Leptin augments the inflammatory effect of Interleukin 1 Beta on synoviocytes mainly through NF-kB and STAT3 prompting its possible implementation in OA pathogenesis

doi:10.21203/rs.3.rs-4878390/v1

Download PDF

Research Article

Leptin augments the inflammatory effect of Interleukin 1 Beta on synoviocytes mainly through NF-kB and STAT3 prompting its possible implementation in OA pathogenesis

https://doi.org/10.21203/rs.3.rs-4878390/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Increasing evidences have shown high osteoarthritis (OA) incidence in both weight and non-weight bearing joints among obese patients. Since leptin level in synovial fluid of obese OA patients was elevated compared to that of healthy people, leptin may be a crucial contributing factor in pathogenesis of OA, especially for non-weight bearing joints, in obese patients. This study aimed to examine the effect of leptin on synovial inflammation in the presence or absence of IL-1β, known inducer of joint inflammation, and the plausible intracellular mechanisms in SW982 synoviocytes.

Methods and results

Leptin, in the physiological concentration, as low as 1 ng/mL, can induce the expression of inflammatory cytokines, IL-6 and IL-8, in synoviocytes by activating p65, p38, JNK, STAT1, and STAT3. In addition, the lowest pathological concentration of leptin can enhance the inflammatory effect of IL-1β resulting in significantly higher production of IL-6 and IL-8 via activation of p65 and STAT3, highlighting its role in pathogenesis of inflammation-related OA.

Conclusions

Our research discovered crosstalking of p65 and STAT3, it may have a significant role in the development of OA in the joints of obese people by enhancing inflammation through the activation of p65 and STAT3.

leptin

obesity

IL-1β

synoviocyte

osteoarthritis

OA is a joint degenerative disease characterized by synovium inflammation (synovitis) and articular cartilage degradation [1]. Fibroblast-like synoviocyte (FLS) is one of the cell types in the synovium responsible for joint homeostasis [2]. However, under pathological conditions, it can be a major source of pro-inflammatory cytokines and degradative enzymes leading to OA progression rapidly [3]. Moreover, synovitis has been documented to occur at all stages of OA, even at the early stage [4].

Overweight and obese patients with increased mechanical loading, one of risk factors in OA, are prone to develop OA at load-bearing joints, such as the knee and hip [5]. Nevertheless, OA can also be found in non-weight bearing joints of those patients such as hand. Excessive fat distribution across the body of those patients results in low-grade systemic inflammation, driven by adipocytes in fat tissue generating pro-inflammatory cytokines [6]. Leptin, an adipokine-like hormone produced by adipocytes to regulate food consumption and energy expenditure, has been established as one of the major contributors accounting for the systemic inflammation in obese patients [7]. Leptin levels in synovial fluid and plasma were found to be positively linked with BMI [8–10], and its level in obese OA patients was higher than that in healthy [11]. Comparing with reported physiological leptin levels in healthy synovial fluid, 1.0-4.6 ng/mL, its level in non-obese OA patients [11] and obese OA patients could be as high as 0.5–15.8 ng/mL and 40.22±12.66 ng/mL (pathological concentrations) [12] respectively. In certain cases, leptin levels in synovial fluid of obese OA patients could even approach 120 ng/mL [13]. These data are prompting leptin may have a role in the development of OA, especially, in non-weight bearing joints.

In agreement with the speculation, previous studies found that leptin can induce the production of cartilage degradative enzymes such as matrix metallopeptidase-3 (MMP-3), MMP-13, disintegrin, and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) via the NF-κB and MAPK pathways in human articular cartilage (HAC). Interleukin-1-β (IL-1β) is a pro-inflammatory cytokine known to involve in OA pathogenesis and has been widely used for induction in OA models [3]. In this IL-1β mediated OA induction model as well, leptin enhanced the activity of IL-1β resulting in increased synthesis of those degradative enzymes [14]. Similarly, the inflammatory and destructive effects of leptin were also demonstrated in human OA cartilage; leptin itself induced the expression of pro-inflammatory cytokines such as IL-6, IL-8, nitric oxide (NO), and prostaglandin E2 (PGE2) and degradative enzymes such as MMP-1, -3, and − 13 via NF-κB and MAPK, or amplified the effects of IL-1β resulting in a higher production of the pro-inflammatory cytokines and degradative enzymes [15, 16]. However, all those studies were done in either articular cartilage cells or cartilage. Since synovitis, inflammation of synovium, is the crucial component of OA pathogenesis in addition to cartilage destruction, it is a necessity to study the effect of leptin in synovial cells particularly in the presence of known inflammatory source to mimic the actual environment of an inflamed OA joint. Though leptin alone was reported to increase IL-6 and IL-8 expression in synoviocytes [17, 18], the effect of leptin has not yet elicited in synoviocytes using IL-1β based inflammatory model. Therefore, this study aimed to determine the effect of leptin in combination with IL-1β on the pro-inflammatory cytokine production in SW982 synoviocytes and investigate the signaling pathways involved. Additionally, we studied the effect of Omega-3 fatty acid (ω-3 fatty acid), DHA or EPA, on the leptin and IL-1β induced synoviocytes whether they can protect against the synovial inflammation.

