Asymptomatic Hyperuricemia – A silent activator of the innate immune system

Abstract

Asymptomatic hyperuricemia affects approximately 20 percent of the general population in the USA, with variable rates in other countries. Historically asymptomatic hyperuricemia was considered a benign laboratory abnormality with little clinical significance in the absence of gout or kidney stones. Yet, there is increasing evidence that asymptomatic hyperuricemia can predict the development of hypertension, obesity, diabetes mellitus and chronic kidney disease. One potential mechanism by which asymptomatic hyperuricemia may contribute to disease is by stimulating inflammation. While urate has been classically viewed as an anti-oxidant with beneficial effects, more recent studies suggest both crystalline and soluble urate may activate various inflammatory pathways. Herein we review the role of urate in the inflammatory response. Further research is needed to define the role of asymptomatic hyperuricemia in these proinflammatory pathways.

Introduction

Hyperuricemia is classically defined as a serum urate of more than 7.0 mg/dL in men or more than 6.0 mg/dL in women. Today approximately 20 percent of the US population have hyperuricemia.¹ The well accepted consequence of hyperuricemia is an increased risk for gout and kidney stones, and currently treatment of asymptomatic hyperuricemia is recommended only in people with these established conditions.² Yet, people with asymptomatic hyperuricemia are also at risk for developing a variety of other conditions, including hypertension, acute and chronic kidney disease, obesity, metabolic syndrome, fatty liver and diabetes mellitus.^3,4 While it was often viewed that the hyperuricemia develops secondary to these conditions, most studies show that the hyperuricemia precedes these diseases^5–7. Indeed, a recent study in the Japanese population showed that the presence of hyperuricemia in healthy, lean, normotensive people still carries an increased risk of cardiometabolic disease⁸ and the treatment of asymptomatic hyperuricemia above 8 mg/dL is recommended in Japan.⁹ Given these findings, we herein review studies examining the role of uric acid in driving inflammation.

Throughout this paper, we use the generic term “uric acid” for the end-product of purine catabolism in humans and to describe data from experimental models in which exogenous uric acid was dissolved and administered in order to study inflammatory effects. Knowing that uric acid is predominantly found in ionized form in the circulation, we use the term “urate” when referring to circulating plasma or serum levels¹⁰ and discuss inflammatory effects of both crystalline and soluble uric acid. In line with the consensus gout labels¹¹, hyperuricemia represents the elevated circulating levels of urate of 7 mg/dl in men and 6 mg/dl in women. Asymptomatic hyperuricemia is defined as elevated urate levels in absence of episodes of inflammation triggered by urate crystals (gout flares).

Uric acid and Susceptibility to Hyperuricemia and Gout in Humans

Uric acid is a purine degradation product generated from the breakdown of nucleic acids, purine-containing compounds such as adenosine triphosphate (ATP), and guanine and adenine. Uric acid can also be generated from ribose phosphate by a series of enzymatic reactions. In most mammals, circulating urate (the form in the blood) is relatively low (1-3 mg/dl) due to the presence of urate oxidase (uricase), an enzyme in the liver (or occasionally the kidney) that degrades uric acid to 5-hydroxyisourate, and eventually allantoin (Figure 1).¹² However, a number of species lack functional uricase, including birds and most reptiles. In these species, serum urate is higher, and the excretion of uric acid is the primary means for eliminating excess nitrogen. In fact, it has been proposed that the loss of functional uricase in these species provided an evolutionary benefit for living in terrestrial versus marine environments, as the uric acid could be precipitated in the cloaca and excreted with the minimal loss of water.¹³

Figure 1. Regulation of serum urate.

Following sequential mutations in the uricase gene, humans and higher primates do not express functional uricase, needed to metabolize uric acid to allantoin. Serum urate can interact with reactive species generating substances or radicals such as allantoin, 6-aminouracil or triuret. However, most of the serum urate will be excreted via the kidney or, alternatively, via the gut. Both proximal tubule and intestinal cells express several urate transporters (collectively known as the transportasome) that are responsible for the excretion and/or reabsorption of urate. Several main urate transporters are illustrated: URAT1, GLUT9, OAT4 and OAT10 are involved in urate reabsorption at the level of the apical membrane (brush border) of proximal renal tubular cells; GLUT9 is responsible for urate transport out of the cell via the basolateral membrane into the blood; ABCG2 is a unidirectional transporter mediating the secretion of urate via the apical membrane; OAT1 and OAT3 localized on the basolateral membrane are involved in urate excretion. OAT, organic anion transporter; URAT1, urate transporter 1; GLUT9, glucose transporter 9; ABCG2, ATP-binding cassette super-family G member 2.

