, and deterioration of joint integrity [15].

Gout is a urate-based metabolic condition coexisting with multiple comorbidities, including metabolic syndrome, hypertension, diabetes mellitus, and cardiovascular and renal impairment, leading to premature death [1,7,19]. A causal relationship between increased urate concentrations and gout with comorbidities is not proven, and the existence of a link has been consistently proposed [19,20,21,22,23]. The primary mechanism for eliminating urate is through renal excretion, yet this system is not well-suited to function effectively under hyperuricemia, potentially increasing the likelihood of nephrolithiasis formation, according to a recent study [24,25].

Increased serum urate concentrations could have implications for chronic inflammatory processes, which may result in maladaptive immune responses. Urate-induced inflammation has been experimentally described in peripheral blood mononuclear cells, and exposure to this factor can induce long-lasting inflammatory reprogramming [26,27]. This observation leads to the hypothesis that urate may be an inducer of trained immunity, a process consisting of a particular type of immunological memory mediated by epigenetic reprogramming and metabolic changes in innate immune cells [28]. Trained immunity is characterized by a more potent inflammatory response upon secondary exposure to stimuli after initial exposure to an aggressor such as pathogens or alarmins [6,28,29,30,31,32]. This behavior was studied in relation to epigenetic processes such as DNA methylation, histone modifications, and non-coding RNA-mediated mechanisms [33]. In urate-exposure settings, cells undergo changes at the genomic level that can persist by altering the expression of genes involved in inflammatory pathways [26,34]. By using inhibitors of histone methyl transferases in vitro, the urate inflammatory priming of mononuclear cells was reverted, suggesting the implication of epigenetic remodeling in soluble urate-induced inflammation [35]. Additionally, MSU crystal stimulation is also proposed to alter the epigenetic landscape of monocytes or macrophages [7]. As discussed above, functional changes in the immune cells may predispose to a more pro-inflammatory phenotype, contributing to the inflammatory variability in patients with gout. Understanding and better integrating the knowledge into long-term phenotypic alterations in response to urate may further elucidate whether trained immunity is a driving process in the development and progression of gout.

In this review, we focus on understanding the role of epigenomic reprogramming in urate-induced inflammation and the mechanistic pathways of action.

2. Local Versus Systemic Inflammation

Nowadays, gout is recognized as both an autoinflammatory condition and a metabolic disorder [36]. A gout flare is defined as a self-limited innate immune reaction that contains three different stages: initiation, amplification, and resolution. Different factors can modulate these three phases, and they are related to both host and environmental factors, such as genetic, epigenetic, cell metabolism, nutrition, or circadian rhythm. Locally, resident macrophages react to MSU crystals and secrete pro-inflammatory cytokines and chemokines, such as interleukin IL-1β, IL-6, or CXC ligand 8 (CXCL8) [37]. For IL-1β secretion, an additional step of activation through caspase-1 is required, which cleaves the inactive pro-IL-1β into its active form (Figure 1). The recruitment of monocytes and neutrophils through these inflammatory mediators leads to the production of more pro-inflammatory cytokines, consequently sustaining the inflammatory response and causing damage to the local tissue. Resolution is promoted by neutrophil apoptosis and cells that have acquired an anti-inflammatory phenotype, such as M2 macrophages, and secretion of anti-inflammatory cytokines like TGF-β [38].

Beyond local inflammation, gout is also characterized by a systemic inflammatory response. Urate has been implicated in various biological processes, leading to increased responsiveness of the immune system through inflammatory reprogramming via transcriptional and epigenetic changes. In vitro studies proved that soluble urate has the potential to induce epigenetic changes such as histone modification [26,35,39]. Mechanistically, urate signaling in monocytes is linked to the AKT-PRAS40 pathway, followed by mTOR activation and transcriptional control of IL1-Ra and IL-1beta production (Figure 1).

Recent studies investigating circulating inflammatory markers have described systemic pro-inflammatory signatures in both gout and hyperuricemia without gout.