2.1. Cell culture

SW982, a cell line isolated from synovium, was obtained from ATCC^→ No.HTB-93^™. Cells were grown in Leibovitz's L-15 (L-15) medium containing 10% fetal bovine serum (FBS) in, 200 units/ml of penicillin, 200 mg/ml of streptomycin and 50 µg/ml of gentamicin. Cells were incubated at 37°C without CO₂.

2.2. Treatment conditions

Gene and protein expression levels were examined by RT-qPCR and ELISA respectively. Cells were starved in serum-free media for 8 h before being cultured in serum-free media containing leptin (1-100 ng/mL), IL-1β (0.1 ng/mL), or a combination of leptin and IL-1β for 24 h. Then, cell lysate and media were harvested at respective time points.

To confirm the signaling pathway by using commercial inhibitors, cells were pre-treated with either individual or combination of the inhibitors, 10 µM BAY11-7082 and 1 µM ruxolitinib for 2 h before being induced with combination of leptin and IL-1β.

To study the protective effect of fatty acids in SW982 induced with combination of leptin and IL-1β, the free fatty acid-BSA complex was firstly prepared in molar ratio of 5:1 (FA: BSA). Specifically, 1.3% BSA-fatty acid free (BSA-FAF) (Capricorn scientific, Cat: CCS-FAF-1U, Lot# CP19- 2768) in media was mixed with 200 mM of fatty acids, and the mixture was warmed at 37°C and vortexed every 10 minutes until the mixture became clear. Then, Millex-GS Syringe Filter Unit (0.2 µM pore size) was used to filtrate the mixture before it was diluted with media to get the working concentrations used for the treatment. Cells were pre-treated with FA-BSA complex for 24 h which was followed by induction with cytokines before the cell lysate was collected.

2.3. Cell viability assay

When the cells in the culture flask reached 80–90% confluence, they were detached using trypsin-EDTA and transferred to a 96-well plate (30,000 cells/well) for overnight incubation. After cells were then treated with respective treatments for 24 h, the treatment media were removed, and 100 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 0.5 mg/mL prepared in L-15 medium was added to each well. After incubation for 2 hours, MTT reagent was removed, and the formed formazan crystals were dissolved in 100 µL of dimethyl sulfoxide (DMSO). The wavelength used to measure absorbance is 540 nm.

2.4. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR)

Cells were cultured in a 6-well plate with 450,000 cells per well and incubated overnight. The cells were then serum-starved in incomplete media overnight before being induced in the same conditions as described above for 24 hours. RNA was extracted using Illustra^™ Mini RNA Isolation Kits in accordance with the manufacturing protocol, and mRNA was transcribed to cDNA using Tetro cDNA Synthesis kit (Bioline). Gene amplification was performed by the SensiFASTTM SYBR^® Lo-ROX Kit for 45 cycles with the 7500 Fast Real-Time PCR System (Applied Biosystems): denaturation at 95°C for 0.05 minutes, annealing at 60°C for 0.1 minutes, and extension at 72°C for 0.3 minutes. Primers used were GAPDH; F: 5ʹ-GAA GGT GAA GGT CGG AGT C-3’, R: 5ʹ-GAA GAT GGT GAT GGG ATT TC -3’. IL-6; F: 5’-GGT ACA TCC TCG ACG GCA TCT-3’, R: 5’-GTG CCT CTT TGC TGC TTT CAC-3’. IL-8; F: 5’-CTC TCT TGG CAG CCT TCC-3’, R: 5’-CTC AAT CAC TCT CAG TTC TTT G-3’. TNF-α; F: 5’CCC CAG GGA CCT CTC TCT AAT C-3’, R: 5’GGT TTG CTA CAA CAT GGG CTA CA-3’. GAPDH was used as an internal control, and 2^-ΔΔCt method was utilized to analyze relative gene expression [19].