Uricase is also absent in hominoids due to a series of mutations that affected the great apes and humans, as well as a parallel mutation that eliminated uricase in the lesser apes (gibbons).^14,15 The parallel loss of uricase during the mid-Miocene strongly suggests that there was a natural selection advantage at that time. Moreover, the gene encoding urate transporter URAT1 was found to have coevolved with the sequential reduction of uricase enzymatic activity, enhancing the URAT1 affinity in primates and allowing for better control of higher serum urate levels.¹⁶

The observation that all mammals including humans excrete most of their nitrogen as urea suggests different benefits for why humans and apes lost uricase. Several hypotheses have been proposed. First, uric acid has a chemical structure similar to caffeine, and it has been posited that uric acid may have effects on mental performance, drive, response time, and impulsivity.^17–19 Second, uric acid can function as an anti-oxidant, and it has been hypothesized that its anti-oxidant effects may have been of benefit by blocking the oxidative stress associated with aging and cancer.²⁰ Indeed, it has been proposed that the mutation may have countered the loss of antioxidant activity associated with the mutation in L-gluconolactone oxidase that resulted in the inability to synthesize vitamin C.²¹ Finally, there is emerging evidence that the loss of uricase may have had survival benefits by its ability to help maintain blood pressure, stimulate fat and glycogen stores, and stimulate gluconeogenesis.^14,22–24 It is also possible that the benefit of uricase may involve some aspect of each of these proposed mechanisms.

Regulation of Serum Urate in the Absence of Uricase

In the absence of uricase, the kidney and the gut became the primary means for removing uric acid. Whereas some urate can be degraded by oxidants or nitric oxide (generating allantoin, triuret and 6-aminouracil)^25,26, most urate (two-thirds) is excreted by the kidneys, largely by a series of transporters in the proximal tubules, of which SLC2A9, URAT1, and ABCG2 are especially important (Figure 1). Some urate (one-third) also passes into the gut via SLC2A9 and ABCG2 transporters where the uric acid is degraded by uricolytic bacteria. These excretory pathways are fairly effective, and in people who eat native (nonwestern) diets, serum urate tends to be low, classically in the 3 to 5 mg/dl range.²⁷

Causes of Hyperuricemia

While the absence of uricase only results in a slight increase in serum urate (of 1-2 mg/dl), additional genetic variants in genes encoding urate transporters or enzymes involved in uric acid metabolism have been shown to modify serum urate levels, either through small effects when considering common variants^28–30, or through damaging effects in the case of rare alleles³¹. Environmental factors can also contribute to hyperuricemia as subjects on a western diet tend to have significantly higher serum urate concentrations.^32,33 This is partly driven by diets high in sugar (which contains fructose), purines (especially from animal proteins), and alcohol (especially beer).^33,34 However, there is also reduced fractional excretion of urate in people with obesity, insulin resistance and hypertension ³⁵, and there are also drugs that can impair urate excretion, especially the thiazide and loop diuretics. Hyperuricemia is also common as kidney function fails, and at the time of dialysis initiation approximately half of patients demonstrate hyperuricemia.^36,37

Clinical Consequences of Hyperuricemia

One of the consequences of higher serum urate is the risk for gout, urate kidney stones, and acute kidney injury from urate crystalluria. The prevalence of gout in the US approaches 6 percent in men and 2 percent in women.¹ with a worldwide range of 0.1 to 10% and increasing trend in developed countries³⁸. People with acute gout present with severe joint pain due to the deposition of urate crystals in the synovium associated with a marked inflammatory response.^33,39 Kidney stones from uric acid are also common in people with gout and hyperuricemia^40,41 , and even calcium stones may occur in which the initial nidus is from urate crystal deposition⁴².

Another risk resulting from the uricase mutation is that the renal excretory system is not adapted for large increases in urate excretion that can occur when there is sudden production, such as following the release of DNA and RNA from tissue injury or tumor lysis.⁴³ For example, marked uricosuria with crystal formation, intratubular obstruction and renal inflammation may result when there is a sudden rise in urinary uric acid from rapid tumor death following chemotherapy.⁴⁴ Urate concentrations may also increase in the setting of dehydration and low urine volumes, leading to elevated concentrations that can result in urate crystal formation.⁴⁵ High urinary urate concentrations have also been associated with tubular injury and activation in experimental models even in the absence of documented crystalluria.^46–48

