High-Molecular-Weight Hyaluronic Acid Inhibits IL-1β-Induced Synovial Inflammation and Macrophage Polarization through the GRP78-NF-κB Signaling Pathway

Recent evidence has suggested that synovial inflammation and macrophage polarization were involved in the pathogenesis of osteoarthritis (OA). Additionally, high-molecular-weight hyaluronic acid (HMW-HA) was often used clinically to treat OA. GRP78, an endoplasmic reticulum (ER) stress chaperone, was suggested to contribute to the hyperplasia of synovial cells in OA. However, it was still unclear whether HMW-HA affected macrophage polarization through GRP78. Therefore, we aimed to identify the effect of HMW-HA in primary synovial cells and macrophage polarization and to investigate the role of GRP78 signaling. We used IL-1β to treat primary synoviocytes to mimic OA, and then treated them with HMW-HA. We also collected conditioned medium (CM) to culture THP-1 macrophages and examine the changes in the phenotype. IL-1β increased the expression of GRP78, NF-κB (p65 phosphorylation), IL-6, and PGE2 in primary synoviocytes, accompanied by an increased macrophage M1/M2 polarization. GRP78 knockdown significantly reversed the expression of IL-1β-induced GRP78-related downstream molecules and macrophage polarization. HMW-HA with GRP78 knockdown had additive effects in an IL-1β culture. Finally, the synovial fluid from OA patients revealed significantly decreased IL-6 and PGE2 levels after the HMW-HA treatment. Our study elucidated a new form of signal transduction for HMW-HA-mediated protection against synovial inflammation and macrophage polarization and highlighted the involvement of the GRP78-NF-κB signaling pathway.


Introduction
Osteoarthritis (OA) is the most common reason for total hip and knee replacement [1]. The mechanisms leading to the progression of cartilage degradation and failure have been extensively investigated [2]. Recently, some research has proposed that chronic low-grade inflammation is the most common pathogenesis of OA. In addition, synovitis is now recognized as a characteristic of OA in both its early and late stages and a result of activated macrophages infiltrating the synovial membrane [3]. One widely used treatment option is the intra-articular injection of hyaluronic acid (HA) to replace the synovial fluid that has lost its viscoelastic properties [4]. In osteoarthritic joints, the synovial fluid always contains a lower concentration of HA than healthy joints do [5]. In inflammation and tissue injury, HA plays a central role because it can function as a pro-and anti-inflammatory

IL-1β Treatment Increased Inflammatory Responses and GRP78 Expression
IL-1β is thought to be one of the most important cytokines in the OA inflammatory response. We used IL-1β (10 ng/mL) to treat synoviocytes to mimic the OA condition. To evaluate the expression of inflammatory factors during synovial inflammation, we used the primary synoviocytes from OA patients and then treated them with IL-1β for 24 h. The levels of inflammatory genes IL-6, IL-8, substance P (SP), PGE synthase (PGE-S), TNF-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2) were significantly increased in the group treated with IL-1β compared with controls ( Figure 1a). In addition, IL-6 and PGE2 are important proinflammatory cytokines in OA. The levels of cytokine IL-6 and PGE2 in synoviocytes were significantly increased in the group treated with IL-1β (Figure 1b,c), suggesting that IL-1β treatment can induce the inflammatory state of the synovium. Next, we evaluated the expression of GRP78 during synovial inflammation. We found that GRP78 expression was increased in the synoviocytes treated with IL-1β ( Figure 1d). In the setting of degenerative knee OA, we examined the NF-κB signaling pathway and the inflammatory-associated signaling of p65, which is a subunit of the NF-κB transcription complex that plays a crucial role in biological processes such as inflammation. The phosphorylation of p65 was increased in the group treated with IL-1β (Figure 1e). These findings indicated that IL-1β treatment increased the expressions of inflammatory factors and GRP78. evaluate the expression of inflammatory factors during synovial inflammation, we u the primary synoviocytes from OA patients and then treated them with IL-1β for 2 The levels of inflammatory genes IL-6, IL-8, substance P (SP), PGE synthase (PGE-S), T α, inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2) were significa increased in the group treated with IL-1β compared with controls ( Figure 1a). In addit IL-6 and PGE2 are important proinflammatory cytokines in OA. The levels of cytokine 6 and PGE2 in synoviocytes were significantly increased in the group treated with IL ( Figure 1b,c), suggesting that IL-1β treatment can induce the inflammatory state of synovium. Next, we evaluated the expression of GRP78 during synovial inflammat We found that GRP78 expression was increased in the synoviocytes treated with IL ( Figure 1d). In the setting of degenerative knee OA, we examined the NF-κB signa pathway and the inflammatory-associated signaling of p65, which is a subunit of the κB transcription complex that plays a crucial role in biological processes such as infl mation. The phosphorylation of p65 was increased in the group treated with IL-1β (Fig  1e). These findings indicated that IL-1β treatment increased the expressions of inflam tory factors and GRP78.