Cabău et al. investigated the serum inflammatory landscape in patients with gout and in people with hyperuricemia by targeted proteomic analysis utilizing the Olink inflammation panel. A total of 58 proteins exhibiting differential abundance were identified when comparing asymptomatic hyperuricemia samples to controls. The results reveal an upregulation of mTOR, together with cytokine families (IL-6, IL-10, and TNF), a broad spectrum of chemokines (MCP-1, MCP-2, MCP-3, and MCP-4), and other immunoregulatory proteins (HGF, CSF-1, TGF-α, β-NGF, VEGFA, FGF-21). Another distinct inflammatory signature is the increased level of the inflammatory proteins MMP-1, IL-6, VEGFA, and CCL23 and decreased levels of DNER and CD6 levels in flare vs. non-flare gout patients [40]. Furthermore, various circulating markers, including IL-6, CCL23, and S100A12, exhibited associations with polyarticular involvement or larger joints as opposed to small joints [40].

Another report has linked increased urate concentrations to higher oxidative stress and to a pro-inflammatory state in asymptomatic hyperuricemia individuals compared to healthy controls, as suggested by an augmented systemic production of TNF and IL-6, and ROS markers such as malondialdehyde (MDA) and superoxide dismutase (SOD) [41]. Urate-induced oxidative stress may have a role as a second stimulus, triggering inflammatory signaling pathways to induce ROS-dependent cytokine production [41].

Growing evidence suggests that hyperuricemia acts as an independent risk factor for endothelial dysfunction inducing inflammatory priming due to urate exposure. Vascular lining cells are affected by increased urate levels in the peripheral blood, which generate pro-inflammatory cytokines and create a domino effect of inflammation [23]. In an ex vivo study, urate pre-treated human umbilical vein endothelial cells (HUVECs) exhibited an enhanced production of chemokines and adhesion molecules such as chemoattractant protein-1 (MCP-1), interleukin 8 (IL-8), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) via nuclear factor-kappa B signaling (NF-κB). Their role is essential for the migration of inflammatory cells in the sub-endothelium and subsequently to initiate plaque formation. This inflammatory chain elicits endothelial dysfunction in hyperuricemia-associated disorders and produces atherosclerosis [42]. Although urate is a widely recognized risk factor for gout, asymptomatic hyperuricemia seems to also contribute to a systemic inflammatory state and may be causally implicated in additional medical conditions.

As a self-limited inflammatory attack, a typical gout flare resolves in two weeks and is followed by an asymptomatic (intercritical) period. Patients with gout, whether experiencing symptomatic or asymptomatic phases of the disorder, exhibit the presence of MSU crystals within the joints [1]. Recently, the systemic inflammatory profile of gout patients in three different stages of the disease was characterized using plasma samples collected from a prospective cohort (GOUTROS) [43]. To achieve this goal, the authors used plasma samples collected at the onset of a gout flare, at the intercritical phase, and after reaching the serum urate concentration target. The main finding of this study was the identification of Tumor necrosis factor superfamily 14 (TNFSF14), also named LIGHT, as a new biomarker associated with gout flares. Moreover, TNFSF14 was identified as being produced locally in the inflamed joint through ELISA of synovial fluids of patients during a gout flare. The in vitro results also suggest that TNFSF14 could modulate the inflammatory response during a gout flare and may be considered as a therapeutic target [43]. After a gout flare, patients present an increased risk of major adverse cardiovascular events (MACE) [44]. Numerous studies have shown that TNFSF14 is also involved in atherosclerosis pathogenesis, plaque formation and rupture, and cardiovascular diseases and events [44,45,46,47].

3. Epigenetics in Gout

Epigenetic changes, which do not alter the structure of DNA, have a central role in regulating gene expression involved in inflammatory signaling pathways through key mechanisms such as DNA methylation, histone posttranslational modifications, and non-coding RNAs (small non-coding RNA and long non-coding RNA) (Figure 2) [33]. This area of research could have a major influence on understanding the immune-mediated response to urate by exploring modifications in gene expression and cellular function.