More recently the potential role uric acid may play with chronic inflammatory disorders has been comprehensively investigated, particularly the association of uric acid with obesity, hypertension, metabolic syndrome, diabetes mellitus, kidney disease and cardiovascular disease.⁴ The literature remains inconsistent, for, while epidemiological studies are generally positive, genetic Mendelian randomization studies, that are supposed to better predict whether causality is present, are often negative^49–51. However, a problem with the Mendelian randomization studies is that they often of rely on single nucleotide polymorphisms (SNPs) that modulate urate transport as opposed to urate synthesis, and it is not known how these polymorphisms affect intracellular urate levels that are thought to drive the biological effects⁵². Moreover, only very recently the functional consequence of SNPs have been taken into account to the importance of a certain SNP in inflammation.^53,54 In addition, while pilot studies with urate lowering therapies often suggest benefit, the trials usually enroll a limited numbers participants, are often nonblinded and the beneficial effects are typically modest, while other studies are negative.⁴ Nevertheless, meta-analyses have largely supported a potential benefit of urate lowering in subjects with hypertension and/or chronic kidney disease ^55,56.

At the heart of the controversy is the question of whether a substance that is thought to be an antioxidant can actually be prooxidative and proinflammatory. In the next sections we investigate potential proinflammatory effects of both soluble and crystalline uric acid.

Urate as a Mechanism for Inducing an Inflammatory Response

Several studies have been performed to better understand potential mechanisms whereby urate may induce a proinflammatory response. Below we discuss several proposed mechanisms, including inflammation driven by urate crystals (including both inflammasome-independent and dependent mechanisms) as well as mechanisms mediated by soluble urate.

Urate Crystal-Dependent Inflammation.

Classically, monosodium urate crystals (MSU) activate the innate immune response by triggering inflammasome pathways⁵⁷ that result in the release of interleukin-1β (IL-1β) (Figure 2). Inflammasome-independent mechanisms are also possible contributors to urate crystal induction of cytokine secretion (Figure 3).

Figure 2. Inflammasome-dependent activation of IL-1β in response to MSU crystals.

IL-1β production is initiated by exogenous or endogenous stimuli (PAMPs or DAMPs) which use the PRR machinery to induce an inflammatory response. Engagement of PRR ligands to the receptors leads to the transduction of signal to the nucleus and transcription of the inactive pro-IL-1β precursor. In the intracellular space, pro-IL-1β is proteolytically activated by specific cleavage performed by caspase-1. Caspase-1 is, in turn, present in the cytosol in a precursor form (procaspase-1). MSU crystals act as inducers of the protein platform that cleaves procaspase-1 to caspase-1, thus mediating the activation and release of biologically active IL-1β. In the monocytes (left), caspase-1 is constitutively active, thus MSU crystals potentiate IL-1β maturation. MSU crystals also upregulate mTOR gene transcription, leading to enhanced gene transcription of IL-1β and other inflammatory cytokines, and drive monocyte cell death that could, indirectly, further stimulate the inflammasome. In the macrophages (right), caspase-1 is not constitutively active, MSU crystals drive changes inside the cell that result in the assembly of the inflammasome components and caspase-1 generation. MSU crystals can do this in multiple ways: (1) induce mitochondrial ASC recruitment to the site of inflammasome assembly in a microtubule dependent manner, (2) induce mitochondrial dysfunction and ROS generation, (3) lysosomal dysfunction with impairment of autophagy, increased levels of p62 and ROS generation, (4) AMPK inhibition in an NLRP3 dependent manner. PAMP, Pathogen Associated Molecular Patterns; DAMP, Damage Associated Molecular Patterns; PRR, pattern recognition receptor; ASC, Apoptosis-Associated Speck-Like Protein Containing CARD; ROS, reactive oxygen species; AMPK, AMP-activated protein kinase.

Figure 3. Inflammasome-independent regulation of inflammation in response to MSU crystals.

