How to process synovial fluid samples of gouty arthritis and extract its exosomes for subsequent cytokine analysis

Background:

The comparative analysis of ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation for the isolation of exosomes in gouty arthritis synovial fluid (GASF) is rarely reported, and it is not known whether different isolation methods can influence subsequent cytokine analysis.

Methods:

GA patients were enrolled during a 1-year period from May 2021 to May 2022. Morphology, particle number, size, purity, protein concentration, and biomarker proteins of GASF-derived exosomes in both extraction methods were observed using transmission electron microscopy, nanoparticle tracer analysis, bicinchoninic acid assay, and Western blotting. An ELISA-based assay platform was used to detect the cytokines in exosomes using Meso Scale Discovery.

Results:

Thirty-two cases of fresh GASF were taken and randomly divided between the UC group (n = 16) and the PEG group (n = 16). Transmission electron microscopy images and nanoparticle tracer analysis results showed round vesicles measuring 100 nm on average. The protein expressions of TSG101, CD63, and CD81 in exosomes of the 2 groups were measured via Western blotting. The number and protein concentration of GASF-derived exosome particles from the PEG group were significantly higher than that of the UC group (P < .001). However, in the purity estimation, the UC group reflected significantly higher exosomes extractability (P < .01). Expression of IL-6 and IL-8 in the GASF-derived exosomes were higher in the UC group (P < .05), showing a median of 3.31 (interquartile range, IQR: 0.84–13.16) pg/mL, and a median of 2.87 (IQR: 0.56–13.17) pg/mL, respectively; moreover, IL-1β was mostly undetectable in the PEG group.

Conclusion:

The UC method was found to yield exosomes of a higher purity, albeit at a lower quantity but with more abundant inflammatory cytokines; whereas the opposite was the case for the PEG group. The chemical precipitation method might not be suitable in terms of extracting GASF-derived exosomes for inflammation and immunity studies.

1. Introduction

Gout arthritis (GA) is an inflammation of the joints caused by monosodium urate crystals (MSU) within the joint envelope, synovium, cartilage, bone, or any other periarticular structures.^[1] GA is a complex condition involving interaction between genetic, environmental, and lifestyle factors. To date, biomarkers for the early diagnosis and follow-up of GA are not available, and there remains the need for researchers to develop biomarkers and molecular mechanisms for diagnosing and treating patients with GA.^[2]

In synovial joints, the synovium and cartilage are tightly connected by synovial fluid (SF), which allows the articular tissues to function as one.^[3] Typically, SF is the first to show signs of pathological changes in joints. SF represents a more complex and visualized intra-articular microenvironment, and its diagnostic value may even be superior to that of circulating blood or other body fluids.^[4] In the intra-articular inflammatory microenvironment, exosomes, as protein and nucleic acid carriers for intercellular communication, are widely present in the joint fluid and play a key role in regulating the physiological and pathological processes of the body.^[5] A growing body of evidence suggests that exosomes produced by joint fluid could serve as diagnostic and therapeutic agents.^[6] Among them, some biomarkers, such as proinflammatory cytokines and immune cell infiltrates, may be obtained from the SF and SF-derived exosomes for screening or follow-up purposes.^[7,8]

SF-derived exosomes enrichment is currently accomplished by various methods, including ultracentrifugation (UC) and polymer-based precipitation methods, among others. Recently, scholars^[9,10] have investigated gouty arthritis synovial fluid (GASF)-derived exosomes using the chemical precipitation method (e.g. commercial kits), but few have been concerned with the different extraction methods which determine the purification rate of GASF-derived exosomes. In particular, it is not known whether different extraction methods can significantly influence subsequent inflammatory analysis. Further, it has been reported that exosomes derived from neutrophils continuously stimulated by MSU can inhibit the proliferation of osteoblasts in an in vitro study.^[11] The synergistic effects of MSU and exosomes on osteoclastogenesis might play an important role in GA-related bone destruction. In other words, the existence of hyperuricemia and MSU make the study of GASF different from that of other arthritis-related SF. More research into the effects of MSU in joint fluid on the release of GASF-derived exosomes is required. However, it has been demonstrated in many studies that uric acid concentration, temperature, pH value and various ions including sodium ions in the local environment contribute greatly to the existing form of MSU.^[12] Therefore, the polymer-based chemical precipitation method might significantly influence GASF-derived exosome isolation in the MSU environment. Therefore, this study aims to extract exosomes from GASF using 2 different methods, and to compare their performance in terms of morphology, particle size, purity, marker protein identification, and expression of various inflammatory factors of GASF-derived exosomes.