2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

The media was collected for evaluation of secretory proteins using ELISA (ELISA MAX^™ Deluxe Set Human IL-6, cat. 430504 and IL-8, cat. 431504) by following the manufacturer's instructions.

2.6. Western blot

Cells were treated the same as forementioned, and they were induced for 30 minutes before being collected using RIPA buffer. For p65, p38, and ERK, 3 µg of protein was loaded into each lane of 12% Tris-glycine gel and separated by electrophoresis using a constant voltage of 90 volts for 2 hours. The proteins were then transferred onto nitrocellulose membrane using a trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (BioRad). Then, the membrane was blocked with 5% skim milk before incubation with primary antibodies against the proteins of interest, prepared in 0.1% Tween-PBS (1:1000), overnight at 4°C. After washing 4 times, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG in 0.1% T-PBS (1:1000) for 1 hour at room temperature. SuperSignal ECL Chemiluminescent kit (Thermo Scientific) was used as substrate and ChemiDoc^™ Imagine System (BioRad) was used to detect the band density. In case of JNK, STAT-1 and STAT-3, 30 µg of proteins was used. Apart from the amount of protein, all the other procedures were the same. β-actin was used as the internal control. Band intensity was measured using ImageJ program.

2.7. Statistical analysis

Data from three independent experiments is presented as mean ± standard deviation (SD). One-way ANOVA was chosen for the statistical analysis, and the Turkey test was employed to identify statistically significant group pairings.

3.1. Leptin enhances the effect of IL-1β on increasing of pro-inflammatory cytokines production

The effect of leptin alone and combination of leptin with IL-1β on production of pro-inflammatory cytokines, IL-6, IL-8, and TNF-α, was studied in SW982 cells. Concentrations of leptin were chosen to reflect the physiological (1 ng/mL) and pathological concentrations (10–100 ng/mL). In addition, supra-pathological concentration (500–1000 ng/mL) which was used in many previous studies was also included in this study. For IL-1β induced inflammatory model, 0.1ng/mL concentration of IL-1β was selected to combine with leptin. The effect of leptin alone, IL-1β alone and combination of leptin with IL-1β on cell viability was first investigated using MTT assay, and the findings confirmed that all treatments had no toxic effect on SW982. Meanwhile, combination of leptin at concentration greater than 500 ng/mL (supra-pathological concentration) with IL-1β slightly increased cell proliferation (Fig. 1). According to results of gene and protein expression (Fig. 2), while IL-1β, 0.1ng/mL, significantly elevated the levels of all pro-inflammatory cytokines, leptin alone just slightly increased IL-6, IL-8, and TNF-α expressions both in gene and protein levels, except leptin at 1000 ng/mL which increased the expressions statistically significantly at gene level. However, combination of leptin, at concentration greater than physiological concentration, with IL-1β could amplify the effect of IL-1β-mediated IL-6 and IL-8 upregulation at mRNA level. Moreover, combination of leptin, at concentration greater than pathological concentration, with IL-1β could magnify the effect of IL-1β on expression of IL-6 and IL-8 at protein level as well. Still, leptin did not enhance the IL-1β-mediated TNF-α upregulation.

3.2. Leptin enhances the effect of IL-1β via p65 and STAT3 signaling proteins

In next experiment, western blotting was used to identify the intracellular signaling pathways responsible for augmentative effect of leptin on effect of IL-1β. The result revealed that leptin only slightly increased the phosphorylation of p65, p38, JNK, STAT1, and STAT3 (Fig. 3A-D, F-G) whereas phosphorylation levels of p65, p38, and JNK were all highly increased by IL-1β (Fig. 3A-D) as well as STAT3 (Fig. 3A and G). However, IL-1β had no effect on STAT1 phosphorylation (Fig. 3A and F). Comparing to the individual treatments, combination of leptin with IL-1β significantly activated p65 and STAT3 phosphorylation (Fig. 3A-B, G) and the activation level was greater than that induced by either leptin alone at the same concentration or IL-1β alone. Consequently, p65 and STAT3 might be the major signaling pathways accounting for the enhanced IL-6 and IL-8 expressions in SW982 cell line induced by combination of leptin with IL-1β.