MSU crystal deposition leads to cell death and cellular infiltrate, mostly characterized by neutrophil recruitment at the site of crystal deposition, especially in the affected joints of patients with gout. Dying cells release intracellular molecules (DAMPs, alarmins) in the extracellular space, including precursors of the IL-1 cytokines. Pro-IL-1β is inactive while pro-IL-1α already has biological activity in its precursor form, therefore is able to mediate inflammatory responses in the surrounding tissue. Infiltrating cells, the majority of which are neutrophils, contain and release proteolytic enzymes (such as PR3 or NE, among others) that have the potential to cleave IL-1 precursors at alternative cleavage sites and activate IL-1β or enhance the bioactivity of IL-1α. This mechanism of protease induced activation of precursor cytokines can be limited by anti-proteases such as AAT. Conversely, neutrophils can undergo NETosis (neutrophil extracellular trap formation), which consists in the externalization of chromatin and binding of intercellular molecules. This phenomenon is considered to play an important role in the resolution of inflammation in gout: NETs can bind active cytokines or chemokines and facilitate their enzymatic digestion contributing to the limitation of inflammation; moreover, NETosis can contribute to the sequestration of MSU crystals and encapsulation of crystals within the tophus; neutrophil microvesicles (PMN-Ecto) can downregulate inflammatory pathways in paracrine manner, limiting the magnitude of the cytokine production during the acute phase by inhibiting NLRP3 or activating SOCS3. DAMP, Damage Associated Molecular Patterns; PR3, Proteinase 3; NE, Neutrophil Elastase, NLRP3, NACHT, LRR and PYD domains-containing protein 3; SOCS3, Supressor of Cytokine Signalling 3.

Inflammasome activation.

IL-1 is a potent proinflammatory cytokine, with a tightly regulated production mechanism and low circulating levels. There are two major IL-1 proteins: IL-1α and IL-1β. Whereas IL-1α is present primarily intracellularly and membrane bound, IL-1β can be secreted by stimulated cells ^58,59. This secretion of IL-1β is differentially regulated in myeloid cells: in macrophages it requires a two-step activation, whereas in monocytes it can be directly processed into its bioactive form ⁶⁰ (Figure 2). IL-1β is translated as a precursor protein (pro-IL-1 β) and needs to be activated through protein cleavage by caspase-1. Caspase-1, in turn, needs to be activated in protein platforms called inflammasomes⁶¹.

The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome⁵⁷ and subsequent procaspase-1 cleavage is the most commonly known mechanism to complete the activation and release of active IL-1β upon interaction with MSU. The mechanism of MSU activation of the NLRP3 inflammasome was first described in THP1 cells⁶². Furthermore, MSU crystals have been shown to contribute to NLRP3 inflammasome formation through a mechanism dependent on tubulin polymerization and mitochondrial ASC recruitment at the site of inflammasome component colocalization ⁶³. Interestingly, however, in an in vivo murine model of gouty arthritis induced by intra-articular injection of a combination of MSU crystals and stearate fatty acids, NLRP3 involvement was not observed, despite the inflammatory reaction being dependent on ASC and caspase-1⁶⁴. This suggests (as will be discussed below) that the in vivo effect of NLRP3 can be bypassed or overridden by other proinflammatory systems⁶⁴.

As summarized in Figure 2, the inflammasome protein platform also shows interactions with organelle clearance and autophagy. MSU was found to damage lysosomal integrity and to impair autophagy, leading to p62 accumulation. Autophagy related p62 excess led to Nrf2 activation and translocation to the nucleus, where Nrf2 induced transcription of heme oxygenase-1 and superoxide dismutase⁶⁵, contributing to altered intracellular redox status. In line with this, another report linked p62 expression to enhanced ROS production and, subsequent caspase-dependent apoptosis and IL-1β production⁶⁶. Moreover, MSU dependent IL-1β and CXCL1 production was shown to be mediated by inhibition of AMP-activated protein kinase (AMPK) in a mechanism dependent on both NFκB and NLRP3⁶⁷. These findings suggest that the intracellular homeostasis is disrupted at several different networks by the sterile MSU crystals (autophagy dysfunction, diminished clearance of damaged organelles such as mitochondria, in addition to oxidative stress and, at least in part, AMPK inhibition) culminating in the activation of the inflammasomes (Figure 2, right). A recent study performed in monocytes also showed that MSU crystals lead to inflammatory cytokine production through an mTOR dependent mechanism involving pyroptosis which could further, indirectly, lead to inflammasome activation⁶⁸ (Figure 2, left).

Inflammasome-independent urate crystal induced inflammation.

