Increased Metallothionein-1 Associated with Gout Activity and Tophi

ABSTRACT Background and aims Gout is a chronic self-limiting inflammatory arthritis. An increase in metallothionein-1 (MT-1) has been reported in rheumatoid arthritis and osteoarthritis, and it attenuates inflammation and the pathology of diseases. This study aims to detect MT-1 levels in patients with gout and to explore its correlation with disease activity, clinical indexes, and inflammatory cytokines. Methods The expression of MT-1 messenger RNAs (mRNAs) and protein levels in patients with gout were measured using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Correlations between MT-1 and clinical indexes or inflammatory mediators were analyzed using Spearman’s correlation test. Results Compared with healthy controls (HCs, n = 43), patients with active gout (n = 27) showed higher levels of MT-1 mRNA in peripheral blood mononuclear cells and protein in serum, particularly those with tophi. No significant difference in serum MT-1 levels was observed among patients with inactive gout, HCs, and patients with hyperuricemia without gout. Furthermore, no significant difference was observed between patients with gout with kidney damage and HCs. In addition, serum interleukin (IL)-1β, IL-6, and IL-8 levels were significantly increased in patients with active gout, particularly in those with tophi. The serum MT-1 level was positively correlated with C-reactive protein, as well as with IL-1β, IL-6, and IL-18. Conclusion The higher levels of MT-1 were found in patients with gout, which were correlated with disease activity and gout related pro-inflammatory cytokines. Indicating MT-1 may serve as a new marker for predicting disease activity. Abbreviations: IL-1β: Interleukin 1β; MT-1: Metallothionein-1; CRP: C-Reactive Protein; ROS: Reactive Oxygen Species; IL-10: Interleukin 10; TGF-β: Transforming Growth Factor Beta