IL-1β-Treated Synoviocytes Affected the Polarization of Macrophages
To explore the role of the macrophage phenotype in the OA condition, we collected the supernatant from synoviocytes as conditioned media (CM). CM were then harvested and used to treat macrophages, and the macrophage phenotypes were then examined. Previous research has identified the specific markers expressed by the M2 cytokine markers (IL-8 and IL-10) and the M1 cytokine markers (IL-12 and TNF-α). The M2 and M1 markers were significantly increased in cells polarized with CM plus IL-1β compared with the CM group (Figure 2a). Similarly, the M2 and M1 cytokines were also significantly increased in cells polarized with CM plus IL-1β compared with the CM group (Figure 2b). Flow cytometric analysis was employed to detect the surface markers of the macrophage phenotypes. The expressions of CD206 (an M2 surface marker) and CD16 (an M1 surface marker) were increased by cells polarized with CM plus IL-1β, compared with the CM group (Figure 2c,d). These results showed that the IL-1β treatment increased inflammation in synoviocytes and also increased macrophage M1 and M2 polarization.

IL-1β-Treated Synoviocytes Affected the Polarization of Macrophages
To explore the role of the macrophage phenotype in the OA condition, we collected the supernatant from synoviocytes as conditioned media (CM). CM were then harvested and used to treat macrophages, and the macrophage phenotypes were then examined. Previous research has identified the specific markers expressed by the M2 cytokine markers (IL-8 and IL-10) and the M1 cytokine markers (IL-12 and TNF-α). The M2 and M1 markers were significantly increased in cells polarized with CM plus IL-1β compared with the CM group (Figure 2a). Similarly, the M2 and M1 cytokines were also significantly increased in cells polarized with CM plus IL-1β compared with the CM group (Figure 2b). Flow cytometric analysis was employed to detect the surface markers of the macrophage phenotypes. The expressions of CD206 (an M2 surface marker) and CD16 (an M1 surface marker) were increased by cells polarized with CM plus IL-1β, compared with the CM group (Figure 2c,d). These results showed that the IL-1β treatment increased inflammation in synoviocytes and also increased macrophage M1 and M2 polarization.

Knockdown GRP78 in Synoviocytes Decreased Inflammatory Responses and Promoted M2 Polarization
To explore whether GRP78 is involved in and regulates the IL-1β mediated inflammatory response in synoviocytes in OA, using siGRP78 in synoviocytes, we first showed the knockdown efficiency of GRP78 in synoviocytes (Figure 3a). The inflammatory genes IL-6, IL-8, substance P (SP), PGE-S, TNF-α, iNOS, and COX2 were significantly decreased in the group treated with a combination of IL-1β and siGRP78, compared with the group treated with IL-1β only (Figure 3b). The cytokine levels of IL-6 and PGE2 in synoviocytes were significantly decreased in the group treated with the combination of IL-1β and siGRP78 compared with the group treated with IL-1β only (Figure 3c,d). Moreover, the phosphorylation of p65 was decreased in those treated with the combination of IL-1β and siGRP78, compared with those treated with IL-1β only (Figure 3e). These findings indicated that the knockdown of GRP78 decreased the inflammatory factors and associated inflammatory signaling. We then collected the supernatant from synoviocytes as CM, which we used to treat the macrophages. We then examined the macrophage phenotypes. The M2 cytokines (IL-8 and IL-10) were significantly increased in the cells treated with CM plus IL-1β with siGRP78 compared with the CM plus IL-1β group (Figure 4a). By contrast, the M1 cytokines (IL-12 and TNF-α) were significantly decreased in the cells treated with CM plus IL-1β with siGRP78 compared with the CM plus IL-1β group (Figure 4a). Similarly, the M2 cytokines were also significantly increased in the cells treated with CM plus IL-1β with siGRP78 compared with the CM plus IL-1β group (Figure 4b). The M1 cytokines were also significantly decreased in the cells treated with CM plus IL-1β with siGRP78 ( Figure 4b).
In addition, the expression of CD206 (M2 surface marker) was increased in the cells treated with CM plus IL-1β with siGRP78, compared with the CM plus IL-1β group (Figure 4c), and the expression of CD16 (M1 surface marker) was decreased in the cells treated with CM plus IL-1β, compared with the CM group ( Figure 4d). These data suggest that the knockdown of GRP78 can decrease the inflammatory response and regulate macrophage polarization to promote the M2 phenotype.