In gout, a challenging aspect in the development of flares is understanding why certain patients may experience a single acute attack while others may develop a series. It is well known that untreated gout can progress and result in worse flares, and the more elevated the urate level, the more frequent the flares will happen. While hyperuricemia is a relatively prevalent metabolic condition, only a minority of hyperuricemic individuals progress to clinical gout. In addition, the inflammatory status of individuals with hyperuricemia or gout is believed to be positively correlated with dietary intake, potentially through the alteration of epigenetic mechanisms by nutrient-driven epigenetic modifiers like short-chain fatty acids, omega-3 fatty acids, folic acid, and polyphenolic compounds [48]. Therefore, certain questions remain unanswered regarding the triggers involved in the development of gout.

In gout, innate immune cells have a central role in initiating and maintaining the inflammatory response, which is consequently followed by proteomic and epigenetic modifications, which are discussed in the next paragraphs. Innate immune cells undergo functional adaptation after being exposed to urate in in vitro conditions and determine antagonistic production of IL-1β and IL-1Ra upon LPS stimulation, with changes also observed at the transcriptional level [49]. Consequently, the epigenetic landscape of circulating and tissue-resident cells is adjusted due to persistent urate exposure to a long-lasting inflammatory state [40]. In addition, soluble urate and monosodium urate (MSU) crystals initiate the inflammatory response through various pathways, leading to epigenetic modification [7]. The interplay of genetic susceptibility and epigenetic regulation has the potential to elucidate additional factors involved in the progression from hyperuricemia to symptomatic inflammatory disease [50]. The genetic contribution in describing the link between elevated serum urate concentrations and gout progression is increasingly well assessed in genome-wide association studies (GWAS). The largest GWAS on gout conducted by Merriman et al. identified new candidate genes and pathways involved in the pathogenesis of hyperuricemia and gout, unveiling a total of 148 newly identified loci [51]. An interesting finding was the identification of the XDH locus in association with urate levels, particularly in males, which was attributed to its expression in the urothelial cells. Furthermore, this study provides new insights into epigenetic plasticity as a potential contributor to gout by revealing gout-associated loci involved in the epigenetic control of gene expression [51]. Less than 10% of the risk of developing gout can be ascribed to genetic variation, pointing to the involvement of other mechanisms in its pathogenesis [51].

Netea et al. focused on better understanding the innate immune system by investigating a specific immunological memory known as trained immunity [28,52]. The presence of innate immune memory was suggested to be a characteristic of plants and invertebrates, given their lack of an adaptative immune system [53]. However, extensive studies over the past years proposed that vertebrates, despite having adaptive immune memory, could also possess a particular type of innate immune memory [54]. This immune phenotype was initially studied in monocytes and macrophages, but other cell types such as (natural killer) NK cells, innate lymphoid cells or myeloid cells exhibit this property [52]. Trained cells have the ability to remember previous stimuli and respond robustly, a process mediated by epigenetic reprogramming and metabolic changes that occur subsequent to encounters with inflammatory stimuli [28]. BCG (Bacillus Calmette-Guérin) vaccination is a pivotal model for trained immunity mediated by histone methylation and acetylation, followed by shifting the cellular metabolism towards glycolysis, which supports the immune response [55]. Upon a second exposure, the innate immune response is enhanced independently of adaptive memory by increasing cytokine production. An intriguing aspect is how this inflammatory phenotype persists despite the short lifespan of peripheral circulating cells. The ability to sustain this behavior may be attributed to the bone marrow, where hematopoietic stem (HSCs) and progenitor cells (HSPCs) can store the memory induced by stimuli and maintain it in the differentiated cells [52]. Therefore, we hypothesize here that the process of trained immunity may also be linked to the chances of patients having multiple flares and the progressive severity of these flares. We based our hypothesis on the fact that the epigenetic changes caused by the low-grade inflammation due to the increased urate levels and the inflammatory response during the first flares lead to more responsive cells over time, resulting in a progressive increase in the inflammatory response and tissue damage. In addition to this observation, we hypothesize that myeloid cells of gout patients could undergo training, and this myeloid signature would be preserved in the differentiated cells, thereby contributing to immune activation.