Introduction
Gout, a chronic inflammatory disease, is considered the most common inflammatory arthritis in men aged >40 years (Dalbeth et al. 2019(Dalbeth et al. , 2021. Epidemiological investigation in Asia, Europe, and North America showed that gout incidence ranges from 0.6 to 2.9 per CONTACT Zhizhong Ye yezhizhong2000@163.com; Zhong Huang zhuang809@126.com Joint Research Laboratory for Rheumatology of Shenzhen University Health Science Center and Shenzhen Futian Hospital for Rheumatic Diseases, Shenzhen, China 1000 person-years in adults (Chen-Xu et al. 2019;Dehlin et al. 2016;Desai et al. 2017;Kim et al. 2017;Kuo et al. 2015;Rai et al. 2017;Zobbe et al. 2019). Gout is caused by monosodium urate (MSU) crystal-induced inflammation in joints and surrounding tissues (Desai et al. 2017). MSU crystal formation is caused by the excessive intake of purine-rich food/beverages and various health factors, such as obesity and heart and kidney diseases, which result in serum uric acid (UA) concentration that is higher than physiological saturation (Choi et al. 2004). Hyperuricemia (HUA) is a major risk factor for gout; however, only approximately 10% of the people with HUA have gout symptoms (Campion et al. 1987;Grassi et al. 2013).
Interleukin (IL)-1β plays a pivotal role in gout, and clinical trials with IL-1β inhibitors showed an effective sustained reduction in the recurrent episode of gout arthritis (Terkeltaub et al. 2009). Pathways related to release-activated IL-1β caused by MSU crystals are complicated, the mechanism of which needs to be elucidated. A popular belief is that MSU crystals deposited in joints and surrounding tissues activate the the nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasomes, which cause caspase-1 overactivation and lead to increased production of activated IL-1β and IL-18 (Martinon et al. 2002). However, a study showed that the secretion of activated IL-1β was induced by the interaction of free fatty acid with Toll-like receptor 2 and synergy with MSU crystals, which relied on caspase-1 and an apoptosis-associated spot-like protein containing the adaptor molecule CARD-associated spot-like protein but not on NLRP3 (Joosten et al. 2010). The expression of pro-IL-1β and NLRP3 can be induced by IL-6; IL-1β production induced by MSU crystals drastically increased in cells primed with IL-6, and these results were further confirmed using IL-6 inhibitor tocilizumab (Temmoku et al. 2021). Furthermore, a synergistic effect of upregulating IL-1β secretion in neutrophils was observed in tumor necrosis factor-alpha (TNF-α) and MSU crystals compared with the stimulation of MSU crystals alone (Yokose et al. 2018). Moreover, the cooperation between MSU and reactive oxygen species (ROS) to stimulate the release of activated IL-1β has been reported (Carta et al. 2011;Zhou et al. 2011). Activated IL-1β induces IL-8, which recruits neutrophils, the major inflammatory cells in a gout episode, into the inflammatory joint area; these neutrophils produce proteinase-3, which activates pro-IL-1β (Joosten et al. 2009), which forms a positive forward feedbacked loop and further secretes a large amount of IL-8, attracting an influx of massive neutrophils into articular joints and periarticular tissues, exacerbating inflammation and resulting in a gout attack.
An increase in inflammatory factors and leukocytes causes an acute gout attack. An acute gout attack is characterized by self-limiting inflammation of joints and tissues (Wallace et al. 1977). Until now, the mechanism by which the spontaneous rapid resolution of inflammation in gout occurs is still unclear, but the disturbance of proinflammatory and antiinflammatory cytokines induced by MSU and other factors may be one of them. Metallothionein (MTs) are low-molecular-weight, cysteine-rich proteins that play key roles in the regulation of metal homeostasis and protection against heavy metal toxicity as well as oxidative stress under physiological conditions (Kägi 1991;Subramanian Vignesh and Deepe 2017). Humans have four MT subtypes, namely MT-1, MT-2, MT-3, and MT-4 (Kägi 1991;Subramanian Vignesh and Deepe 2017). MT-1 expression is significantly upregulated in patients with rheumatoid arthritis (Youn et al. 2002) and osteoarthritis (Won et al. 2016), is closely related to disease activity and attenuates the inflammation and pathogenesis of diseases. However, MT-1 expression in patients with gout and the correlations of MT-1 with gout activity, complication, clinical indexes, and proinflammatory cytokines are still unclear.
In this study, we analyzed the expressions of MT-1 messenger RNAs (mRNAs) in peripheral blood mononuclear cells (PBMCs) and protein in the serum of patients with gout, patients with HUA, and healthy controls (HCs) and compared MT-1 levels among patients with inactive gout, patients with active gout, patients with active gout with tophi and without tophi, patients with gout with and without complications, and HCs. In addition, we investigated the correlation of serum MT-1 protein levels with the disease activity, clinical indexes such as UA, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and creatinine (CREA), as well as proinflammatory cytokines such as IL-1β, IL-6, and IL-8 in the serum of patients with gout, which indicated the possible effect of MT-1 on gout.

Patients' recruitment
In total, 49 adults with a gout diagnosis based on the 2018 updated EULAR recommendations for gout diagnosis were consecutively enrolled in the study after obtaining informed consent (Richette et al. 2020). Furthermore, 17 patients without gout with a serum UA value of >420 µmol/L on two separate days were considered to have HUA. All patients were recruited from the Shenzhen Futian Hospital for Rheumatic Diseases, China. The control group consisted of 43 age-and sex-matched healthy volunteers who were enrolled after obtaining informed consent. Table 1 presents the demographic and clinical characteristics of all patients and controls. All clinical manifestations and laboratory findings were recorded on the day of blood withdrawal. Except where otherwise indicated, values are expressed as number (%), mean ± standard deviation (SD). There were no significant differences between gout patients, hyperuricemia patients and healthy controls in terms of age and sex. UA, uric acid; TG, triglyceride; CHOL, cholesterol; HDL, high density lipoprotein; LDL, low density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; BUN, blood urea nitrogen; CREA, creatinine.

