CD39 inhibits increased inflammation and promotes resolution of gout

doi:10.21203/rs.3.rs-2971283/v1

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CD39 inhibits increased inflammation and promotes resolution of gout

https://doi.org/10.21203/rs.3.rs-2971283/v1

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Background

Gout is a self-limiting inflammatory arthritis induced by the formation of urate crystals due to hyperuricemia. Recent studies have shown that the pathogenesis of gout involves the participation of extracellular ATP, and the synergistic effect of MSU and extracellular ATP can cause gout. CD39 is an ATP hydrolase that is distributed on the surface of macrophages and almost all other immune cells, and it can inhibit inflammation caused by ATP. This suggests that CD39 may inhibit the aggravation of gout inflammation and participate in the remission mechanism of gout.

Methods

The expression of CD39 mRNA in PBMCs from gout patients, PBMCS from gout model rats and THP-1 cells stimulated by MSU were detected and analyzed. CD39 knockdown was carried out by lentivirus in a monosodium urate (MSU) induced rat model, and CD39 inhibitor was used in MSU induced mouse model. After CD39 was blocked, the activation level of NLRP3 inflammasome and the incidence of gout were observed.

Results

The results showed that the deletion of CD39 aggravated MSU-induced gouty arthritis and delayed the remission of gout. Furthermore, it promoted the activation of the NLRP3 inflammasome signaling pathway and the release of IL-1βin macrophages.

Conclusions

Our results confirm that CD39 is elevated in the gout inflammatory environment, and its presence inhibits the aggravation of inflammation and promotes the resolution of gout.

Gout

CD39

eATP，IL-1β

NLRP3

Gout is a form of inflammatory arthritis that results from the chronic deposition of monosodium uric acid crystals in the presence of increasing concentrations of uric acid. It is a common autoinflammatory disease in adults that is periodic, self-limiting, recurrent, and transient in nature. Gout is characterized by rapidly progressive mono-articular synovitis in peripheral joints, which manifests as redness, swelling, pain, and limited movement in the affected joints [1]. The activation of the NOD-like receptor protein 3 (NLRP3) inflammasome by monosodium urate (MSU) crystals is the central mechanism that initiates the inflammatory response [1-3]. Upon activation, the NLRP3 protein interacts with apoptosis-associated speck-like proteins containing CARD (ASC), leading to the recruitment of caspase-1 to form the NLRP3 inflammasome. The NLRP3 inflammasome then undergoes cleavage to form active caspase-1 [4], resulting in the secretion of mature IL-1β and IL-18, which elicit an inflammatory effect [5].

Although hyperuricemia is present in almost all patients with gout, most individuals with hyperuricemia are asymptomatic and do not develop gout [2]. Therefore, the deposition of monosodium urate crystals alone does not necessarily cause inflammation. In addition to MSU crystals, it has been shown that metabolic substances in the body, including adenosine triphosphate (ATP), nigericin, glucose, and lipids, contribute to the activation of the NLRP3 inflammasome [6, 7]. Extracellular ATP (eATP) acts as a damage-associated molecular pattern (DAMP) that binds to purinergic receptors and triggers a signaling cascade to induce inflammatory responses [8]. This includes the release of microvesicles from monocytes and macrophages, leading to rapid production of IL-1β and IL-18 [9]. Our previous studies have shown that ATP is involved in the progression of gout [10]. Recently, we have further demonstrated that eATP can synergistically activate the NLRP3 inflammasome with MSU, inducing sufficient IL-1β secretion in gout pathogenesis, while other NLRP3 inflammasome activators cannot synergistically interact with MSU. This suggests that ATP plays an indispensable role in the pathogenesis of gout [11].

Clinically, most patients with acute gout can self-resolve within two weeks, indicating that the pathogenic signals of gout, ATP or MSU, may be attenuated in the gout inflammatory environment. Ectonucleoside triphosphate diphosphohydrolase 1 (CD39) is widely expressed on the surface of macrophages and other immune cells, binds eATP and converts it to extracellular adenosine [12]. In vitro studies in macrophages have shown that activated P2X7 receptor (P2X7R) can upregulate cytosolic CD39 expression by triggering a lipid raft-dependent mechanism [13]. CD39 plays a significant role in transitioning from an ATP-mediated proinflammatory environment to an adenosine-driven immunosuppressive environment [14, 15]. CD39 expression in macrophages limits the pro-inflammatory response mediated by P2X7R, and CD39 gene deletion increases inflammation and exacerbates experimental liver injury induced by sepsis [16, 17]. This indicates that CD39 can not only decompose ATP and promote adenosine production, but also timely control the upregulated inflammatory state of the body and play an inhibitory role in inflammation. CD39 is suggested to inhibit inflammation and promote the remission of gout during the pathogenesis of gout.