3.3. Pro-inflammatory cytokines induced by leptin and IL-1β are suppressed by signaling inhibitors, BAY11-7082 and ruxolitinib

To confirm the primary signaling pathways mediated by IL-6 and IL-8 that are accountable for the elevated levels of inflammatory cytokines by combination of leptin with IL-1β in the SW982, the commercial inhibitors, BAY11-7082 (10 µM) and ruxolitinib (1 µM) were used in this experiment. Regarding mRNA analysis and secretory protein levels in Fig. 4, BAY11-7082 reduced the effects of leptin resulting in decreased IL-6 and IL-8 gene expression, while ruxolitinib did not show any effect. In IL-1β treatment, BAY11-7082 reduced IL-6 and IL-8 expression in both of gene and protein levels whereas ruxolitinib just decreased IL-6 and IL-8 gene expression. As expected, either individual or combination of inhibitors could reduce the effects of combination of leptin with IL-1β on IL-6 and IL-8 expressions while the vehicle control, DMSO, which was used to dissolve inhibitors had no effect on both IL-6 and IL-8 expressions.

Compared to BAY11-7082, ruxolitinib also decreased the effect of leptin, IL-1β, and leptin combined with IL-1β, but with much lesser extent than BAY11-7082. Surprisingly, treatment with combined inhibitors could also reduce the secretion of IL-6 and IL-8 proteins but not as much as BAY11-7082 alone although it could reduce IL-6 and IL-8 mRNA levels more than BAY11-7082 alone did.

3.4 Inhibition of NF-κB can diminish the effect of leptin and IL-1β on induction of pro-inflammatory cytokines

The underlying intracellular signaling of combination of leptin with IL-1β was confirmed by using inhibitors and western blotting. The results showed that BAY11-7082 dramatically reduced leptin effect via inhibition of activation of not only p65 but also STAT3 as same as ruxolitinib. Surprisingly, BAY11-7082 inhibited both phosphorylated-p65 and phosphorylated-STAT3, while ruxolitinib inhibited only phosphorylated-STAT3, regardless of induced conditions, either IL-1β alone treatment or combination of leptin with IL-1β (Fig. 5). Based on this result, it was suggested that NF-κB and STAT3 might have a crosstalk.

Additionally, the combination of inhibitors has less inhibitory effect on p65 phosphorylation than BAY11-7082 alone. Thus, inhibition of p65 may be enough for reduction of IL-6 and IL-8 productions. Other compounds that can suppress both NF-κB and STAT3 or individual NF-κB induced by combination of leptin with IL-1β might be the best candidate for inhibition of the inflammation in this model.

3.5. DHA and EPA prevent inflammation of SW982 induced by combination of leptin with IL-1β

Omega-3 fatty acid (ω-3 fatty acid) becomes a popularized food supplement in the last decade [20]. Most of studies documented its anti-inflammatory property in several inflammatory diseases including osteoarthritis. Integrating ω-3 fatty acids into cell membranes causes low-grade inflammation by converting to inflammatory mediators. Moreover, ω-3 fatty acid can directly cross through cell membrane to inhibit signaling pathways [21, 22]. Previous studies in HAC induced with combination of IL-1β and leptin, EPA or DHA decreased p65 phosphorylation [23]. In addition, EPA was shown to reduce NF-kB phosphorylation in response to other agents that triggered NF-kB signaling. EPA inhibited p38 MAPK and NF-kB signaling in sodium nitroprusside (SNP)-induced normal human chondrocytes-knee [24]. In in vivo model, oral administration of EPA reduced NF-kB phosphorylation in rats intra-injected with complete Freund’s adjuvant (CFA) [25]. Similarly, DHA diminished the activation of NF-kB in SW1353 (26) stimulated by LPS [26]. However, there has been no report whether DHA or EPA can reduce pro-inflammatory cytokines in SW982 induced with combination of leptin and IL-1β. Thus, the effect of both EPA and DHA on anti-inflammation of synoviocytes induced with leptin and IL-1β was also investigated in this study.