Blood collection and PBMCs isolation
The peripheral blood of patients and HCs was collected. PBMCs were isolated using standard Ficoll Paque Plus (TBD science, China) following the manufacturer's instructions. The collected cells were used for RNA extractions. Serum samples were stored at−80°C until cytokines were determined.

Quantitative real-time polymerase chain reaction
Total RNA from PBMCs was extracted using Trizol reagent (Takara, Dalian, China) according to the manufacturer's instructions. Then, RNA quality and purity were validated through absorbance at 260 and 280 nm based on microvolume spectroscopy (NanoPhotometer N50, IMPLEN, Germany). Only samples with ratios from 1.8 to 2.0 were accepted for the next reverse transcription reaction. Complementary DNAs (cDNAs) were prepared using the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, USA). Quantitative real-time polymerase chain reaction (qRT-PCR) amplification reactions were prepared with the SYBR Green PCR kit (TransGen Biotech, China) and performed using the CFX96 RT-PCR system (Bio-Rad, USA). PCR products were verified through melting curve analysis. Primers were synthesized by Sangon Biotech (Shanghai, China): MT-1 forward primer 5′-AGAGTGCAAATGCACCTCCTGC-3′, reverse primer 5′-CGGACATCAGGCACAGCAGCT-3′; β-actin forward primer 5′-TCCTCTCCCAAGTCCACACAGG-3′, reverse primer 5′-GGGCACGAAGGCTCATCA TTC-3′. The relative mRNA level of MT-1 was calculated with normalization to control the housekeeping gene β-actin and was reported using the 2 −ΔΔct method.

Statistical analysis
The results are presented as the mean ±standard error of mean and were analyzed using GraphPad Prism software. Statistical comparisons of two groups were performed using a two-tailed Student's t-test. Spearman's correlation test was used to investigate the correlations between serum MT-1 levels and laboratory values, as well as serum cytokine levels.
A one-way analysis of variance analysis was used for multiple comparisons. Differences with p values<0.05, <0.01, <0.001, and<0.0001 were considered significant.

MT-1 mRNAs and serum protein levels were higher in patients with gout but not in those with HUA
The expressions of MT-1 mRNAs in PBMCs and serum MT-1 protein levels in 49 patients with gout, 17 patients with HUA, and 43 HCs were detected using RT-PCR and ELISA, respectively. The mRNA and protein levels of MT-1 increased significantly in patients with gout when compared with those in HCs (Figure 1a,b). No significant differences in the mRNA and protein levels of MT-1 were observed between HUA and HCs. Thus, MT-1 may be involved in gout pathogenesis.

MT-1 mRNAs and serum protein levels were increased in patients with active gout, particularly in those with tophi
We next investigated whether MT-1 was related to disease activity in patients with gout. Acute episodes of gout characterized by rapid onset of redness, joint swelling, and intense pain are the main clinical indicators to distinguish gout flares from asymptomatic interval gout. In this study, gout flares were considered active gout, and asymptomatic intervals were considered inactive gout (Martinon et al. 2002). The tophus in patients with gout was diagnosed through joint ultrasound or dual-energy Computed Tomography (CT), and nine patients with a tophus with active gout were included in the study. Table 2 presents the demographic and clinical characteristics of patients with active gout, inactive gout, and gout with or without a tophus. The results showed that MT-1 mRNAs and serum protein levels were significantly increased in patients with active gout compared with those in patients with inactive gout and HCs (Figure 2a,c). Moreover, MT-1 was significantly increased in patients with active gout with a tophus compared with that in those without a tophus in terms of both mRNAs and serum protein levels (Figure 2b,d). The receiver operating characteristic (ROC) curve was used to analyze whether the MT-1 level could be used as a potential biomarker for active gout diagnosis, and the result indicated that the MT-1 level could accurately distinguish patients with active gout from HCs (Figure 2e).

MT-1 expression is not related to kidney damage in patients with gout
Because kidney disease is a common comorbid disorder in gout, we next investigated the expressions in patients with gout with kidney damage and those without kidney damage. No significant differences in MT-1 mRNAs and serum protein levels were found between kidney damage and HCs, illustrating that MT-1 may not be related to kidney disease in patients with gout (Figure 3a,b).

