Advance in Pathogenesis of Gout and Related Gene Polymorphism

Lamei ZHOU1, Wen XU1, Chunqing LI

1. Department of Rheumatology and Immunology, Wuxi Hospital of Traditional Chinese Medicine, Wuxi 214000, China; 2. Department of Nephrology, Wuxi Integrated Traditional Chinese and Western Medicine Hospital, Wuxi 214000, China

Abstract Gout is an independent risk factor for hypertension, diabetes mellitus, hyperlipidemia, coronary heart disease and cerebral infarction. At present, the studies on the mechanism of gout at home and abroad have mainly focused on immune inflammation, gene polymorphism and related studies. Uric acid deposition or crystal precipitation activates phagocytes, fibroblasts and mast cells in synovium, produces IL-1β, TNF and chemokine IL-8 (CXCL8), thereby activating neutrophils, urate crystal polyanion surface can be coated with immunoglobulins and other serum proteins as substrates for complement activation, complement substitution pathways and classical complement pathways to activate complements. Uric acid crystals activate inflammation-related signal transduction pathways including the activation of inflammation-related signal transduction pathway by uric acid crystals and TLRS/MyD88 signal transduction pathways. Gene polymorphism is related to inflammation and signaling pathway, EGF gene is closely related to gout inflammation, which may be involved in the regulation of gout inflammation, among which NLRP3 inflammatory signaling pathway and gene polymorphism have been deeply studied in the pathogenesis of gout, which is the main therapeutic target of anti-inflammatory and uric acid lowering.

Key words Gout, Uric acid crystals, Inflammation-related signal transduction pathway, Gout-related gene polymorphism

1 Introduction

Gout is a metabolic and inflammatory/immune disease. Its incidence has been increasing year by year in recent years, and it has become the second major rheumatic disease after osteoarthritis. Gout is an independent risk factor for hypertension, diabetes, hyperlipidemia, coronary heart disease and cerebral infarction. Among 5 707 gout patients in the United States, 74% had hypertension, 71% had chronic kidney disease, 53% had obesity, 26% had diabetes, 24% had kidney stones, 14% had myocardial infarction, 11% had cardiac insufficiency, and 10% had stroke; among patients with serum uric acid (>10 mg/dL), 86% had chronic kidney disease, 66% had hypertension, 65% had obesity, 33% had cardiac insufficiency, 33% had diabetes, 23% had myocardial infarction, 12% had stroke, and 3-33 times as much as those with serum uric acid <4 mg/dL[1]. At present, the number of patients with hyperuricemia (HUA) in China has been as high as 20% of the population, and urate deposition has been found in the tissues. Gout/HUA is often accompanied by hypertension, hyperlipidemia and hyperglycemia, which interact to induce metabolic syndrome. Gout patients increased their cardiovascular risk at the first visit[2]to their doctors, patients requiring regular uric acid lowering therapy and receiving uric acid lowering measures were less than 5%.

Uric acid has certain biological functions and is the main endogenous adjuvant of damaged cells, urate is involved enough to precipitate crystals to form crystals and induce inflammation. Urate crystal forms chalkstone, which is composed of urate crystals and debris in the center, the central area being surrounded by coronal area, which is composed of macrophages, mast cells and plasma cells. Macrophages in the coronal and central areas express surface markers associated with pro-inflammatory activation, inflammation and immune response in the body are closely related to gout. Gout inflammation begins with the emergence/release of urate crystals (MSU), and the local/intra-articular inflammatory mediators react with uric acid to gradually form chalkstone, resulting in long-term inflammation and renal involvement. At present, the studies on the pathogenesis of gout at home and abroad have mainly focused on immune inflammation, gene polymorphism and related researches.

2 Activation of neutrophils by urate crystals and complement activation

2.1 Activation of neutrophils by urate crystalsUric acid deposition or crystallization activates phagocytes, fibroblasts and mast cells in synovium to produce IL-1β, TNF and chemokine IL-8 (CXCL8), these cytokines enable neutrophils to adhere to and pass through endothelial cells, act on vascular endothelium, promote its expansion and leakage, and promote the expression of adhesion molecules such as selectin and intercellular adhesion molecule (ICAM) on the vascular endothelium, these cytokines promote the activation of neutrophils in the blood stream. Neutrophils flood into the joint and migrate to MSU and phagocytose it, at the same time, MSU stimulates neutrophils to produce a large number of inflammatory mediators and neutrophil chemokines such as IL-8 and LTB4,etc., releasing a series of tissue-damaging substances, including oxygen free radicals and metalloproteinases, resulting in incomplete/ineffective phagocytoses of crystallization and lysosome lysis, and the release of components into damaged cells. Neutrophil-mediated production of IL-1β promotes the recurrence of gout, causing joint damage, severe pain and fever[3]. Low levels of inflammation are common in chronic gout and chalkstone, which causes macrophages to produce cytokines and proteases continuously, thus promoting cartilage and bone destruction.