To determine the anti-inflammatory effect of DHA and EPA on pro-inflammatory cytokines production and molecular mechanism, SW982 cells were pre-treated with various concentrations (1.25-10 µM) of either DHA, EPA and BSA complex before induction with combination of leptin and IL-1β. According to Fig. 6, the reductive ability of DHA on IL-6 and IL-8 expressions was showed at low concentrations, 1.25 and 2.5 µM, both in mRNA levels and secretory proteins of IL-6 and IL-8. In contrast, EPA had the highest reduction efficacy on IL-6 and IL-8 expression at high concentration, 10 µM. In addition, the BSA-complex had no effect on both IL-6 and IL-8 expression.

Based on our western blot results showed in Fig. 7, DHA had the most significant effect on the suppression of p65 and STAT3 phosphorylation at the lowest concentration of 1.25 µM. While EPA showed statistically reduction p65 and STAT3 phosphorylation at highest concentration of 10 µM. Comparing the reductive effect of ω-3 fatty acids with BAY11-7082, ω-3 fatty acids had similar inhibiting activities as BAY11-7082, even though their effects were lower. Hence, these results indicated that ω-3 fatty acids which can suppress p65 and STAT3 activation, may alleviate IL-6 and IL-8 production in SW982 induced by combination of leptin and IL-1β.

Our findings suggested that leptin at concentrations 1, 10, 100, and 500 ng/mL, could modestly trigger the production of pro-inflammatory cytokines such IL-6, IL-8, and TNF-α, but not considerably. Only exception was leptin at supra-pathological concentration, 1000 ng/mL, which promoted pro-inflammatory cytokines production, IL-6 and IL-8. These findings agree with the study done in the temporomandibular joint osteoarthritis synovial fibroblasts (TMJ-SFs) which reported leptin at pathological concentrations did not significantly increase IL-6 production at the gene and protein levels [27]. Furthermore, supra-pathological leptin concentrations were reported to increase IL-6 expression in TMJ-SFs and OASF [18, 27] and IL-8 expression (leptin was used at 160 g/mL) in OASF [17].

Regarding TNF-α expression, leptin did not significant upregulate TNF-α gene even at 1000 ng/mL in this study. Relevant to Yamazaki et al., they reported that the expression of TNF-α mRNA was not upregulated in SW982-induced with IL-1β at 1 ng/mL [28]. Furthermore, TNF- α level in obese OA patient’s synovial fluid was reported as low as 15 pg/mL, and that level was not different compared with healthy people [29]. Based on these findings, leptin may be ineffective for inducing TNF-α or the concentration of leptin used in this study may not be the effective dose for upregulation of TNF-α in the SW982. Therefore, in this study, leptin at supra-pathological concentration showed a significant inductive effect on the expression of pro-inflammatory cytokines, IL-6 and IL-8 but not TNF-α, in accordance with the findings of previous studies [16, 30–32].

Moreover, our study showed that leptin at lowest pathological concentrations could enhance the effect of IL-1β on the expression of mRNA of IL-6 and IL-8. Consistent with the findings of Vuolteenaho K., et al., that leptin at concentrations of 100 and 1000 ng/mL also amplified the effect of IL-1β by inducing the synthesis of IL-6 and IL-8 in human OA cartilage [16]. Agreeing with the current study, Phitak et al., found that leptin showed an additive effect with IL-1β in enhancing the expression of MMP-3, MMP-13, and ADAMTS-4 in HAC [23] and in downregulation of ECM components such as ACAN, COL2A1, and COL1A1 in chondrocyte pellet system [14]. Hence, our results confirmed and indicated that synovium is a source of pro-inflammatory cytokines, IL-6 and IL-8. Leptin either alone or combination with IL-1β could drive IL-6 and IL-8 expression and leptin could enhance production of IL-6 and IL-8 substantially in the presence of IL-1β. This finding suggested that in obese people who already have systemic low-grade inflammation related to high leptin level, whenever any additional inflammation occurs in joints and synovium, leptin may accelerate the OA pathogenesis resulting in detrimental effects on joints by augmenting the effect of IL-1β mediated IL-6 and IL-8 production.

During OA development, there are multiple pathways involved, including NF-κB, MAPK, JAK/STAT, AMP-activated protein kinase (AMPK), and other pathways. Our results revealed that all leptin concentrations used in this study could activate p65, p38, JNK, STAT1, and STAT3 in SW982 agreeing with many previous studies in other cell types or different sources of cells such as OASF [17], TMJ-SF [27], and chondrocytes [23, 33, 34]. Nonetheless, there were contradictory results in some reports as well. They found that leptin 100 ng/mL failed to induce STAT3 in obese patients' chondrocytes [34]. For IL-1β we used the low dose, however it still had the same efficiency as previous studies [30, 31, 35].