3.1. DNA Methylation

DNA methylation is a mechanism involved in gene expression through the addition of a methyl group to the C-5 position of the cytosine nucleotide in the DNA sequence, usually in proximity to guanine (referred to as CpG) [56]. This mechanism has been implicated in urate-driven inflammation, and the persistent effect of urate exposure on innate immune cells is likely to be modulated by epigenetic modifications, which can facilitate an inflammatory profile [26,56].

Several targeted reports have assessed DNA methylation at interest loci in the context of gout. Zhong et al. have described a potential link between the DNMT1 rs2228611 polymorphism and the susceptibility to develop gout. This polymorphism was assessed in the Chinese Han population, resulting in changes in the activity of the DNMT1 enzyme and potentially affecting the status of DNA methylation as a result [57]. Additionally, research on the chemokine CCL2 contribution in the evolution of gout reveals a positive correlation between hypomethylation of the promoter region and the enhanced inflammatory response in gout males of Chinese Han ethnicity [58].

Through integrative analyses combining genotyping with DNA methylation, expression of the gout-associated gene NRBP1 (nuclear receptor binding protein 1) was evaluated. In vivo and in vitro studies revealed elevated expression of the NRBP1 gene in gout-associated SNP (rs780093), which was attributed to hypomethylation of the promoter. Despite not fully understanding the extent of this gene’s impact, the encoded protein has a role in modulating the expression of the ABCG2 urate transporter and is likely to be associated with gout [59]. Another protein that depends on its methylation status is uromodulin glycoprotein (UMOD), which shows a higher expression in gout patients than in the control group due to its hypermethylated status [60].

Another interesting study linked the dopaminergic system to gout by enhancing glomerular filtration and leading to increased urate excretion in conditions of homeostasis [61]. Dopamine undergoes enzymatic degradation by catechol-O-methyltransferase (COMT), which is highly prevalent in the kidneys [62]. In male gout patients, the hypomethylation of the COMT promoter is observed compared to healthy controls [61]. Consequently, this leads to a decline in urate excretion and offers an epigenetic perspective.

Forthcoming data indicate the implication of epigenetic methylation patterns of various cell lineages in the IL-1 signaling pathway, opening a new era for cell-type targeted therapies. Investigation of the monocyte-methylation profile showed a strong association of nine different CpG regions mapped to eight genes (PRKCZ, CIDEC, VDAC1, CPT1A, BIRC2, BRCA1, STK11, and NLRP12) with gouty inflammation. Furthermore, methylation of PRKCZ and STK11 genes has been correlated with the familial aggregation of gout [63].

Prior research by Wang et al. identified various differentially methylated loci (DML) in gout-affected individuals compared to a healthy control group, with analysis conducted in two separate cohorts. Twenty-two gout-risk genes overlapping the DMLs described in the study were involved in the gout phenotype via immune-modulatory responses. Genes encoding proteins involved in linking innate to specific immune systems (ILR23), in ion transport (SLC2A9, ABCC9), in modulation of inflammation via AMPK (PRKG2), and in neutrophil recruitment via the bradykinin receptor (BDKRB2) exhibited hypomethylation in individuals with gout [64]. In addition, a new meta-analysis of epigenome-wide association studies (EWAS) reveals a link between previously acknowledged genetic variants and urate-associated differentially methylated CpGs. At the site of the main serum urate transporter gene SLC2A9, which has been identified as having the most substantial impact in genome-wide association studies (GWAS), four CpGs were correlated with altered gene expression. Extensive analysis linked CpG cg11266682 with increased urate concentration, while the other three were associated with the opposite effect. Furthermore, a significant causal effect of the CpG cg03725404 at SLC2A9 was proven to link to gout [65].

3.2. Histone Modifications

Epigenetic control via posttranslational modifications of histones can modulate the accessibility of transcription factors to the DNA sequence by influencing chromatin configuration that results in reduced or boosted gene expression. Several types of histone modifications have been described so far; among them, acetylation and methylation are recognized as crucial modifications in terms of transcriptional activity regulation in gout.