Knockdown GRP78 in Synoviocytes Decreased Inflammatory Responses and Promoted M2 Polarization
To explore whether GRP78 is involved in and regulates the IL-1β mediated inflammatory response in synoviocytes in OA, using siGRP78 in synoviocytes, we first showed the knockdown efficiency of GRP78 in synoviocytes ( Figure 3a). The inflammatory genes IL-6, IL-8, substance P (SP), PGE-S, TNF-α, iNOS, and COX2 were significantly decreased in the group treated with a combination of IL-1β and siGRP78, compared with the group treated with IL-1β only (Figure 3b). The cytokine levels of IL-6 and PGE2 in synoviocytes were significantly decreased in the group treated with the combination of IL-1β and siGRP78 compared with the group treated with IL-1β only (Figure 3c,d). Moreover, the phosphorylation of p65 was decreased in those treated with the combination of IL-1β and siGRP78, compared with those treated with IL-1β only (Figure 3e). These findings indicated that the knockdown of GRP78 decreased the inflammatory factors and associated inflammatory signaling. We then collected the supernatant from synoviocytes as CM, which we used to treat the macrophages. We then examined the macrophage phenotypes. The M2 cytokines (IL-8 and IL-10) were significantly increased in the cells treated with CM plus IL-1β with siGRP78 compared with the CM plus IL-1β group ( Figure 4a). By contrast, the M1 cytokines (IL-12 and TNF-α) were significantly decreased in the cells treated with CM plus IL-1β with siGRP78 compared with the CM plus IL-1β group ( Figure 4a). Similarly, the M2 cytokines were also significantly increased in the cells treated with CM plus IL-1β with siGRP78 compared with the CM plus IL-1β group ( Figure 4b). The M1 cytokines were also significantly decreased in the cells treated with CM plus IL-1β with siGRP78 ( Figure 4b). In addition, the expression of CD206 (M2 surface marker) was increased in the cells treated with CM plus IL-1β with siGRP78, compared with the CM plus IL-1β group (Figure 4c), and the expression of CD16 (M1 surface marker) was decreased in the cells treated with CM plus IL-1β, compared with the CM group ( Figure 4d). These data suggest that the knockdown of GRP78 can decrease the inflammatory response and regulate macrophage polarization to promote the M2 phenotype.