2.2 Activation of complements by urate crystalsThe surface of urate crystal polyanion can be coated with immunoglobulin and other serum proteins, which act as the substrate for complement activation, complement substitution pathway activates C3 and C3b activates downstream component C5a; urate crystals can also activate C1q through classical complement pathways, namely, immunoglobulin, CRP-dependent pathway, or antibody binding with IgG specifically activates C1q, thereby activating C5a; C5a activation forms soluble complement membrane attack complex, which promotes local inflammation. Complement C5a can effectively stimulate the activation of CD18 and the adhesion of neutrophils, neutrophils and monocytes adhere tightly to the vascular endothelium, and exude blood vessels due to the chemotaxis of C5a complement gradient, leading to contact and phagocytosis of urate crystals.

3 Activation of inflammation-related signal transduction pathway by uric acid crystals

3.1 NLRP3 inflammatory complex signal transduction pathwayNLRP3 is an important member in the NLRs receptor family, and is produced by T and B lymphocytes. As an intracellular pattern recognition receptor, uric acid crystals of pathogen-related molecular pattern (PAMPs) or DAMPs-related molecular pattern (DAMPs) are identified to oligomerize NLRs protein, recruit apoptosis-related speck-like protein (ASC), and activate caspase-1 from caspase activation recruitment domain (ARD) to form NLRP3 inflammatory body. Urate crystallization induces injury, which leads to intracellular potassium outflow and low potassium activates inflammatory complex; the crystals activate the oxidase of phagocytes, which produces reactive oxygen species (ROS) and is directly or indirectly perceived by inflammatory complexes; the activation state of urate crystals due to incomplete or ineffective phagocytosis contributes to the activation of inflammatory complexes; the phagocytized uric acid crystals can lyse and phagocytize lysosomes, which leads to intracellular acidification and intracellular release of cathepsin B cells, and become an activator of NLRP3 inflammatory complex. The activation of caspase-1 splicing of NLRP3 inflammatory effector protein makes pro-IL-1β an active form, and the intracellular complex of NLRP3 inflammatory complex molecule generates interleukin IL-1β, which then stimulates the activation of neutrophils, and the release of TNF-α, IL-6 and other pro-inflammatory cytokines and inflammatory chemokines. Sodium urate crystals trigger gout inflammation, and NALP3 inflammasome plays an important role, NALP3 can recognize urate crystals released by apoptotic cells, activate inactive NALP3 signaling pathway and induce inflammatory reaction, and express inflammatory factors such as NALP3, ASC, Caspase-1, IL-1β and IL-18[4]. IL-1β is an important cytokine in the process of acute gouty arthritis, its production is closely related to NLRP3 complex, the production of inflammation and cascade amplification of inflammation. NLRP3 inflammatory body is a key link in the process of gouty arthritis reaction, and gout is also known as NLRP3 inflammatory body-related disease[5].

3.2 Activation of TLRS/MyD88 signal transduction pathway by uric acid crystalsToll receptors (TLRs) are recognition receptors of human innate immune system, which can recognize pathogen-related molecular patterns (PAMPs) and damage-related molecular patterns (DAMPs), and are mainly expressed in monocytes, DCs, granulocytes, macrophages,etc. The pathogenesis of gout is also closely related to TLRS/MyD88 signal transduction pathway, MSU can be specifically recognized by TLR2 and TLR4 and regulate inflammatory response by mediating the signal transduction of the upstream proteins TLR2, TLR4, MyD88, NF-κB P65 and the downstream inflammatory factors IL-1β and IL-8. MSU can induce the increase of TNF-α and IL-1β levels and up-regulate the expression of TLR2, TLR4, MyD88, NF-κB and TRIF genes in this signaling pathway[6].

4 Study on gout-related gene polymorphism

A series of genetic polymorphism studies were carried out from the aspects of region, race and gender.

4.1 RegionThree polymorphisms of PDZ protein kinase 1(PDZK1) rs1967017, insulin-like growth factor-l receptor (IGF1R) rs6598541 and liver leukemia factor (HLF) rs7224610 have been confirmed to be associated with gout and hyperuricemia in the European population. In the meta-analysis of China, SLC2A9 (rs3733591, rs16890979, rs6855911, rs13124007 and rs1014290) and ABCG2(rs2231142) polymorphism are correlated with primary gout susceptibility[7]. The polymorphisms of rs6598541 and rs1967017 loci are associated with primary gout in male Han Nationality population in Shandong, and the allele T of rs1967017 may be a risk factor for gout[8]. High frequency of hyperuricemia, high total cholesterol, ApoAI-CIII-AIV gene cluster-75 loci allele A, poAI-CIII-AIV gene cluster+83 loci allele T, and ApoAI-CIII-AIV gene cluster Xmn I loci mutation allele X2 in immediate family members are rick factors for gout[9].