While inflammasome-dependent maturation and secretion of IL-1β is paramount in monocyte and macrophage mediated innate immune responses, inflammatory diseases which are characterized by polymorphonuclear leukocyte (neutrophil) infiltrates are commonly independent of inflammasome activation⁶⁹. In gout, the inflammatory reaction has been shown to be mediated by inflammasome activation in response to MSU, although this is likely to be short-lived due to the subsequent clinical picture determined by rampant neutrophil infiltrate. While neutrophils are capable of transcribing IL-1β ⁷⁰, an even greater inflammatory process is mediated by intracellular serine-proteases (such as neutrophil elastase (NE), cathepsin G (CG), proteinase 3 (PR3)) which are released as a consequence of cell influx, MSU mediated damage and cell death. Neutrophil-derived serine proteases, such as proteinase-3 (PR3) and elastase (NE) can process the inactive IL-1β precursor at alternative proteolytic cleavage sites, leading to bio-active IL-1β ⁷¹. Moreover, due to cell damage, IL-1α is also released from dying cells in the extracellular space and functions as an alarmin (Figure 3). ^58,59. This can explain why in many in vivo scenarios, including but not restricted to gout, the inflammatory outcomes remain IL-1 driven, while the inflammasome involvement is limited or bypassed^64,69. In line with this, a promising therapeutic option for gout and MSU dependent inflammation is represented by alpha-1-antytripsin (AAT), the natural plasma inhibitor of serine-proteases, which has been shown to limit inflammation through upregulation of IL-1 receptor antagonist (IL-1Ra) and through inhibition of PR3 dependent IL-1 activation⁷² (Figure 3).

Conversely, neutrophils have been shown to be involved in the spontaneous resolution of acute attacks of gout. Neutrophil-derived micro vesicles (PMN-Ecto) have been reported to be released in the peritoneum of mice injected with MSU and to downregulate IL-1β production in response to complement 5a via upregulation of SOCS3⁷³. Moreover, neutrophils can undergo oxidative burst and form extracellular traps (NETs) which contribute to resolution of inflammation through several mechanisms. These include the immobilization of MSU crystals and proteolytic degradation of cytokines or chemokines^74,75, which are protected from the activity of antiproteases while associated with aggregated NETs ⁷⁶. Nevertheless, other proinflammatory cell types are likely to be entrapped in the NETs, contributing to the variability of the anti-inflammatory outcomes in NETosis^77,78. Consequently, NETs are presumed to contribute to the local tolerance towards the remaining urate crystals, as aggregated NETs and subsequent granuloma formation drive the development of tophaceous deposits and tissue remodeling^79,80(Figure 3).

Soluble Urate Inflammatory Pathways.

While early studies focused on the effects of crystalline uric acid on inflammatory cell activation, more recent studies show that soluble uric acid has many proinflammatory and prooxidative effects in the intracellular environment. Some of these pathways are discussed below, including oxidative stress, inflammatory signaling, autophagy and intracellular immunometabolic sensors (Figure 4).

Figure 4. Intracellular pathways involved in proinflammatory effects of soluble uric acid.

Soluble uric acid has been shown to modulate cytokine production and inflammatory outcomes via several pathways. Uric acid enhances AKT phosphorylation, leading to PRAS40 phosphorylation, and resulting in autophagy inhibition; this is in line with downregulation of expression in genes involved in FOXO pathway. Uric acid was shown to inhibit AMPK phosphorylation, which is consistent with mTOR activation. Several transcription factors can be induced by uric acid signaling (NFκB or AP-1) following p38 and ERK MAPK activation, leading to enhanced transcription of innate cytokines in addition to the remarkable downregulation of natural IL-1 antagonist, IL-1Ra. In vascular endothelial cells, smooth muscle cells or adipocytes, enhanced transcription of vasoactive substances is known to lead to elevated angiotensin II, MCP-1, endothelin and low availability of nitric oxide which can mediate hypertension. In vascular cells, growth factors such as PDGF are upregulated, leading to smooth muscle cells proliferation and promotion of atherosclerosis. Soluble uric acid has also been shown to induce oxidative stress, alter the redox status of the cell which, on the one hand, activates signaling pathways for gene transcription and, on the other hand, lead to ASC speck formation and inflammasome activation, thus contributing to cleavage of pro-IL-1β. The transcriptional rewiring of uric acid stimulated cells is reminiscent of innate immune memory developed in response to endogenous sterile stimuli, leading to a scenario in which soluble uric acid acts as a silent stimulus that drives epigenetic reprogramming in circulating or tissue resident cells, thereby causing persistent inflammatory effects in response to continuous exposure to uric acid. We propose a model in which cells exposed to high uric acid levels are more prone to develop a strong proinflammatory response when exposed to specific triggers due to innate immune memory, epigenetic changes in exposed cells or persistence of exposure to high uric acid levels.

Oxidative Stress.