2. Materials and methods

2.1. Human subjects and ethical issues

From May 2021 to May 2022, consecutive hospitalized patients with GA who were subject to treatment via invasive needlescope for removal of intra-articular gout stones were recruited from the Department of Chinese Medicine at General Hospital of Southern Theater Command. All patients suffered from conditions matching the EULAR/ACR diagnostic criteria for gout.^[13] The ages of GA patients suffering from joint effusion ranged from 18 to 60 years. Patients diagnosed with various cancers, other immune-related or immune diseases, cardiovascular diseases, metabolic diseases, other serious chronic illnesses, pregnancy, recent infections, and severe traumas were excluded from this study. All patients who participated in the study had provided written consent, and an ethical committee at General Hospital of Southern Theater Command approved the proposed study (NZLLKZ2022141). The protocol was preregistered in the Chinese Clinical Trial Registry (ChiCTR2300068301) and the Open Science Framework (https://osf.io/qcujv/).

2.2. Sample collection and grouping

A sample of knee synovial fluid (10 mL) from each GA patient was placed in an ethylenediamine tetraacetic acid (EDTA, Ningbo Siny Medical Technology Co., Ltd., Zhejiang, China) tube. Next, the samples were centrifuged at 3000 rpm/min for 20 minutes at 4°C. Further, the supernatant was transferred to a sterile EP tube (Jiangsu Kangjian Biotechnology Co., Ltd., Jiangsu, China) and stored in a refrigerator at −80°C.^[14] Afterwards, the participants’ knee synovial fluid was randomly assigned to the 2 groups: the UC group, and the PEG group.

2.3. Main instruments and reagents

Beckman Coulter (Brea, CA) provided a Beckman Optima MAX-XP ultracentrifuge; nanoparticle tracer analysis (NTA) was conducted using ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany); a transmission electron microscopy (TEM; Hitec-Science and Technology H-7650, Tokyo, Japan) was used to obtain the images; the PEG 6000 device was purchased from Sigma-Aldrich (St. Louis, MO); Pall Corporation, Port Washington, NY, provided the 0.22&0.45 μm Pall Supor filters to filter the GASF samples; BioRad (Richmond, VA) provided the electrophoresis reagent; mouse anti-human TSG101, CD63, and CD81 monoclonal antibodies and horseradish peroxidase-labeled goat anti-mouse IgG were purchased from Abcam (Cambridge, UK); and Millipore Inc. (Billerica, MA) supplied the polyvinylidene difluoride membranes.

2.4. Separation and extraction of GASF-derived exosomes

Following thawing, 2 mg/mL hyaluronidase^[14] was added to each sample, and the samples were shaken at 200 rpm for 30 minutes at room temperature. The supernatant liquid of GASF had been pooled and filtered through 0.45- and 0.22-micron pore-size Millipore^TM membranes.

With the PEG group, for the purpose of further separation, PEG 6000 (8 g) and NaCl (2.922 g) were dissolved in 50 mL of ddH₂O to produce a 16% stock solution that was filtered using 0.45- and 0.22-micron pore-size Millipore^TM membranes. A volume equal to 16% PEG 6000 solution was incorporated into the extracted GASF supernatant (with a final solubility of 8% PEG 6000), and the resulting mixture was incubated overnight at 4°C. As a next step, the mixture was centrifuged for 45 minutes at 4°C at 12,000 g. Afterward, the supernatant was discarded, and the precipitate was resuspended in 200 μL of 1 × phosphate buffer saline (PBS). Collected samples were kept at −80°C.