To verify the role of CD39 in promoting gout remission, we used CD39 knockdown lentivirus and CD39 inhibitor to investigate whether CD39 mediates gout inflammation. We found that CD39 plays an inhibitory role in MSU-induced activation of the NLRP3 inflammasome in macrophages, and lack of CD39 exacerbates and prolongs MSU-induced acute gouty arthritis.

Patients

The study compiled with the declaration of the Helsinki and was approved by the Ethics Committee of The First Affiliated Hospital of University of Science and Technology of China. All subjects gave written informed consent. Gout patients, meeting ACR/EULAR criteria[18], and healthy controls were recruited from the Department of Rheumatology and Immunology at The First Affiliated Hospital of University of Science and Technology of China between June 2022 and March 2023. The characteristics of acute gout patients (n = 18) can be found in Supplementary Materials Table 1. Acute gout group: gouty patients with gout flare and the recurrence is less than six times within half a year and self-remission.

Cell culture and stimulation

THP-1 cells (2 × 10⁵ cells/mL) were cultured in RPMI 1640 medium (HyClone AG29775810) supplemented with 10% FBS. They were then stimulated overnight with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma P1585) to generate THP-1-derived macrophages.Peripheral blood mononuclear cells (PBMCs) were isolated from stable gout patients using lymphocyte isolation and erythrocyte lysis (Haoyang LTS1077, NH4CL 2009). The isolated cells were inoculated in RPMI 1640 medium (2 × 10⁶ cells/mL) and cultured overnight. The following day, the supernatant was removed to obtain crude extracted macrophages.

Flow Cytometry Analysis of Cell pyroptosis

Cell pyroptosis was measured with Annexin V-FITC apoptosis detection kit (BD Biosciences，USA)According to the manufacturer's instructions, cells were incubated with 100 μL binding buffer containing 5 μL Annexin V-FITC and 5 μL propidium iodide at 37 °C for 15 min Cell pyroptosis was analyzed by the A00-1-1102 flow cytometer (Beckman Coulter, USA)

Western blotting

Western blotting was used to detect protein levels in PBMC lysates from gout patients and joint grinds from rat gout models. Electrophoresis was performed using a segmented constant voltage method, with the concentrated gel run at a constant voltage of 80 V. The voltage was then increased to 120 V until the bromophenol blue dye reached the bottom of the gel. The membrane was rotated at a constant flow of 200 mA in an ice bath and blocked with 5% skim milk powder for 1 hour. Primary antibodies against IL-1β (CST D3A3Z, 1:1000), caspase-1 (CST D7F10, 1:1000), NLRP3 (CST D4D8T, 1:1000), and β-actin (CST 8H10D10, 1:1000) were incubated overnight at 4°C. HRP-conjugated secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG, Abbkine A21020, A21010, 1:5000) were then incubated for 1 hour at room temperature, followed by development.

Gouty arthritis induced by monosodium urate crystals in rats

Sprague-Dawley (SD) male rats weighing 200 g were obtained from the Laboratory Animal Center of the First Hospital of USTC and housed in a pathogen-free environment. The classical Coderre method (Coderre and Wall, 1987) was used to establish a rat model of gout, which was then randomly divided into three groups. The rats were treated as follows: 1) Control rats were injected with 100 μl of sterile saline into the right ankle joint cavity. 2) Rats in the MSU group received an intra-articular injection of 100 μl of MSU crystal suspension (0.1 g/ml) into the right ankle joint cavity. 3) Rats in the MSU+POM-1 group received an intraperitoneal injection of 500 μl of POM-1 solution (25 mg/kg) 30 min prior to the intra-articular injection of MSU crystal suspension. The injected ankle perimeter of rats with string was determined at 0, 2, 4, 8, 12, 24 h,48h after MSU stimulation. Animal processing and data analysis were performed blindly. Each measurement was duplicated reproducibly. Rat ankle joints were obtained from each group, and tissue homogenates were prepared according to the manufacturer's instructions.

LDH assay

Culture supernatants were collected from MSU - primed THP 1 cells treated with POM - 1 (5 mg/ml) for 30 min and stimulated with ATP(3mmol). LDH levels were measured using the Lactate Dehydrogenase (LDH) Assay kit (Solarbio, cat# BC0685) following the manufacturer’s instructions.