Uric acid is an antioxidant that can react with superoxide anion to generate allantoin, peroxynitrite to form triuret, and nitric oxide to form 6-aminouracil^25,81. However, the reactions of uric acid with peroxynitrite will also generate the aminocarbonyl radical and the triuretcarbonyl radical.²⁶ Uric acid will also form radicals when reacted with myeloperoxidase.⁸² Moreover, inside the cell uric acid is prooxidative, and this has been shown in vascular smooth muscle cells, endothelial cells, adipocytes, hepatocytes, pancreatic islet cells and renal tubular cells.^83–89 The mechanism involves activation of NADPH oxidase, and in some cell types the NADPH oxidase may translocate to the mitochondria.^86,89,90 Interestingly, the effect of soluble urate on mononuclear cells is more complex, as priming of PBMCs with urate results in enhanced IL-1β release in response to LPS, but lesser ROS release⁹¹ while another study found no statistically significant effects on IL-1β, SOD2 gene transcription nor total antioxidant capacity of the cell⁹².

Inflammatory Signaling.

Soluble uric acid has also been found to activate a series of mitogen activated protein kinases (MAP kinases), including p38 and ERK, leading to activation of transcription factors such as NF-κB and AP-1.^85,93,94 This is associated with the production of vasoconstrictive substances (including (pro)renin receptor⁹⁵, endothelin, thromboxane and angiotensin II), a loss of vasodilatory substances (such as nitric oxide), an increase in growth factors (platelet-derived growth factor), and an increase in chemokines (such as monocyte chemoattractant protein-1, MCP-1).^{84,93,94,96,97} Additionally, reports from experimental studies have provided evidence that elevated uric acid concentrations have proinflammatory effects in various tissues and organs: neutropenic mice have a higher cytokine production upon LPS challenge compared to control animals due to hyperuricemia ⁹⁸; NFκB was shown to be activated in mice injected intraperitoneally with uric acid in renal or pancreatic cells ^47,99; uric acid was revealed to induce NFκB activation in hypothalamic cells and promote inflammation and gliosis with possible consequences of altering the hypothalamic functions in metabolic regulation ¹⁰⁰.

Autophagy and inflammasome.

The interplay between inflammasome and autophagy in the post-translational processing and release of IL-1β is also implicated in soluble urate induced effects, in addition to the most widely studied MSU induced inflammation. In vitro data using primary human mononuclear cells revealed a shift in IL-1β and IL-1Ra production at uric acid concentrations ranging from 12.5 to 50 mg/dL¹⁰¹. RNA-sequencing (RNAseq) data in highly pure human monocytes treated for 24h with medium or uric acid confirmed effects such as IL-1β or IL-6 upregulation and IL-1Ra decrease in both RNA and protein levels. Pathway enrichment analysis suggested involvement of the AKT pathway involving mTOR (upregulated genes) and FoxO transcription factors (downregulated genes). AKT phosphorylation was enhanced and PRAS40 phosphorylation was also induced in the presence of uric acid. PRAS40 is a molecule that exerts inhibitory roles on mTOR in non-phosphorylated state: once phosphorylated, PRAS40 dissociates from the Raptor protein, releasing the inhibitory signal on mTOR ¹⁰². This coincided with inhibition of autophagy in uric acid primed cells⁹¹, in line with mTOR signaling acting as an autophagy inhibition mechanism ¹⁰³. In line with these data, a recent report described a significant positive correlation between mTOR gene expression circulating serum urate levels in patients with gout⁶⁸.

Another report in bone marrow derived macrophages in an in vivo model of obstructive nephropathy in mice, showed that soluble urate (180 μM), released in hypoxic conditions, leads to mitochondrial ROS production, ASC speck formation and caspase-1 activation, thereby inducing IL1-β production in an NLRP3 and MyD88 dependent manner¹⁰⁴. Soluble uric acid has also been shown to induce ROS in mitochondria from both endothelial cells and hepatocytes^86,90. The finding that basal autophagy levels are diminished, is reminiscent of the enhanced cytokine production observed in situations where autophagy was inhibited, therefore showing that autophagy could be a possible new source for intervention in gout. While, based on previously cited reports, autophagy defects are a known cause for IL-1β overproduction in both inflammasome dependent and inflammasome independent manners, it is still not completely understood how IL-1Ra is uncoupled from IL-1β. Pharmacological inhibition of autophagy can diminish IL-1Ra levels but further studies are necessary to show whether these two effects are mechanistically related ¹⁰⁵. Moreover, since often the concentrations of uric acid surpass in vivo conditions, new studies are required to validate these cytokine profiles and to assess the relevance in clinical situations.

Intracellular immunometabolic sensors.