4.2 RaceForty-seven single nucleotide polymorphisms (SNPs) were screened from genome-wide association analysis, and it was found that gout patients with SLC2A9, WDR1, CLNK, PKD2 and ABCG2 gene variations in Han and Tibetan populations in Tibet Plateau might affect gout susceptibility[10]. Polymorphism of TGF-β1 gene rs1800470 in Chinese Han male population may be closely related to gout/chalkstone formation[11]. NLRP3 gene rs10754558G>C and TLR4 gene rs2149356T>G single nucleotide polymorphism may be related to the incidence of primary gout in Chinese Han population, GG and TT genotypes are risk factors for the incidence of gout, and rs10754558 haplotype CGA may be related to gout[12].

4.3 GenderThe genetic polymorphisms of Rs13124007(G/C), rs6850166(G/A) and rs2231142(C/A) loci are closely related to the susceptibility to gout in Chinese Han females, and rs1183201 locus is most closely related to female primary gout[13]. SNP in exon region of MOVIOLI gene has a protective effect on genetic susceptibility to gout by regulating testosterone secretion and expression in Chinese Han males[14].

Urate transporter genes are hyperuricemia and gout susceptibility genes locked by genome-wide scanning in recent years, such as SLC22A12, SLC2A9 and ABCG2 genes,etc., which encode proteins related to renal urate transport system and participate in renal reabsorption and secretion of uric acid. Currently, the targeted urate transporter gene can explain about 7% of the variation in blood uric acid level, and influential loci are expected to be found. Previous studies have applied these susceptible genes to pharmacogenomics and Mendelian randomization, which is a new direction in the future. Previous studies showed that the combination of uric acid transporter hURAT1 and ABCG2 gene detection predicted the susceptibility of hyperuricemia/gout. Based on the support of the Six Kinds of Talents Peak Project in Jiangsu Province (2015-WSN-089), our results showed that the mutation rates of rs7932775 locus of ABCG2 gene and rs3825015 locus of hURAT1 gene in gout and hyperuricemia patients were 60.7% and 50%, respectively, which were higher than those in control group statistically. These results suggested that uric acid transporter hURAT1 combined with ABCG2 gene has gene SNP polymorphism, which can be used as a therapeutic regulatory target[15].

5 Association of gene polymorphism with inflammation and signaling pathway

EGF gene is closely related to gout inflammation and may be involved in the regulation of gout inflammation[16]. Sodium urate crystals are recognized by Toll-like receptors (TLRs) to activate the nuclear transcription factor pathway to mediate inflammation. Several polymorphic sites of TLR2 are not associated with gout, and the polymorphism of TLR4 gene rs2149356T>G may be involved in the regulation of TLR4mRNA and TLR4-IL1β signaling pathway in patients with gout[12]. The interleukin family plays an important role in immune regulation of gout, and NLRP3 inflammasome is associated with gout risk factors. The rs10754558G>C polymorphism of NLRP3 gene may be involved in regulating the expression of NLRP3mRNA and the inflammasome signaling pathway of NLRP3[12], monosodium urate crystallization stimulates NLRP3 gene deletion in mouse models or mouse macrophages, NLRP3 is essential for the inflammatory response induced by urate crystallization. NLRP33’-UTRrsl0754558 polymorphism is associated with gout in Chinese Han population. Changes in different TCM syndromes may be related to MSU regulating the expression of some NLRP3 inflammasome genes and mRNA expression of its transcription spliceosome[17]. Mengetal. conducted a study on the correlation between NLRP3 gene polymorphism and primary gouty arthritis and found that the difference in rs7512998 was statistically significant. NLRP3-4 transcription variant is closely related to primary gout, and it may provide new targets for the diagnosis and treatment of primary gout[18].

6 Conclusions

The studies on immune response and gene polymorphism in gout attack have mainly focused on the activation of neutrophils, macrophages, NLRP3 inflammatory complexes, Toll-like receptors, P38 mitogen-activated protein (MAP), SyK, JNK inflammation-related signaling pathways, complement activation, gene polymorphism and inflammation signal transduction pathway by urate crystals/chalkstone, among which NLRP3 inflammatory signaling pathway and gene polymorphism have been deeply studied in the pathogenesis of gout, which is the main therapeutic target of anti-inflammatory and uric acid lowering.