Histone acetylation can be catalyzed by two main families of proteins that exert opposing effects, namely histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs enhanced gene expression through the transfer of an acetyl group to the histone tail lysine residues. This process leads to the removal of the positive charge to a negative charge on the histones, making the interaction between histones and DNA weaker and the chromatin less compatible and more accessible to the transcriptional machinery. In contrast, HDACs can repress gene expression through the removal of acetyl groups from the histone tail lysine residues [66,67].

In the context of gout, it was observed that stimulation of macrophages with MSU crystals can increase glycolysis and, consequently, the production of acetyl-CoA, an important product responsible for acetylation and deacetylation of histones [68]. The presence of acetylation of lysine 27 (H3K27ac) and trimethylation of lysine 4 (H3K4me3) in histone 3 was explored in urate-exposed monocytes [26]. The authors observed variability in twelve genes (MED24, CSF3, TAF1C, DNAAF1, HCAR2, ACO73072.5, IDO1, RP11-44 K6.2, RP11-370F5.4, RP11-44 K6.5, SNRPC, and APOE) for both histone modifications. Importantly, both histone marks were enriched in genes responsible for encoding cytokines related to crystal-mediated inflammation, in this case, IL1B and IL1A genes, responsible for encoding IL-1β and IL-1α cytokines, respectively. The opposite was observed in the gene encoding APOE (apolipoprotein E), in which H3K4me3 and H3K27ac were downregulated. This finding is relevant since APOE has been reported to coat MSU crystals and inhibit the inflammatory response [69]. Therefore, the authors suggested that the APOE locus could be involved in the progression of hyperuricemia to gout; however, to prove this hypothesis, more studies should be conducted. Of note, it was demonstrated that treatment of PBMCs with histone deacetylase inhibitors can suppress the inflammatory response after stimulation with C.16.0 (palmitic acid) and MSU [70]. This finding emphasizes the potential of using inhibitors related to histone modification to assist in the treatment of gout.

Unlike acetylation, histone methylation does not alter the electrostatic bond with DNA; instead, it can influence the recruitment and binding of different regulatory proteins to chromatin. The proteins involved in the process of methylation are histone methyltransferases (HMTs), which include lysine methyltransferases (KMTs) and arginine methyltransferases (PRMTs). HMTs can promote mono-, di- or tri-methylation of ε-amino groups of lysine residues [66]. The use of methyl transferase inhibitors, such as 5′-deoxy-5′-methylthioadenosine (MTA), has demonstrated efficiency in reducing urate-induced inflammation [35], which suggested the possibility that histone methylation-related processes play a role in gout. As discussed above, tri-methylation of histones, specifically H3K4me3, was shown to display variability at a limited number of genes in monocytes treated with urate in vitro [26]. The demethylation of histones by histone demethylases remains to be explored in gout. The enzymes involved in the process of demethylation can be classified into two groups. The first group includes lysine-specific demethylase 1-(LSD1), which removes mono- or di-methyl groups from H3K4. The second group includes Jumonji C-(JmjC) domain-containing demethylases, which remove the tri-methylated modification [71].

A limited number of studies have been conducted to investigate the effects of histone modifications in the context of gout and other crystal-related diseases. It is worth noting that there are other types of histone modifications that remain to be explored in gout, such as lactylation, phosphorylation, and ubiquitination. Among these, histone lactylation has recently been suggested as a possible mechanism in the regulation of the macrophage response to MSU [72] and variation in intracellular lactate production has been observed in MSU-stimulated macrophages in vitro [73]. Histone ubiquitination and phosphorylation can work in conjunction with other histone modifications and, therefore, impact the transcription of genes. Histone ubiquitination is involved in protein translocation, DNA damage signaling, and transcriptional regulation. Monoubiquitination of histone H3 can regulate the acetylation of the same histone [74]. Histone phosphorylation is a crucial modification associated with several cellular processes, including DNA damage repair, control of chromatin compaction, transcriptional regulation, and apoptosis. The phosphorylation of serin 10 in histone H3 promotes acetylation of the lysine 14 in the same histone [75]. None of these modifications have been explored in the context of gout, but they may play a role in the inflammatory process in conjunction with other previously reported modifications.