Correlation of MT1 levels with UA and other gout clinical indexes
To survey potential correlations between MT-1 levels and clinical indexes in patients with gout, we examined the relationships between serum MT-1 protein levels and the values of UA, CRP, ESR, and CREA. The results showed that serum MT-1 levels were positively correlated with CRP (R 2 = 0.2026, p = .0012; Figure 4a). However, we did not observe the correlation of serum MT-1 levels with ESR (R 2 = 0.01345, p = .4274; Figure 4b), CREA (R 2 = 0.03672, p = .1920; Figure 4c), and UA (R 2 = 0.05461, p = .1061; Figure 4d).  Each symbol represents an individual Gout patient. Spearman's rank correlation analysis was used to calculate significance. n < .05 represents a significant difference.

Increased serum levels of proinflammatory cytokines in patients with gout, particularly in those with a tophus
Proinflammatory cytokines play an important role in gout inflammation. We examined the serum levels of IL-1β, IL-6, and IL-8 in 49 patients with gout and 43 HCs. The serum protein levels of IL-1β, IL-6, and IL-8 were significantly higher in patients with gout without a tophus than in HCs ( Figure 5). Interestingly, IL-1β, IL-6, and IL-8 expressions were much higher in patients with gout with tophi than in those without a tophus.

Correlation of serum MT-1 levels with proinflammatory cytokines IL-1β, IL-6, and IL-8 in patients with gout
To determine whether MT-1 levels are related to gout inflammation, a correlation analysis between MT-1 and IL-1β, IL-6, or IL-8 was performed through Spearman's correlation test.