Immunometabolic sensors are increasingly well characterized in disorders associating inflammation, as intracellular metabolites, energy consumption or production are shown to have an interplay with cytokine biology. In this system, AMPK senses variation in AMP to ATP or ADP to ATP ratios and promotes catabolism¹⁰⁶. The downstream phosphorylation of substrates induced by AMPK can, however, also impact on inflammatory pathways, such as mTOR signaling or autophagy, therefore AMPK is considered a master regulator of cytokine production in relation to the cellular energy status¹⁰⁷. In line with findings above, AMPK signaling was studied in renal and pancreatic cells and was shown to be inhibited by elevated uric acid levels through ROS and mTOR induction¹⁰⁸. Furthermore, the effect of uric acid to inhibit AMPK has also been shown in the liver where it is thought to have a role in driving gluconeogenesis, stimulating intrahepatic fat accumulation, and causing insulin resistance^24,109.

This effect of AMPK inhibition by urate has also been reported in THP1 human monocytic cell line, which also translated to higher CD68 expression, foam cell formation in response to oxidized LDL cholesterol and activation of NFκB¹¹⁰. In contrast, human primary monocytes were not found to show elevated ROS production, nor was AMPK phosphorylation significantly modified in response to uric acid ⁹¹.

In line with AMPK inhibition by uric acid, this effect could be reversed in pancreatic cells by zurampic (inhibitor of urate transporter URAT1)¹¹¹. Moreover, the oral antidiabetic drug metformin, a known AMPK inducer, rescued the reduced glucose uptake and uric acid induced insulin resistance in muscle cells¹¹² and liver cells¹⁰⁹. These results indicate that AMPK is a valid target in gout and hyperuricemia as well. While monocyte data is conflicting, AMPK inhibition is consistent across tissues which are affected by hyperuricemia-associated diseases, such as the kidney, pancreas, muscle and liver.

Innate immune memory in hyperuricemia and functional consequences

The innate branch of the immune system possesses an epigenetically encoded memory of previous insults¹¹³. The hypothesis that the immunological memory is not restricted to the adaptive branch of the immune system, was originally introduced in 2011 under the name “trained immunity” ¹¹³ and was based on a large body of previous evidence. Trained immunity is now a widely studied process that is based on the epigenetic reprogramming of myeloid or lymphoid cells involved in innate immunity. The general concept behind trained innate immunity refers to the fact that an initial exposure of cells to a stimulus can promote changes in the transcriptional program of the cell, which lead to elevated inflammatory responses in subsequent stimulations. Trained immunity has been mechanistically deciphered for several pathogenic stimuli and has relevance for susceptibility to infection, immunological tolerance or vaccine biology^114–116. In addition, sterile stimuli, acting via the same signaling machinery as pathogens, could also induce innate immune memory¹¹⁷. The long-term effects of sterile stressors could be of great importance for a wide range of inflammatory diseases. For example the involvement of epigenetic memory is already well known and extensively studied through the concept of “glycemic memory” in diabetes mellitus ¹¹⁸. Moreover, trained immunity has been shown to be a mechanism that promotes atherosclerosis, in experimental designs using oxidized LDL or Lipoprotein(a) oxidized phospholipids^119,120, with epigenetic changes that are persistent in vivo in patients with familial hypercholesterolemia, despite the pharmacological normalization of lipid levels¹²¹.

Studies in patients with hyperuricemia have shown that elevated serum urate levels are associated with inflammatory markers such as white blood cells, C reactive protein and circulating innate immune cytokines such as IL-6, IL-1Ra, TNF or IL-18¹²². CD14+ monocyte counts have been reported to be elevated in hyperuricemic individuals, who displayed higher plasma CCL2 levels and higher expression of adhesion molecule CD11b¹²³. These data indicate that innate immune inflammatory pathways, especially monocyte or macrophage dependent, are upregulated in presence of hyperuricemia. One report on asymptomatic hyperuricemia in male individuals described an inverse correlation between serum urate levels and Natural Killer cell counts¹²⁴ showing that lymphoid cells could also be associated to changes in urate levels. However, the functional consequences are yet unexplored and further studies on different lymphoid populations are needed.

Having described evidence linking soluble urate inflammatory mechanisms to similar pathways that drive innate immune memory and knowing that hyperuricemia is associated to inflammatory markers, we propose that stimuli such as uric acid or MSU crystals could drive a memory of the innate immune system. Similar to other autoinflammatory conditions, gout patients exhibit a higher cytokine production profile in response to ex vivo stimulations ^92,101,125. One study showed that the ex vivo cytokine production was associated to serum urate concentrations in gout patients¹⁰¹, while another study showed a statistically non-significant correlation between serum urate levels and cytokine production capacity⁹². In vitro pre-exposure to soluble uric acid of peripheral blood mononuclear cells or enriched monocytes potentiates proinflammatory cytokine secretion to a subsequent stimulus in a way that is reversible by broad methylation inhibitors¹⁰¹. In vivo intravenous administration of uric acid in healthy human volunteers was consistent with higher levels of plasma IL-6 after acute lipid ingestion, whereas potently lowering of serum urate with rasburicase did not have a significant effect on IL-6 plasma values after lipid oral tolerance test¹²⁶. These observations enforce the finding that soluble uric acid works as a silent modulator on monocytes, that, alone, has no remarkable effects on the inflammatory cytokine production in vitro or in vivo but when subsequently challenged, culture supernatants or plasma of healthy volunteers depict elevated proinflammatory cytokines. In line with this hypothesis, a case-control study in gout patients with more than two flares in 12 months versus patients with less than two flares in 12 months found that serum urate values are associated with frequent gout attacks¹²⁷.