HMW-HA Treatment Improves Synovium Inflammation and Macrophage Polarization through GRP78 Expression
HMW-HA (M W 1000-2000 kDa) is used to treat OA, most commonly for OA of the knee. Next, we tried to explore the effect of HMW-HA treatment for synovial inflammation processes and macrophage polarization through GRP78 signaling in synoviocytes. First, we examined the viability of synoviocytes and GRP78 expression after treatment with different dosages of HMW-HA. We found that different HMW-HA dosages did not affect cell viability (Figure 5a), but GRP78 expression was decreased in a dose-dependent manner based on HMW-HA (Figure 5b). Second, we examined the inflammatory factors in synoviocytes after the HMW-HA treatment (1 mg/mL). The inflammatory genes IL-6, IL-8, SP, PGE-S, TNF-α, iNOS, and COX2 were slightly decreased after the HMW-HA treatment and were significantly more decreased with IL-1β plus HMW-HA in the siGRP78 group, compared with the group treated with IL-1β plus HMW-HA (Figure 5c). The cytokine levels of IL-6 and PGE2 in synoviocytes were slightly decreased after the HMW-HA treatment and were significantly more decreased in the IL-1β plus HMW-HA with siGRP78 group, compared with the IL-1β plus HMW-HA group (Figure 5d,e). Moreover, there was a significant decrease in the phosphorylation of p65 in the IL-1β plus HMW-HA group and the IL-1β plus HMW-HA with siGRP78 group as compared to the IL-1β group (Figure 5f). This result indicated that HMW-HA might suppress the inflammatory responses partially through GRP78. To investigate whether HMW-HA treatment can improve inflammatory responses and regulate macrophage polarization, we collected the supernatant from synoviocytes as CM, which we then used to treat macrophages and examine macrophage phenotypes. The M2 cytokines (IL-8 and IL-10) were slightly increased after the HMW-HA treatment and were significantly increased in the group treated with CM plus IL-1β with HMW-HA and siGRP78 compared with the CM plus IL-1β with HMW-HA group (Figure 6a). In contrast, the M1 cytokines (IL-12 and TNF-α) were slightly decreased after the HMW-HA treatment and were significantly decreased in the CM plus IL-1β with HMW-HA and siGRP78 group compared with the CM plus IL-1β with HMW-HA group (Figure 6a). Similarly, the M2 cytokines were slightly increased after the HMW-HA treatment and were also significantly increased in the CM plus IL-1β with HMW-HA and siGRP78 group compared with the CM plus IL-1β with HMW-HA group (Figure 6b). The M1 cytokines were slightly decreased after the HMW-HA treatment and were also significantly decreased in the CM plus IL-1β with HMW-HA and siGRP78 group compared with the CM plus IL-1β with HMW-HA group (Figure 6b). In addition, the expression of CD206 (an M2 surface marker) was increased in the CM plus IL-1β with HMW-HA group and was further increased in the CM plus IL-1β with HMW-HA and siGRP78 group, compared with the CM plus IL-1β with HMW-HA group (Figure 6c), and the expression of CD16 (an M1 surface marker) was decreased in the CM plus IL-1β with HMW-HA group and was further decreased in the CM plus IL-1β with HMW-HA and siGRP78 group, compared with the CM plus IL-1β with HMW-HA group (Figure 6d). Finally, we analyzed the inflammatory cytokines in the synovial fluid before and after the HMW-HA treatment from OA patients. The levels of the cytokines IL-6 and PGE2 were significantly decreased after the HMW-HA treatment (Figure 7a,b). The above results showed that the HMW-HA treatment decreased the GRP78 expression in synoviocytes and then affected the inflammatory cytokines and macrophage polarization toward the M2 phenotype.