Furthermore, a few studies have identified epigenetic modifiers that modulate the inflammatory pathways and decrease cytokine production. For instance, romidepsin is a potent inhibitor of MSU-driven inflammation in PBMCs at the protein and transcriptional level. This compound acts as a class I histone deacetylase inhibitor (HDAC1/2 inhibitor) by increasing acetylation followed by downstream upregulation of the SOCS1 gene, which in turn limits the transcription of IL-1beta [39]. Similarly, butyrate was tested for its capacity to inhibit cytokine production in in vitro studies and in ex vivo cells from gout patients and showed a dose-dependent action by decreasing the mRNA level of IL-1beta possibly via inhibition of HDAC1, HDAC2, and/or HDAC3 [70]. All this evidence reinforces the role of histone modifications and suggests a new target for therapeutical implications.

3.3. Non-Coding RNAs (MicroRNA and Long Non-Coding RNA)

A novel area in epigenetics involves uncovering the role of long non-coding RNA (lncRNA) within cellular mechanisms. LncRNA are non-coding transcripts with an approximate length of 200 nucleotides, which can modulate gene expression at various stages by interfering with chromatin architecture and transcriptional and posttranscriptional processes [76]. Within a growing area of research, it has been highlighted that immune cells exhibit specific expression of lncRNAs, influencing the inflammatory response and, consequently, offering insights into immune-mediated diseases. This is a burgeoning field currently under exploration in connection with gout, with the potential to influence its development. Up to now, a few studies investigating gout-related lncRNA have been published, and in the present review, we aim to consolidate the findings of primary research. Emerging scientific findings have revealed novel processes based on the interaction of the 3D chromatin structure with immune genes via chromosomal looping engaging lncRNAs. The subset of lncRNAs that engage with immune genes and contribute to their epigenetic and transcriptional regulation in the context of innate immune memory are referred to as immune-gene priming lncRNAs (IPLs) [77].

In gout, inflammation is driven by IL-1β, and a very recent study has comprehensively described the chromatin-regulated circuit involving the IL1B gene. Of note, IL1B is part of the same topologically associating domain (TADs) with the IL37 gene encoding the anti-inflammatory cytokine IL-37, which has also been previously shown to play a key role in gout [78]. A recently described IPL was shown to control the antagonistic circuit of pro-inflammatory IL-1β and anti-inflammatory IL-37 and was therefore named AMANZI (A MAster Non-coding RNA antagoniZing Inflammation). AMANZI-dependent cytokine regulation was linked to the SNP variant rs16944 A genotype and was associated with increased levels of anti-inflammatory cytokine IL-37 and decreased levels of pro-inflammatory cytokine IL-1β. This observation could be relevant for understanding the innate immune memory induced by urate and open a new field of therapeutic approaches based on IPLs [79].

While prior research described AMANZI-mediated inflammation, another paper gained attention by suggesting a possible link of gout to IL-37 cytokine production. Four rare variants clustered in the functional domain of exon 5 of the IL-37 encoding gene were identified in gout patients. Among these, variant p.(C181*) induces a premature stop codon, resulting in structural and functional modifications of IL-37 protein assessed using an in vitro model of gout, which revealed diminished anti-inflammatory action. In addition, all the variants were associated with early onset and severe forms of gout in the study cohort [78]. By integrating AMANZI lncRNA and IL-37 variants, new perspectives on the importance of the anti-inflammatory cytokine IL-37 in the pathogenesis of gout are underscored.