Immunohistochemistry

The ankles of the isolated rats were fixed in tissue fixative (containing 10% neutral formalin) for 24 hours and subsequently placed in decalcification solution (containing formalin and EDTA) with shaking for 4-6 weeks to ensure adequate decalcification. Adequate decalcification was determined by smooth penetration of the needle through the entire bone tissue. After washing, dehydration, and decalcification, the ankle tissue was fixed in paraffin and sectioned for hematoxylin and eosin (HE) staining.

Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from whole blood using a whole Blood RNA isolation kit (Simgen), total RNA was isolated from cultured cells using Hipure total RNA mini kit (Magen). Isolated total RNA was reverse transcribed with the BioRT Master Hisensi cDNA strand synthesis kit (BioFlux, cat# BSB40M1). ABI Prism 7500 Sequence Detection System (Applied Biosystems) was used for RT-PCR. SYBR Green PCR Master Mix (Muma, cat# A4004M) was used to perform PCR of rat CD39 and, IL-1β, NLRP3, caspase-1, and ACTB. The relative level of gene expression is determined by the comparative threshold cycle method described by the manufacturer, in which the data for each sample is normalized to ACTB constituent genes and expressed as a fold change compared to the control. PCR primers can be found in Supplementary Materials Table 2.

Statistical Analysis

All results are presented as mean ± standard error of the mean. Independent-sample t-tests and one-way analysis of variance (ANOVA) were used to compare differences between two groups and multiple groups, respectively. Significant differences revealed by one-way ANOVA were further analyzed using Dunnett’s t-test. The level of statistical significance was set at p < 0.05. All bar graphs were generated using GraphPad Prism 8.0 software (GraphPad Software).

In an inflammatory state of gout, the expression level of CD39 is elevated

To determine the mRNA expression levels of CD39 in gout patients, peripheral blood mononuclear cells (PBMCs) were collected from 18 gout patients (Gout group) and 8 healthy controls (HC group). CD39 expression was found to be significantly higher in PBMCs of gout patients (Fig. 1A). Furthermore, an increase in CD39 gene expression levels was observed in THP-1 cells stimulated by MSU in vitro, as well as in MSU-induced rat PBMCs (Fig. 1B,C). These results indicate that CD39 expression is up-regulated in gout inflammation.

CD39 deficiency enhances NLRP3 inflammasome activation and pyroptosis in vitro

To examine the role of CD39 in limiting NLRP3 activation induced by MSU in macrophages, we used POM-1, a potent inhibitor of CD39, to block CD39. Macrophages were pretreated with a high concentration of ATP (1 mmol) and then stimulated with MSU. We then assessed the expression of NLRP3, caspase-1 (p20), and IL-1β in macrophages. As expected, Western blotting and q-PCR results showed that MSU-induced NLRP3 inflammasome activation was enhanced by extracellular ATP stimulation at high concentrations and was significantly augmented by POM-1 treatment (Fig. 2A, B).

As pyroptosis plays a critical role in the pathogenesis of gouty arthritis induced by MSU, we performed pyroptosis assays. PI staining was used to detect pyroptosis-induced changes in cell membrane permeability[19, 20]. Flow cytometry data showed a significant increase in the proportion of macrophages from PI double-positive cells compared to the control group (Fig. 2C). In addition, pyroptosis-induced membrane disruption is the main mechanism for the rapid release of cell contents, and we tested the levels of the pyroptosis marker LDH in gout. The results showed that extracellular LDH release was increased, and intracellular caspase-1 expression was upregulated after MSU crystal stimulation (Fig. 2D). In the absence of CD39, MSU-induced THP-1 cells showed an increased pyroptosis rate in the pyroptosis assay. These results indicated that the loss of CD39 activity aggravated MSU-induced pyroptosis.

Additionally, we detected IL-1β in the supernatant of THP-1 culture, which was dependent on inflammasome activation. Our findings revealed that IL-1β secretion was significantly higher after stimulation with high concentrations of ATP and treatment with POM-1 compared to the control group (Fig.2E). The levels of other inflammatory factors, such as IL-6 and TNF-α, were also significantly increased by ATP stimulation but were not enhanced by POM-1 treatment, likely because they are not dependent on the NLRP3 inflammasome (Supplementary Fig. S1A, B). Overall, our results suggest that extracellular ATP concentration is crucial for the MSU-induced macrophage inflammatory response, and CD39 enzyme activity is essential for limiting inflammasome activation.