For precipitation of GASF-derived exosomes using the UC method, the supernatant was centrifuged at 300 g for 10 minutes, 2000 g for 45 minutes, and 10,000 for 10 minutes at 4°C, separately. Following removal of cell debris and apoptotic bodies, the supernatant was centrifuged twice at 100,000 g for 70 minutes at 4°C. Lastly, 200 μL of 1 × PBS was used to realize resuspension of the precipitate after the supernatant had been discarded. For further experiments, the extracts were stored at −80°C (Fig. 1).

Figure 1.

The scheme for the GASF-derived exosomes enrichment and separation. GASF = gouty arthritis synovial fluid.

In order for the extraction process to satisfy aseptic principles, all materials requiring direct contact with GASF were sterilized.

2.5. GASF-derived exosome morphology observation with TEM

Copper grids (mesh 400) with carbon-coated surfaces were used to hold 20 μL of GASF-derived exosomes suspension. The GASF-derived exosomes were stained in suspension in 2% phosphotungstic acid for 10 minutes, and the solution was absorbed on the copper mesh using filter paper after 5 minutes. For 15 minutes, a 60 W incandescent lamp was placed 15 cm away from the copper mesh. Thereafter, the images were collected using a H-7650 electron microscope.

2.6. WB analysis of TSG101, CD63, and CD81 proteins in GASF-derived exosomes

We quantified GASF-derived exosomes proteins using a bicinchoninic acid Protein Assay Kit (Beyotime Biotechnology Inc., Shanghai, China) after lysing with exosomes dedicated lysate (UR33101, Shanghai Umibio Co., Ltd., Shanghai, China). Prior to loading onto 10% SDS-polyacrylamide gels, these protein samples were boiled in loading buffer for 10 minutes at 100°C. During the electrophoresis, the samples were run at 80 V (30 minutes) and 120 V (60 minutes). Then, the proteins were transferred onto PVDF membranes using a transfer equipment of wet means operating at 200 mA for 120 minutes. After blocking for at least 1 hour at room temperature with the block solution (containing 5% skimmed milk powder and 0.1% Tween 20) on a shaker, these membranes were rinsed with distilled water. Primary antibodies, TSG101, CD63, and CD81 from Abcam all with a 1:1000 dilution were then added to the membranes overnight at 4°C. On Day 2, a TBST wash was performed 3 times, in each instance for 10 minutes. Incubation with secondary antibodies (1:1000 goat anti-mouse IgG) was carried out on the PVDF membranes for 1 hour at room temperature and analyzed by the Bio-Rad Protein Imaging System using ECL developing solution (Thermo Scientific, Rockford, IL).

2.7. Size and concentration identification of GASF-derived exosomes using NTA

For all GASF-derived exosomes samples, the size and concentration of particles were determined using ZetaView PMX 110 and its application software ZetaView 8.04.02. For analysis of particle size distribution and concentration of GASF-derived exosomes, 1 × PBS buffer was used to dilute the exosome samples appropriately. There were 11 positions where NTA measurements were taken and analyzed. Using 110 nm polystyrene beads at a constant temperature of 25°C, the ZetaView was calibrated.

2.8. Analysis of cytokines in GASF-derived exosomes

The GASF-exosome was first lysed using RIPA lysis buffer (UR33101, Shanghai Umibio Co., Ltd.) prior to collection of related proteins. Subsequently, cytokine secretion was evaluated using the V-PLEX Human Cytokine 10-Plex Kit, and cytokines detected using the Meso QuickPlex SQ120 (Meso Scale Discovery, MSD, Rockville, MD) instrument. These cytokines included interferon-gamma (IFN-γ), interleukin-1β (IL-1β), IL-2, IL-4 IL-6, IL-8, IL-10, IL-12p70, IL-13, and tumor necrosis factor-α (TNF-α).