Discussion
Previous studies have shown that IL-1β induces an unfolded protein response and that it is associated with cartilage and synovial cells during the induction of OA, such as C/EBP homologous protein (CHOP) and X-box binding protein 1 (XBP1) [27,28]. XBP1 and CHOP increase the release of matrix metallopeptidase (MMP-3) and the related inflammatory cytokines, resulting in the inflammation of the synovial cells and the degradation of chondrocytes [12]. However, under some experimental conditions [28], XBP1s also have a beneficial effect on the survival of chondrocytes. An in vivo surgically induced instability model of knee OA in mice revealed that the knockout of CHOP partially protected against chondrocyte apoptosis and cartilage degradation [29]. In this study, we first found that the GRP78 in synovial cells showed an increasing trend in an IL-1β-induced mimic OA microenvironment. The increased expression of GRP78 was also accompanied by an increase in the secretion of inflammation-related cytokines and activation of the inflammatory pathway. The elimination of GRP78 can reduce the inflammatory condition of synovial cells. The NF-κB pathway was most prominent in human disease and OA models, suggesting a key regulatory role for stress and inflammatory signaling via the NF-κB pathway in OA [30,31]. Abnormal NF-κB pathways cause a loss of chondrocyte growth arrest, and they also produce pro-degrading hormones, including aggrecanase and MMP, which induce cartilage degradation, as well as proinflammatory cytokines [31]. The results of this study show that knockdown GRP78 in the synovial cells reduced the phosphorylation of p65, indicating that GRP78 affected the NF-κB inflammatory pathway and then the proinflammatory cytokines.
Recent studies have shown that macrophages play an important role in the mechanism of OA and RA. In OA, synovial inflammation and cartilage destructiveness were associated with the involvement of macrophages, and these effects were mediated through the cytokines IL-1β and TNF-α [32]. A previous study used a mouse model induced by clodronate liposome treatment to deplete the macrophages of the synovial lining, which resulted in a significant reduction in osteophyte formation as well as in TGF-β, BMP-2, and BMP-4, which are thought to be implicated in osteophyte formation [33]. Numerous in vivo and in vitro studies have since suggested that macrophage polarization from M1 to M2 and vice versa could be a potential treatment strategy for a number of diseases [34]. The studies in RA, OA, gout synovitis, and spondyloarthritis indicate that the balance between M1 and M2 macrophages significantly affects synovial inflammation [35,36]. Synovial macrophages polarized into M1 through the expression of R-spondin-2 (Rspo2) cause OA [37]. In a high-fat-diet-induced obesity mice model of OA, it was also found that the M1 phenotype was increased [38]. In clinical studies, the ratio of M1/M2 in the synovial fluid of patients with OA was higher than that in healthy individuals [39]. In recent years, the relationship between ER stress and polarization of macrophages has been found to be correlated [40]. One study showed that ER stress inositol-requiring enzyme 1α (IRE1α) inhibited the polarization of a macrophage into M2 and then impaired energy expenditure in obesity [41], and the knockdown of CHOP in adipocytes was found to increase the polarization of a macrophage into M2 [42]. However, studies on the relationship between ER stress and the polarization of synovial macrophages are still lacking. In this study, we first found that increased GRP78 expression in IL-1β-induced synovial cell inflammation caused the production of related inflammatory factors, which, in turn, affected the polarization of macrophages. Furthermore, although we knocked down GRP78, the related inflammatory factors were decreased, and the macrophages were polarized into M2.
Injecting HA into the joint is still one way to treat OA. The traditional mechanism is to provide viscoelasticity to the synovial fluid, reducing friction and stress. HA can reduce the destruction of chondrocytes by absorbing internal pressure and vibration [43], and HMW-HA can reduce friction and achieve a therapeutic effect as a result of its greater viscosity [43]. Studies have shown that HA treatment can reduce the IL-1β-induced inflammatory response and result in anti-inflammatory effects [44]. The HA treatment of OA is typically conducted by inhibiting the production of inflammatory cytokines, including IL-8, IL-6, and PGE2 [8]. Other studies have shown that HA of different molecular weights affects the polarization of macrophages. HMW-HA increases the M2 phenotype, whereas HA of a low molecular weight increases the M1 phenotype [45]. One previous study indicated that, in ER stress, inflamed cells bind to the HA-rich extracellular matrix and increase chronic inflammation [46]. However, only a few studies have focused on the interaction between HMW-HA and ER stress regulators. In our study, we found that HMW-HA can inhibit the expression of GRP78, which causes the inflammatory factors to be altered in synovial cells, hence affecting the polarization of macrophages. Moreover, after treatment with both HMW-HA and siGRP78, we found a significantly synergistic effect of reducing the activation of related inflammatory factors and inflammatory pathways and increasing the M2/M1 ratio. Our clinical synovial fluid samples also confirmed the in vitro data, indicating that HMW-HA treatment can reduce inflammatory cytokines, especially IL-6 and PGE-2 levels. Transmembrane protein 2 (TMEM2) can degrade HA and increase the ER stress expression [47], whereas ER stress also induces HA deposition that is conducive to leukocyte binding [46]. CD44 is the major cell surface hyaluronan receptor that is highly expressed by alveolar macrophages. M1 to M2 polarization occurs due to the high affinity of HA to the CD44 receptors of macrophages [48]. Consistent with our results, the HA treatment of synoviocytes affects the macrophages' phenotype forward M2, and it does so through the regulation of the ER stress marker, GRP78. TGF-β stimulated HA production and reduced NF-kB activation and apoptosis in human fibroblast [49]. The study indicated cross talk among GRP78 and NF-κB in prostate cancer cells [50], and the abrogation of GRP78 blunts the activation of NF-κB through the ATF6 branch of the UPR [51]. Blocking the activation of NF-κB signaling can promote the polarization of M1 macrophages to M2 macrophages [21]. Consistent with our results, the HA treatment reduced GRP78 and NF-κB, and increased M2 macrophages.
Our study had limitations. First, IL-1β was used to induce OA, but IL-1β can also indirectly cause p65 phosphorylation; therefore, this part should have used a p65 phosphorylation inhibitor to verify the role of IL-1β in synovial cells. Second, we used the over-expression of GRP78 in primary synovial cells to evaluate the effects on an IL-1βinduced inflammatory response and macrophage polarization, as well as the relatively significant effects of GRP78 knockdown in our study. Third, HA alone can also inhibit the expression of inflammatory genes and p-p65, while the combined use of HMW-HA and siGRP78 can enhance the inhibitory effect, but some still need more experiments to confirm this. Fourth, in vivo animal experiments would be necessary in the future to support the in vitro data in our study.
In conclusion, the present study elucidates the role of GRP78 in OA-related synovial inflammation and macrophage polarization. In addition, our study provides a new vision of HMW-HA for OA treatment. Our results indicate the possible mechanism by which HMW-HA can reduce synovial inflammation through the inhibition of the GRP78/NF-κB inflammatory pathway and proinflammatory cytokines, thereby affecting the polarization of synovial macrophages, increasing the polarization of M2, and achieving anti-inflammatory effects.