Discussion
Gout is a chronic inflammatory arthritis characterized by a sudden self-limiting episode of inflammation and is caused by the precipitation of MSU crystals in joints and surrounding tissues (Dalbeth et al. 2021). Proinflammatory cytokines undoubtedly play a key role in gout attacks (Cavalcanti et al. 2016;Kingsbury et al. 2011;So and Martinon 2017). In line with this view, published data have shown increased proinflammatory cytokines in patients with gout (Kienhorst et al. 2015;So and Martinon 2017;Zha et al. 2022). Consistent with previous studies, our study revealed that the serum levels of IL-1β, IL-6, and IL-8 were higher in patients with gout than in HCs. Moreover, proinflammatory cytokine levels were much higher in patients with gout with tophi than in those without tophi ( Figure 5), suggesting that proinflammatory cytokines may be induced by MSU crystals in gout.
Mounting evidence indicates that the MT-1 level increases in chronic inflammatory diseases, such as multiple sclerosis (Haase and Linker 2021;Pegoretti et al. 2020), atopic Figure 5. Elevated serum IL-1β, IL-6 and IL-8 levels in gout patients compared to HCs, especially in patients with tophi. The protein levels of IL-1β(a), IL-6 (b) and  in serums from patients with gout without tophi (gout-, n= 40) and patients with gout with tophi (gout+, n = 9) and HC (n=43) were measured by ELISA. Each symbol represents an individual gout patient and HC. Horizontal lines indicate median values. Differences between two groups were performed with one-way ANOVA multiple comparisons.
dermatitis (Guo et al. 2018), inflammatory bowel disease (Tsuji et al. 2013), and osteoarthritis (Wang et al. 2019). Moreover, our previous study demonstrated that MT-1 was upregulated in patients with rheumatoid arthritis and collagen-induced arthritis (Sun et al. 2018). However, the correlation between MT-1 and gout remains unelucidated. In this study, we found that the levels of MT-1 mRNA in PBMC and protein in serum were significantly increased in patients with gout (Figure 1), particularly in those with active gout (Figure 2a,c). Furthermore, the MT-1 level was higher in patients with active gout with tophi than in those without tophi (Figure 2b,d). However, we did not observe a significant difference between the amount of MT-1 in patients with inactive gout and HCs or patients with HUA (Figure 1). MT-1 levels significantly increased in patients with active gout with tophi but not in patients with HUA without gout, which indicates that the increased MT-1 level may be induced by the inflammation caused by precipitated MSU crystals in joints and periarticular tissues.
Recovery from acute gout is spontaneous, suggesting a negative feedback loop is involved in mediating the inflammatory and immunological response to MSU crystals. The balance between proinflammatory and anti-inflammatory cytokines is one of the major mechanisms to resolve inflammation (Steiger and Harper 2014). MT-1 suppresses proinflammatory cytokines closely related to gouts, such as IL-1β, IL-6, IL-8, and TNF-α (Kienhorst et al. 2015;So and Martinon 2017). Considering that MT-1 reduces proinflammatory cytokines and disease activity in chronic noninfectious inflammatory diseases (Guo et al. 2018;Penkowa et al. 2005;Sun et al. 2018;Tsuji et al. 2013;Wang et al. 2019) and, as revealed from our study, that the MT-1 level is positively correlated with the disease activity and proinflammatory cytokine concentration (Figures 4 and 6), we hypothesized that increased MT-1 in patients with active gout might participate in gout inflammation regulation.
The overexpression of MT-1 attenuates neuron apoptosis through oxidative stress reduction (Giralt et al. 2002), exogenous recombinant MT-1 drastically reduces Aβ1-42 peptide-induced oxidative stress by scavenging ROS (Kim et al. 2012), and these were further confirmed by the knockout of MT-1 and −2 in an experimental autoimmune encephalomyelitis mouse model, which enhanced the disease severity by increasing oxidative stress (Penkowa et al. 2003). MT-1 has a powerful ability to quench ROS through its thiol group of cysteine residues; therefore, it can effectively alleviate oxidative stress in inflammatory diseases (Giralt et al. 2002;Kim et al. 2012;Penkowa et al. 2003;Tachibana et al. 2014). Our study showed that MT-1 is significantly increased in patients with active Figure 6. Associations between serum levels of MT-1 and pro-inflammatory cytokines in patients with gout. Serum MT-1 levels were positively associated with IL-1β (a), IL-6(b)and IL-8(c). Each symbol represents an individual gout patient. Spearman's rank correlation analysis was used to calculate significance. P < .05 represents a significant difference. gout and is positively correlated with serum IL-1β, IL-6, and IL-8 (Figures 2 and 6), suggesting MT-1 may be involved in the regulation of ROS levels in patients with active gout.

Conclusions
MT-1 is upregulated in patients with active gout, particularly in those with tophi, and positively correlated with disease activity. In patients with gout, proinflammatory cytokines IL-1β, IL-6, and IL-8 significantly increase and have positive correlations with MT-1. Further studies are required to elucidate resolution by MT-1 in acute gout attacks and to explore the mechanism by which MT-1 regulates the inflammation and immune response induced by MSU crystals, which will provide a new target for gout inflammation intervention.

Authors' contributions
Yanmei Ma performed the experiments and wrote the manuscript. Xinmin Huang, Xinpeng Chen and Lighua Zou contributed to data collection and analysis. and Hanying Dai, Junxiao Cong and Dandan Wu prepared the figures Zhihua Yin designed the study. Zhizhong Ye and Zhong Huang edited the manuscript and supervised the study. All authors discussed the results and commented on the manuscript.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was supported by grants from Shenzhen Science and Technology Basic Research Project JCYJ2019073015124040376, JCYJ20180504170414637, JCYJ20180302145033769 and JCYJ201908 0915120563. Guangdong Provincial Key Laboratory of tissue and organ regional immunity and disease 2019B030301009. Shenzhen Futian Public Welfare Scientific Research Project FTWS2021006 and Sanming Project of Medicine in Shenzhen SZSM201602087.

Consent for publication
Not applicable.

Ethics approval and consent to participate
This study was approved by the Ethics Committee of Shenzhen Futian Hospital for Rheumatic Diseases. Written informed consent was obtained from all patients.