2.9. Statistical analysis

Data analysis was carried out using commercial software (SPSS version 24.0; SPSS Inc., Chicago, IL) and graphs were produced using GraphPad Prism 8.0 (GraphPad Prism Software, San Diego, CA). All data were expressed as the mean value ± standard deviation (SD) or median, and first and third quartiles. In order to assess the differences between the 2 groups, we applied the t test (normally-distributed data), Mann–Whitney U test (skewed data), and Chi-square test. The difference between the 2 groups was considered to be statistically significant when P < .05.

3. Results

3.1. Basic information of the subjects

The GA patients had been randomly divided between the 2 groups (UC method versus PEG method; n = 16 per group). A comparison of each selected group and its original study population showed no statistically significant differences in terms of age, sex, blood uric acid level, routine inflammatory assessments or baseline GA history (P > .05, see Table 1).

Table 1.

Characterization of the subjects.

Parameters	UC group	PEG group	P value
Male/female	12/4	15/1	.33
Age (yr)	39.81 ± 8.96	38.63 ± 9.27	.72
GA history (yr)	3.19 ± 1.42	3.44 ± 1.86	.67
ESR (mm/h)	39.17 ± 16.04	37.07 ± 17.46	.73
CRP (mg/L)	44.26 ± 24.41	37.86 ± 21.72	.44
SUA (μmol/L)	482.75 ± 142.60	485.38 ± 124.22	.96

CRP = C-reactive protein, ESR = erythrocyte sedimentation rate, GA = gouty arthritis, PEG = polyethylene glycol, SUA = serum uric acid, UC = ultracentrifugation.

3.2. Characterization of GASF-derived exosomes

As shown in Figure 2A–D, the electron microscopy results reveal typical cup-shaped morphology (the lipid bilayer membranes) in the exosomes, which had been purified from GASF via UC and to a size range of 50 to 150 nm. In PEG group, The TEM image shows mostly round or oval structures, as well as the presence of protein impurities. The results of the GASF-derived exosomes diameter analysis are shown in Figure 2E. In comparing the 2 extraction methods, it can be seen that the particle sizes of the UC group and PEG group are 108.74 ± 16.62 nm and 111.04 ± 20.23 nm, respectively, with no significant difference (P > .05). Analysis of total protein concentration of extracted exosomes in the 2 groups was carried out using the bicinchoninic acid method. The protein content of exosomes in the PEG group was significantly higher than that in the UC group (820.27 ± 253.75 μg/mL vs 354.23 ± 216.53 μg/mL, P < .001, Fig. 2F). The UC method yielded a much lower number of GASF-derived exosomes, compared to the PEG method [(4.11 ± 1.48) × 10¹⁰ particles/mL vs (7.20 ± 1.32) × 10¹⁰ particles/mL, P < .001, Fig. 2G]. The purity of exosome extraction is expressed by the ratio of the number of exosome particles to total protein^[15]: the higher the ratio, the higher the purity of exosome extraction. For the UC group, the ratio of the number of exosome particles to total protein was higher than that for the PEG group [(1.34 ± 0.39) × 10⁸ particles/μg vs (0.91 ± 0.15) × 10⁸ particles/μg], P < .01, Fig. 2H], and the differences were all statistically significant. In the Western blotting (WB) analysis, both groups of GASF-derived exosomes were positively represented by exosomal-specific markers TSG101, CD63 and CD81 (Fig. 2I).

Figure 2.

Identification of the GASF-derived exosomes. (A and B: Transmission electron microscope photographs; C–E: Size determination and distribution; F–H: Bicinchoninic acid results; I: Western blot results. The significance levels indicated by **P < .01, and ***P < .001 were statistically significant). GASF = gouty arthritis synovial fluid, PEG = polyethylene glycol, UC = ultracentrifugation.