Furthermore, one of the goals of this study is to determine the molecular mechanism of the potentiating effect of leptin over effect of IL-1β on the production of pro-inflammatory cytokines. The result showed that leptin combined with IL-1β further induced the activation of p65 and STAT3 comparing with the IL-1β alone referring that leptin combined with IL-1β enhanced the IL-1β induced IL-6 and IL-8 expression via p65 and STAT3 pathways in SW982. Relevant to our findings, previous study in HAC and human juvenile costal chondrocyte cell line T/C-28a2, leptin also showed an additive effect with IL-1β on expression of protease enzymes through NF-κB [36] and JNK [23]. Additionally, our result showed that BAY11-7082, which is specific to inhibit IκBα phosphorylation in NF-κB, can inhibit both of p65 and STAT3 phosphorylation similar to Baptiste et al [37]. It indicates that there was crosstalk between p65 and STAT3 and it may be the key regulators of pro-inflammatory cytokine productions in SW982. Hence, p65 and STAT3 may be the potential therapeutic targets for management of OA in obese patients.

Numerous studies demonstrate the protective effects of ω-3 fatty acids against several inflammatory disorders, such as rheumatoid arthritis, and obesity, where the main pathway responsible for generating pro-inflammatory cytokines is mediated by NF-κB [38]. Furthermore, there is document that suggests STAT3 has a role in the signal transduction of inflammatory cell [39, 40]. Our findings confirmed that DHA at 1.25 µM (low dose) and EPA at 10 µM (high dose) could reduce pro-inflammatory cytokine productions, both IL-6 and IL-8, induced by leptin combined with IL-1β via suppression of p65 NF-κB and STAT3. EPA has less inhibitory effect since it could be converted to DHA by enzymes within the cells [41]. However, it must be confirmed by measurement of PUFA composition in the model. These results are similar to the previous studies on joint-resident cells. In equine synoviocyte, pre-treatment of DHA or EPA before stimulation with IL-1β suppressed the expression of cytokines, IL-6, IL-1β, and catabolic enzymes, ADAMTS-4, MMP-1, -13 and COX-2 [42]. Additionally, in IL-1β induced SW1353 and a rat AIA model, DHA could inhibit MMP-13 expression through suppression of p38 phosphorylation [43]. Individually pre-treatment DHA or EPA reduced the production IL-1α, IL-1β, TNF-α, ADAMTS-4,-5, COX-2, and MMP-3 in bovine chondrocyte induced by IL-1. In human chondrocytes induced by leptin combined with IL-1β, both DHA and EPA could decrease ADAMTS-4 secretion via inhibition of NF-κB and JNK activation [23].

Our study suggested that DHA and EPA which can inhibit p65 and STAT3 activation, could ameliorate the production of IL-6 and IL-8 induced by leptin combined with IL-1β in the SW982. However, further studies in animal model are warranted, and the effective doses need to be considered. Due to the fact that ω-3 fatty acid has both pro- and anti-inflammatory effects, the dosages of these fatty acid should be investigated further in in vivo models. Taken together, compounds that have ability to inhibit both NF-κB and STAT3 might be the best candidates for OA management and prevention of OA in joints of obese patients.

Our results indicate the combination of leptin and IL-1β is mainly regulated by p65 and STAT3 in synovial fibroblasts of obese patients who may developing osteoarthritis.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethical approval

No applicable.

Funding

This work was supported by Faculty of Medicine Research Fund, Chiang Mai University (to Thanyaluck Phitak, grant number 009/2564 and 039/2562).

Author Contribution

A.W.; Perform and co-design experimemts, Software and Writing-Original draft preparation. P.K. and P.P; Supervision. T.S.; Editing. T. P.; Design experiments, Data curation, Conceptualization, Writing-Reviewing and Editing. All authors read and approved the final manuscript.

Acknowledgement

The authors would like to thank the Thailand Excellence Center for Tissue Engineering and Stem Cells, Faculty of Medicine, Chiang Mai University, Thailand for providing the instruments used in this study.