Source and Culture of Human Primary Synoviocytes
Human synovial tissue samples were obtained from knee joint patients with OA undergoing a total knee replacement (n = 39, mean age, 70 years; range 58-82 year). All experimental protocols were approved by Taipei Medical University (IRB-CRC-01-10-03), and all patients gave written informed consent. Synovial fluids were obtained by needle aspiration from patients before and after an HMW-HA (Hya-Joint Synovial Fluid Supplement (SciVision Biotech Inc., Kaohsiung, Taiwan) with 650-1200 kDa.) injection and then centrifuged at 14,000× g for 20 min. Supernatants were stored at −80 • C until use. For human primary synoviocytes, synovial tissues were minced, stirred with 3 mg/mL of blend collagenase type H (Sigma) in serum-free DMEM (Gibco) for 6 h before filtering through nylon mesh, and washing extensively. The synoviocytes were maintained in DMEM, supplemented with 10% FBS (Sigma), 100 I.U./mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco). Cells between passages 3 and 5 were used. In this study, 722S, 845S, and 457S were mainly used, all from different patients [53].

Preparation of Conditioned Medium (CM) from Synoviocytes
The synoviocytes were seeded at a density of 1 × 10 4 cells/cm 2 for 72 h. When cultures reached 80% to 90% confluence, cells were treated with IL-1β and HA. The medium was then replaced with fresh serum-free medium for 24 h, and then, cells were pelleted, first at low-speed centrifugation (250× g at 4 • C for 10 min), and then using high-speed centrifugation (1000× g at 4 • C for 10 min) to remove cell debris [54]. This was followed by filtration with a 0.22-mm filter (Millipore, Billerica, MA, USA). The CM was preserved at −80 • C for further study [55].

Quantitative Polymerase Chain Reaction Analysis
Total RNA was isolated using Trizol (Invitrogen, Grand Island, NY, USA). Complementary DNA synthesis was performed using a SuperScript ® III Reverse Transcriptase kit (Invitrogen, Grand Island, NY, USA). We then performed a real-time polymerase chain reaction (PCR) for the gene of interest and SYBR green dye (Thermo, Wilmington, DE, USA) using the LightCycler ® 480 System (Roche). The reaction mixture containing reversetranscribed cDNAs was preheated for 2 min at 95 • C to activate the Taq polymerase. Forty cycles of PCR, each consisting of a 10-s denaturation step at 95 • C and a 30-s annealing step at 60 • C (two-step real-time (RT)-PCR), were then performed. Throughout the RT-PCR analysis, product identities were confirmed using melting curve analysis. The ratio of the amounts of target mRNA to the amount of the internal standard (GAPDH) mRNA was determined as an arbitrary unit [52].

Enzyme-Linked Immunosorbent Assay (ELISA)
The THP-1 cells (5 × 10 4 cells per well of a 96-well plate) were incubated with CM. After a 48-h incubation, the supernatants were collected, followed by the determination of human IL-8, IL-10, IL-12, and TNF-α using the Ready-Set-Go ELISA kit (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions. The synoviocytes (5 × 10 4 cells per well of a 96-well plate) were treated with IL-1β and HA. After treatment for 48 h, the supernatants were collected, followed by the determination of human IL-6 and PGE2 assay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Fluorescence-Activated Cell Sorter Analysis
The THP-1 cells (1 × 10 6 cells per well of a six-well plate) were incubated with CM. After a 48-h incubation, cells were collected using a scraper and blocked with an incubation buffer (dissolved in 0.5 g of bovine serum albumin in 100 mL of phosphate-buffered saline) for 45 min and then incubated with an FITC-conjugated CD16 antibody (11-0168 (1:100 dilution) eBioscience, San Diego, CA, USA) and a PE-conjugated CD206 antibody  (1:100 dilution); eBioscience, San Diego, CA, USA) for 60 min at 25 • C. Following the final washing step, labeled cells were analyzed using flow cytometry on a FACScan flow cytometer using CellQuest software (Becton-Dickinson, Franklin Lakes, NJ, USA). The total number of cells to be harvested was set at 2 × 10 5 , and the collection speed was controlled at 200 to 300 cells/s.

Statistical Analysis
Data are expressed as the mean ± SD, unless otherwise noted. The differences between groups were analyzed using a two-tailed Student's t test when only two groups were present, and the null hypothesis was rejected at the 0.05 level.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study were available on request from the corresponding author.