3.3. Inflammatory cytokines expressed in GASF-derived exosomes

Analysis of the 10 inflammatory cytokines in exosomes derived from GASF in the UC and PEG groups was conducted (Fig. 3A). For the PEG group, 9 inflammatory cytokines were clearly detected, while IL-1β in the PEG group was detected in only 56% of the GASF-derived exosomes samples. With the UC group, all inflammatory cytokines were detected, among which IL-6 and IL-8 were significantly elevated compared to their equivalents in the PEG group (3.31 [0.84, 13.16] pg/mL vs 0.15 [0.09, 2.91] pg/mL, respectively, P = .006; 2.87 [0.56, 13.17] pg/mL vs 0.47 [0.14, 0.97] pg/mL, P = .01). In both extraction methods, the expressions of IL-6, IL-8, TNF-α and IFN-γ were higher than those of other inflammatory cytokines (Fig. 3B).

Figure 3.

Inflammatory cytokines expressed in GASF-derived exosomes. (A: Ten inflammatory cytokines expressed in GASF-derived exosomes; B: The volcano plot of 10 inflammatory cytokines in the GASF-derived exosomes. The significance levels indicated by *P < .05, and **P < .01 were statistically significant). GASF = gouty arthritis synovial fluid, PEG = polyethylene glycol, UC = ultracentrifugation.

4. Discussion

GA is a chronic metabolic disease characterized by metabolism disorders of purine, and an imbalance between the production and excretion of uric acid, resulting in the accumulation of monosodium urate crystals within the synovial fluid of joints, and occurrence of inflammatory response and tissue destruction at the local level. Although a universally accepted gold standard for the diagnosis of GA has existed for a number of years, the risk of misdiagnosis and overlooked cases is considerable. Also, current research has yet to provide an adequate explanation for the pathogenesis and progression of GA. Clinically, GA can be diagnosed via analysis of uric acid levels in the blood, although these values can be hysteretic.^[16] Among patients with early-onset gout, uric acid levels might not have increased significantly.^[17] Therefore, in GA research, one of the key objectives is to pinpoint further biomarkers that can be used to predict which forms of early gout are most likely to produce uncontrolled severe GA. There is the possibility that joint effusion associated with GA might serve as a biomarker throughout disease progression and therapy response, and not just for diagnosis.^[18] There occurs delirious metabolism of uric acid, and accumulation of MSU in the GA synovium and joint fluid. It is possible that a strong inflammatory response may be activated in articular tissues stimulated by higher blood uric acid and MSU, triggering several inflammatory factors such as IL-1β, IL-6, and so on to launch a cytokine storm, contributing to GA progression. Therefore, GA disease activity metrics can be calibrated using multiple cytokines to serve as an internal control and biomarker during tracking of joint changes.^[19] Furthermore, GA joint fluid contains a range of biomarkers. As a novel diagnostic marker, synovial fluid-derived exosomes secrete a wide range of inflammatory factors, which are involved in different energy and substance metabolisms, which have important potential applications in GA clinical diagnosis and treatment. It has been demonstrated in a recent study that the separation and purification of synovial fluid-derived exosomes can be achieved using the ExoQuick chemical reagent method, whereby 69 differentially expressed proteins such as lysozyme C and protein S100-A9 in GASF-derived exosomes were isolated, and compared with osteoarthritis, axial ankylosing spondylitis, and rheumatoid arthritis synovial fluid-derived exosomes.^[9]