These studies document that both crystalline and soluble uric acid may have a key function in triggering the immune system to a pro-inflammatory pathway that may result in epigenetic imprinting and can facilitate inflammatory reactions in response to subsequent insults. While this is nowadays a concern in the context of chronic inflammatory diseases, there is some data that uric acid may have a protective role in the host response to infections, such as malaria^128,129. It is possible that the uricase mutation may have also been a mechanism for protecting against infections in the past ^23,130, nevertheless a recent population study failed to show a negative association between hyperuricemia and community acquired pneumonia, urinary tract infection or infection-related mortality ¹³¹.

Hyperuricemia and chronic inflammation

Gout.

Despite general knowledge that the progression from soluble uric acid to MSU crystals and generation of inflammation is the causal mechanism of gout, some questions remain open in relation to the clinical observations related to gout and hyperuricemia. First, hyperuricemia is a relatively common metabolic condition, yet only a minority of 10-15% of hyperuricemia patients developing symptomatic gout ¹³². Thus, the factors that trigger the symptomatic inflammatory response still need to be elucidated. Second, when an attack resolves, crystals still remain in the tissues (intercritical gout), that might be contributing to a low grade systemic inflammatory response in the absence of symptoms ¹³³. In addition, as many as 15 or more percent of subjects with hyperuricemia have crystal deposition without symptoms ^134,135. Thus, neither hyperuricemia nor MSU deposits are sufficient to trigger full blown inflammation. Moreover, experimental designs usually cannot exclude that in vivo or in vitro effects of high concentrations of uric acid are independent of microcrystal formation. Nevertheless, the parallels between soluble uric acid and crystalline uric acid in inducing the inflammatory response is remarkable and suggests the involvement of a pro-inflammatory state that may involve local as well as systemic effects. Studying these responses in patients has the potential of helping to understand the variability of the phenotype in gout and hyperuricemia and of identifying the missing links that determine the progression from hyperuricemia and crystal deposition to clinically symptomatic gout.

Systemic Conditions Beyond Gout.

The recognition that uric acid has such key proinflammatory roles in innate immunity raises key questions of its importance in other diseases. Indeed, hyperuricemia is a powerful predictor of obesity, metabolic syndrome, nonalcoholic fatty liver and diabetes, and experimental studies tie these inflammatory pathways to the mechanism of disease^3,6,24,86. Similar studies have linked serum urate with acute and chronic kidney disease, hypertension, preeclampsia, and cardiovascular disease. ^4,5,136 However, a challenging finding is that most Mendelian randomization studies evaluating the relationship of serum uric acid to cardiovascular endpoints have been negative, and clinical trials have also been mixed.⁴ Nevertheless, there is increasing evidence that lowering uric acid may be beneficial, especially in hyperuricemic individuals with chronic kidney disease who are showing deteriorating renal function.¹³⁷ Clearly further studies are needed, but the recognition of the involvement of urate in metabolic inflammation offers the potential for novel interventions.

Conclusions

Asymptomatic hyperuricemia is common but has been largely viewed as a metabolic abnormality that increases the risk for gout and kidney stones and is innocent otherwise. Recent studies, however, suggest that the people with asymptomatic hyperuricemia may be at significant risk for cardiometabolic diseases, and that the uric acid may play a contributory role in causing these conditions. The studies summarized here document a major role for both crystalline and soluble urate in driving metabolic inflammation, activating innate immunity and triggering epigenetic pathways that can amplify responses. These studies challenge the concept that hyperuricemia is an “innocent bystander” and raises the possibility that conditions in which hyperuricemia is present may benefit not only from urate lowering therapies, but with ones targeting various aspects of the immune system, including IL-1, autophagy, and epigenetic modifiers. Further studies are needed to better understand how these pathways may influence disease, the genetic factors controlling these responses, and the mechanisms transitioning hyperuricemia to gout and beyond.