CD39 deficiency exacerbates gouty arthritis and promotes NLRP3 inflammasome activation in rat model of gout

To further investigate the role of CD39 in regulating gout flare, we evaluated the inflammatory manifestations in traditional gout model rats. In this model, MSU crystals were injected into the ankle joint cavity of rats to induce gouty arthritis [21], which may result in ankle swelling and marked inflammatory infiltration. MSU-treated rats exhibited continuous swelling in ankle circumference during the experiment, and POM-1-treated rats showed a significant increase in ankle circumference compared to controls, indicating exaggerated joint swelling. The POM-1 group also showed significant differences in the inflammatory infiltration of synovial tissue after MSU exposure treatment (Fig. 3A-C). Additionally, we knocked down CD39 in rats by lentivirus delivery of ShRNA, and induced acute gout in rats at 72 hours after lentivirus treatment. Compared to the Lv-shcon group, the Lv-shCD39 rats showed more obvious ankle inflammation and more severe inflammatory cell infiltration in synovial tissue (Fig. 4A-C). These findings suggest that CD39 deficiency exacerbates the inflammatory manifestations of MSU-induced arthritis in rats.

To further confirm the involvement of CD39 in the pathogenesis of acute gouty arthritis, Western blotting and q-PCR were used to investigate the expression of NLRP3 inflammasome components. Consistent with the results of in vitro experiments, MSU induced the activation of NLRP3 inflammasome and increased the expression of NLRP3, active caspase-1 (p20) and its downstream activity IL-1β. When CD39 was inhibited or knocked down, MSU-induced inflammasome activation was more intense, and NLRP3 and active caspase-1 were significantly upregulated (Fig. 3E, F and Fig. 4E, F). Furthermore, ELISA of rat peripheral blood serum showed that CD39 deficiency was accompanied by a higher level of IL-1β release (Fig. 3D and Fig.4D). These results suggest that CD39 has an inhibitory effect on the activation of the inflammasome in acute gouty arthritis.

CD39 deficiency impairs gout resolution in rat model of gout

As ATP is implicated in the pathogenesis of gout, CD39 promotes ATP degradation to facilitate gout remission. This suggests that CD39 plays a vital role in the spontaneous resolution of gout. To investigate the effect of CD39 on gout resolution, we extended the observation period of the rat gout model. Our findings demonstrate that while joint swelling in normal rat gout models returned to normal levels after 72 hours, joint swelling in CD39-deficient rat gout models persisted at the peak stage of inflammation (Fig. 5A, B). Furthermore, CD39-deficient rats still had higher levels of IL-1β, NLRP3, and caspase 1 expression after 72 hours (Fig. 5C). Even after a prolonged observation period of 120 hours, joint swelling persisted in CD39-deficient rats (Fig. 5A, B). These results suggest that CD39 may promote gout resolution and play a vital role in the self-limiting effect of gout.

In patients with hyperuricemia, during the early stage of a gout attack, necrotic, pyroptotic, apoptotic, and inflammatory cells release significant amounts of ATP outside the cell via annexin or nexin channels [22]. This ATP then binds to P2X7 receptors on the cell membrane, causing potassium ion outflow or calcium ion inflow to activate the NLRP3 inflammasome. Studies have indicated that the activation of the P2X7R by ATP plays a critical role in promoting IL-1β secretion in inflammatory cells [10, 23]. P2X7R is a major functional eATP receptor on immune cells and a key factor in NALP3 inflammasome activity. P2X7 receptors are ATP-gated ion channel receptors [24], and their function can be affected by SNP polymorphisms in their genes[14]. Our previous study found that the pathogenesis of gout requires the synergistic action of MSU and ATP[11]. Therefore, patients with impaired P2X7 receptors are less susceptible to gout attacks, which may explain why most patients with hyperuricemia do not experience gout attacks.