Ingale D et al (2021) Synovium-Synovial Fluid Axis in Osteoarthritis Pathology: A Key Regulator of the Cartilage Degradation Process. Genes, 12(7)
Kapoor M (2015) Pathogenesis of Osteoarthritis, in Osteoarthritis. pp. 1–28
Chow YY, Chin K-Y (2020) The Role of Inflammation in the Pathogenesis of Osteoarthritis. Mediators of Inflammation, 2020: pp. 1–19
Mathiessen A, Conaghan PG (2017) Synovitis in osteoarthritis: current understanding with therapeutic implications. Arthritis Res Therapy 19(18):1–9
Thijssen E, Caam Av, Kraan PMvd (2015) Obesity and osteoarthritis, more than just wear and tear: pivotal roles for inflamed adipose tissue and dyslipidaemia in obesity-induced osteoarthritis. Rheumatology 54(4):588–600
Tsuchiya H, Fujio K (2022) Emerging role of leptin in joint inflammation and destruction. Immunological Med 45(1):27–34
Leon-Cabrera S et al (2013) Hyperleptinemia is associated with parameters of low-grade systemic inflammation and metabolic dysfunction in obese human beings. Front Integr Neurosci 7:62
Vuolteenaho K et al (2012) Leptin levels are increased and its negative regulators, SOCS-3 and sOb-R are decreased in obese patients with osteoarthritis: a link between obesity and osteoarthritis. Ann Rheum Dis 71(11):1912–1913
Vuolteenaho K, Koskinen A, Moilanen E (2014) Leptin - a link between obesity and osteoarthritis. applications for prevention and treatment, vol 114. Basic & Clinical Pharmacology & Toxicology, pp 103–108. 1
van Dielen FMH, Schols CvtVAM, Soeters PB, Buurman WA, Greve JWM (2001) Increased leptin concentrations correlate with increased concentrations of inflammatory markers in morbidly obese individuals. Int J Obes 25:1759–1766
Ku JH et al (2009) Correlation of synovial fluid leptin concentrations with the severity of osteoarthritis. Clin Rheumatol 28(12):1431–1435
Abdel-Rahman AAH et al (2020) Impact of obesity on leptin, leptin receptor gene polymorphism, and some adipokines in egyptian patients with knee osteoarthritis. Indian J Rheumatol, 15(2)
Gandhi R et al (2010) Relationship between body habitus and joint leptin levels in a knee osteoarthritis population. J Orthop Res 28(3):329–333
Phitak T et al (2020) Effect of Leptin Alone and in Combination withIL1β on Human Chondrocytes in a Pellet Culture System, vol 19. Chiang Mai University Journal of Natural Sciences, 4
Koskinen A et al (2011) Leptin enhances MMP-1, MMP-3 and MMP-13 production in human osteoarthritic cartilage and correlates with MMP-1 and MMP-3 in synovial fluid from OA patients. Clin Exp Rheumatol 29(1):57–64
Vuolteenaho K et al (2009) Leptin enhances synthesis of proinflammatory mediators in human osteoarthritic cartilage–mediator role of NO in leptin-induced PGE2, IL-6, and IL-8 production. Mediators of Inflammation, 2009: p. 345838
Tong K-M et al (2008) Leptin induces IL-8 expression via leptin receptor, IRS-1, PI3K, Akt cascade and promotion of NF-kappaB/p300 binding in human synovial fibroblasts. Cell Signal 20(8):1478–1488
Yang W-H et al (2013) Leptin induces IL-6 expression through OBRl receptor signaling pathway in human synovial fibroblasts. PLoS ONE 8(9):e75551
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408
Thomas S et al (2018) What is the evidence for a role for diet and nutrition in osteoarthritis? Rheumatology 57(suppl4):iv61–iv74
Knott L et al (2011) Regulation of osteoarthritis by omega-3 (n-3) polyunsaturated fatty acids in a naturally occurring model of disease. Osteoarthritis Cartilage 19(9):1150–1157
Raphael W, Sordillo LM (2013) Dietary polyunsaturated fatty acids and inflammation: the role of phospholipid biosynthesis. Int J Mol Sci 14(10):21167–21188
Phitak T et al (2018) Leptin alone and in combination with interleukin-1-beta induced cartilage degradation potentially inhibited by EPA and DHA. Connect Tissue Res 59(4):316–331