Another study demonstrated differential expression of lncRNA in PBMCs isolated from acute gout patients, intercritical gout patients, and healthy controls. Notably, three lncRNAs (TCONS_00004393, NR_029386, and ENST00000566457) exhibited significant expression in acute gout samples compared to controls, and subsequently, TCONS_00004393 and ENST00000566457 were suggested as potential biomarkers in identifying gout [80]. Interestingly, TCONS_00004393 maps to the IL1A-IL1B locus on chromosome 2, making it interesting to study in the context of gene interactions at this locus or at the TAD involving ILB. Emerging data indicate that lncRNAs play a role in macrophage-mediated inflammatory responses and cytokine expression associated with gout. A change in the polarization status of macrophages to an anti-inflammatory profile was associated with lncRNA-MM2P [81]. The expression of lncRNA-MM2P was downregulated after exposure to MSU and LPS, and cytokine secretion (IL-1β, TNF, IL8) was increased. After transfection with a vector carrying the gene for this long non-coding RNA, the opposite effect was noticed by overexpression of lncRNA-MM2P and diminished cytokine production, therefore identifying lncRNA-MM2P as a potential regulatory player in gout inflammation [81].

In addition to lncRNA, small non-coding RNAs represent another category of transcripts that do not encode proteins but have the potential to modulate inflammation, of which microRNAs(miRNAs) have been recently studied. MiRNAs can be aberrantly expressed in inflammatory pathologies by interfering with mRNA and can be associated with the development of various diseases, including gout [82,83,84]. The molecular mechanism of miRNAs involved in gout targets inflammatory signaling pathways, consequently dysregulating the immune response. From an inflammatory perspective, upregulation of miRNA302-b in THP-1 cells and mouse tissues can suppress IL-1beta expression at both transcriptional and protein levels after MSU crystal exposure by directly targeting interleukin-1 receptor-associated kinase 4 (IRAK4) and Eph receptor A2 (EphA2). In addition, serum measurements of miRNA302-b showed enhanced expression in gouty arthritis patients compared to controls [85]. Furthermore, to address the regulatory role of miRNAs on cytokine production, W. Zhou et al. investigated the 3′-untranslated region (UTR) of the IL-1beta gene and proved that the upregulation of miR-488 and miR-920 leads to transcriptional repression of IL-1beta in MSU-stimulated THP1 cells [86]. Moreover, miRNA 221-5p was found to be downregulated in acute gout patients, and its overexpression in THP-1 cells has been shown to inhibit cytokine release, particularly affecting IL-1beta production. Another report proposes the involvement of miR302F by modulating the inflammation from gout through the regulation of gene expression, but further studies are required [87]. In new findings, miR-20 emerged as undergoing regulation in synovial fluid mononuclear cells (SFMCs) from patients with gouty arthritis and in THP-1 cells pre-exposed to MSU crystals. The increased expression of miR-20 is associated with the reduction of NLRP3 activity, leading to decreased inflammation. In patients with gout, the HOX transcript antisense RNA (HOTAIR) has a negative regulatory effect on miR-20, NLRP3 expression, and cytokine production, progressing toward a pro-inflammatory state via epigenetic regulation [88].

4. Conclusions

Hyperuricemia is a common metabolic condition with a rapidly increasing incidence worldwide, especially in developed countries, emphasizing the need to understand its pathophysiology and pathologic consequences. In this review, we summarize recent findings supporting the hypothesis of epigenomic reprogramming in urate-induced inflammation and gout. Although the local effect of MSU crystals is well established, our understanding of the role of elevated serum urate concentrations in systemic circulation remains limited. Hyperuricemia has the potential to prime the innate immune cells and induce epigenetic changes that initiate, maintain, and enhance the inflammatory response after restimulation. Persistent exposure of immune cells to soluble urate could trigger a state of low-grade chronic inflammation, which may predispose individuals to develop other inflammatory-related diseases. Soluble urate may be considered a silent agent that alters the epigenetic landscape and shifts the immune profile towards a more pro-inflammatory state. The studies discussed above highlight a new intriguing area of research focused on DNA methylation, histone modifications, and lncRNA in the context of gout and urate-induced inflammation, which can provide new insights into addressing this condition. Additional research is required to better describe the interrelation between urate exposure and the maladaptive response of the immune system mediated by epigenetic imprinting.