However, other improved methods for extracting synovial fluid-derived exosomes from GA knee should be taken into account alongside further research into GASF-derived exosomes. Polyethylene glycol is a hydrophobic multimer that binds and co-precipitates with hydrophobic protein and lipid molecules. Adding PEG precipitates to the sample can be an efficient means of purifying large quantities of extracellular vesicles. Almost all normal commercial isolation kits for purifying exosomes include PEG. Rider et al,^[20] found that an 8% PEG concentration resulted in better exosome extraction compared with commercial reagent methods, and demonstrated that those extracted exosomes could subsequently be used for further experiments, such as genetic and proteomic studies. In spite of the fact that many research groups have adopted proprietary extraction kits that purify exosomes, sequential UC is considered the gold standard method for separating exosomes. Unlike chemical methods, UC does not produce biochemical binding reactions, thus reducing the likelihood of exosome destruction. Early in the experiment, we found that GASF differs greatly from SF of other forms of arthritis. When the quantity of urate crystals being deposited and dissolved in articular fluid is abundant, the precipitation of PEG appears to be affected. However, studies on the extraction of GASF-derived exosomes are primarily based on commercial kits such as chemical precipitation methods. To date, no studies comparing the characteristics and inflammatory effects of GASF-derived exosome among different extraction methods have been published.^[9]

Therefore, in this study, we have introduced 2 enrichment methods for characterizing exosomes obtained from GASF with regard to identification, yield concentration, morphological size, and inflammation level: 8% PEG precipitation, and differential UC. The WB and NTA results reveal that with both methods the GASF contained extracellular vesicles. The TEM images were used to examine the GASF-derived exosome structure and sizes. For the UC group, the images reveal a clear lipid bilayer structure in the vesicles, whereas the PEG group images show multiple intercalated heteroproteins. Our results show that the concentration of GASF-derived exosomal proteins in the PEG group was much greater than that of proteins in the UC group. Also, the number of observed particles in the UC group was smaller than that in the PEG group. Lower yields of exosomes were obtained in the UC method, but higher purity was achieved. Therefore, both the UC and PEG methods could extract exosomes from GASF, while the PEG extraction might yield proteins with more impurities. In addition, the UC method appeared to enable better preservation of relevant inflammatory cytokines in GASF, such as IL-6, IL-8, and IL-1β, while with the other group there occurred partial loss of expression of inflammatory cytokines. However, expression profiling of inflammatory cytokines in GASF-derived exosome is influenced by experimental parameters, clinical parameters, and sample heterogeneity. On that note, it should be mentioned that one important limitation in the present study is the small sample size.

In recent years there have occurred promising advances in arthritis research concerning exosomes, and their clinical application has become a major focus among scholars in the field. However, methodological challenges such as the variations among the many exosome isolation methods complicate the journey towards a consensus. This paper has detailed the potential application of 2 exosome isolation methods (UC and PEG) by exploring their advantages and disadvantages in terms of purity, yield, and inflammatory cytokine production of GASF-derived exosome. Both methods appear to have their limitations, and researchers should carefully choose a purification method on the basis of the context in which it is conducted.

5. Conclusion

The study has summarized the merits and demerits of the PEG method and the UC method in terms of extraction of GASF-derived exosome. We have employed a comparative experimental approach to explore the separation and purification capacity via 8 key aspects: particle count, size, protein concentration, purity, morphology, specific antibody, concentration, and characterization of 10 inflammatory cytokines. It was found that the UC method is better in terms of removing protein from the GASF-derived exosomes, and is suitable for inflammatory and immune-related studies of SF-derived exosomes in GA.

Acknowledgments

We were grateful to Malcolm Sutherland (from University of Glasgow) for supporting this study as the English language editors.

Author contributions

Conceptualization: Shaowei Li, Zhihuang Chen, Song Wei.

Data curation: Shaowei Li, Shudan Zhang.

Funding acquisition: Zhihuang Chen, Xianxian Zhang, Song Wei.

Investigation: Rui Ou.

Methodology: Xianxian Zhang, Song Wei, Yiwen Xu, Kaixin Chen.

Project administration: Zhihuang Chen.

Resources: Yiwen Xu.

Software: Shaowei Li, Rui Ou, Yingwan Liu.

Supervision: Shudan Zhang, Kaixin Chen.

Validation: Yingwan Liu, Kaixin Chen, Zhouyi Chen.

Visualization: Zhouyi Chen.

Writing – original draft: Shaowei Li.

Writing – review & editing: Shaowei Li, Zhihuang Chen, Xianxian Zhang, Xinnong Shu.