As a dihydrolase of ATP, CD39 is upregulated in the gout inflammatory environment through various mechanisms. During tissue hypoxia in acute inflammation, CD39 expression is induced by the hypoxia-induced transcription factor Sp1[25, 26]. Additionally, activated P2X7 receptors can upregulate CD39 expression and hydrolase activity [16]. CD39 can set the activation threshold for P2X7-dependent inflammatory cell death and associated IL-1β release [27]. Therefore, upregulation of CD39 in acute gout can help prevent the progression of gout inflammation. Our results indicate that increased expression of CD39 is observed in an inflammatory environment after the onset of gout. The absence of CD39 exacerbated ankle inflammation and enhanced NLRP3 inflammasome activation in the synovial tissue of rats, suggesting that the response of CD39 to MSU may act as a protective mechanism to limit the inflammatory response. The regulatory effect of CD39 on acute gout attacks may be attributed, on one hand, to its hydrolysis of eATP, which limits P2X7R activation and thus affects NLRP3 inflammasome activation. On the other hand, the absence of CD39 could not hydrolyze ATP, which affected the downstream adenosine signaling pathway, resulting in the absence of downstream anti-inflammatory signal response and aggravating the inflammatory response induced by MSU. Additionally, we noted that CD39 deficiency affected the resolution of joint inflammation in rats, which took more time to recover to normal levels compared to the control group. This suggests that CD39 is involved in the mechanism of gout remission and plays an important role in gout self-limitation.

In addition to hydrolyzing ATP, CD39 can influence the outcome of inflammation through various pathways. For example, research findings suggest that the resolution of joint inflammation is associated with the migration of neutrophils after a gout attack [28-30]. Some studies have found that blocking CD39 may prevent neutrophil chemotaxis [31]. In addition to its effect on innate immunity, CD39 also suppresses inflammation by affecting adaptive immunity. CD39 is expressed on T cells and B cells [12], and is abundantly expressed on the surface of Treg cells, which appears to be a mode of control by Treg cells over effector cells. CD39-deficient mice have impaired Treg cell suppression [32-34]. Downregulation of CD39 expression has been shown to impair the immunosuppressive effect of Tregs in LPs-induced lung inflammation [35]. Studies have noted that the splenic Treg and Th17 cells of gout model rats change with the progression of gout [36], indicating that CD39 may also be involved in the course of gout by affecting Treg cells.

Although inflammation can promote CD39 expression and facilitate the resolution of inflammation in most cases, the expression of CD39 is often affected by the disease course and other factors. In acute inflammation, tissue hypoxia can induce the upregulation of CD39 expression[25, 26]. However, chronic hypoxia and other factors may lead to the opposite effect of downregulation of ATPDase activity, resulting in chronic inflammation[37-39]. This may also be one of the mechanisms of the conversion from acute gout to chronic gout. Whether there is a reduction in CD39 in patients with chronic gout needs further confirmation.

In summary, these findings strongly suggest that CD39 plays a crucial role in gout inflammation. CD39 may serve as a potential therapeutic target for human gouty arthritis, and a promising therapeutic approach is to administer soluble apyrase or a truncated form of CD39 (which mimics exonuclease activity) pending clinical development of these agents[17]. Inhibition of P2X7R is also a therapeutic strategy to limit the progression of acute gout and treat established inflammatory diseases. Although several P2X7R antagonists have been clinically applied [40, 41], the desired therapeutic effects have not been achieved in the treatment of inflammation-related diseases. The combination of CD39 truncation and P2X7 receptor blockers may be more effective in limiting the monocyte-macrophage response in the acute phase of gout.

Ethics statement

Our protocols for the collection and processing of animal and human samples were approved by the Animal Ethics Committee (2021-N(A)-041) and Medical Research Ethics Committee(2021 KY No.162) of the First Hospital of the University of Science and Technology of China (Anhui Provincial Hospital), respectively. All persons gave their informed consent before their inclusion in the study.

Data Availability

The data presented in this study are available on request from the corresponding author.

Funding

This work was supported by the Fundamental Research Funds for the Central Universities (YD9110002017).

Disclosure statement: The authors have declared no conflicts of interest.

Acknowledgments

JT and CL contributed to the conception and design of the study. CL , XL ,JG ,YL and HS conducted the experiments and analyzed the data. CL drafted the manuscript. All authors contributed to manuscript revision and approved the submitted version.

We also thank Yujie Tang, Hongliang Zhang, Yuting Li for their technical help.

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No competing interests reported.

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CD39 inhibits increased inflammation and promotes resolution of gout

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Abstract

Figures

Introduction

Materials and Methods

Patients

Cell culture and stimulation

Western blotting

Gouty arthritis induced by monosodium urate crystals in rats

LDH assay

Immunohistochemistry

Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Statistical Analysis

Results

In an inflammatory state of gout, the expression level of CD39 is elevated

CD39 deficiency enhances NLRP3 inflammasome activation and pyroptosis in vitro

CD39 deficiency exacerbates gouty arthritis and promotes NLRP3 inflammasome activation in rat model of gout

CD39 deficiency impairs gout resolution in rat model of gout

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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