Sakata S et al (2015) Oxidative stress-induced apoptosis and matrix loss of chondrocytes is inhibited by eicosapentaenoic acid. J Orthop Res 33(3):359–365
Morin C, Blier PU, Fortin S (2015) Eicosapentaenoic acid and docosapentaenoic acid monoglycerides are more potent than docosahexaenoic acid monoglyceride to resolve inflammation in a rheumatoid arthritis model. Arthritis Res Ther 17:142
Jin X et al (2022) Dietary Fatty Acid Regulation of the NLRP3 Inflammasome via the TLR4/NF-kappaB Signaling Pathway Affects Chondrocyte Pyroptosis. Oxid Med Cell Longev, 2022: p. 3711371
Xiong H et al (2019) Elevated leptin levels in temporomandibular joint osteoarthritis promote proinflammatory cytokine IL-6 expression in synovial fibroblasts. J Oral Pathol Med 48(3):251–259
Taisuke Yamazaki TY, Akatsu H, Tukiyama T, Tokiwa T Phenotypic characterization of a human synovial sarcoma cell line, SW982, and its response to dexamethasone. In Vitro Cellular & Developmental Biology - Animal 2003. 39: pp. 337–339
Pearson MJ et al (2017) IL-6 secretion in osteoarthritis patients is mediated by chondrocyte-synovial fibroblast cross-talk and is enhanced by obesity. Sci Rep 7(1):3451
Cheng B-F et al (2016) Hydroxysafflor yellow A inhibits IL-1beta-induced release of IL-6, IL-8, and MMP-1 via suppression of ERK, NF-kappaB and AP-1 signaling in SW982 human synovial cells. Food Funct 7(11):4516–4522
Lee YS, Choi EM (2010) Myricetin inhibits IL-1β-induced inflammatory mediators in SW982 human synovial sarcoma cells. Int Immunopharmacol 10(7):812–814
Yang J et al (2019) IL–1beta increases the expression of inflammatory factors in synovial fluid–derived fibroblast–like synoviocytes via activation of the NF–kappaB–mediated ERK–STAT1 signaling pathway. Mol Med Rep 20(6):4993–5001
Yaykasli KO et al (2015) Leptin induces ADAMTS-4, ADAMTS-5, and ADAMTS-9 genes expression by mitogen-activated protein kinases and NF-kB signaling pathways in human chondrocytes. Cell Biol Int 39(1):104–112
Stéphane Pallu P-JF, Cécile Guillaume P, Gegout-Pottie P, Netter (2010) Didier Mainard, Bernard Terlain and Nathalie Presle, Obesity affects the chondrocyte responsiveness to leptin in patients with osteoarthritis. Arthritis Res Therapy 12(3)
Chenga B-F et al (2017) Anti-inflammatory effects of pitavastatin in interleukin-1β-induced SW982 human synovial cells. Int Immunopharmacol 50:224–229
Conde J et al (2018) E74-Like Factor (ELF3) and Leptin, a Novel Loop Between Obesity and Inflammation Perpetuating a Pro-Catabolic State in Cartilage. Cell Physiol Biochem 45(6):2401–2410
Baptiste Dumétier AZ, Berthelet J, Causse Sébastien, Allègre J, Bourgeois P, Cattin F, Racoeur C, Garrido CPC et al (2023) cIAP1/TRAF2 interplay promotes tumor growth through the activation of STAT3. Oncogene 42(3):198–208
Calder PC (2010) Omega-3 fatty acids and inflammatory processes. Nutrients 2(3):355–374
Chiu CF et al (2021) Eicosapentaenoic Acid Inhibits KRAS Mutant Pancreatic Cancer Cell Growth by Suppressing Hepassocin Expression and STAT3 Phosphorylation. Biomolecules, 11(3)
Donatella D’, Santoni A, Velotti F (2017) Docosahexaenoic acid (DHA) promotes immunogenic apoptosis in human multiple myeloma cells, induces autophagy and inhibits STAT3 in both tumor and dendritic cells. Genes Cancer 8:426–437
Whelan J (2009) Dietary stearidonic acid is a long chain (n-3) polyunsaturated fatty acid with potential health benefits. J Nutr 139(1):5–10
Caron JP et al (2019) Omega-3 fatty acids and docosahexaenoic acid oxymetabolites modulate the inflammatory response of equine recombinant interleukin1beta-stimulated equine synoviocytes. Prostaglandins Other Lipid Mediator 142:1–8
Wang Z et al (2016) Docosahexenoic acid treatment ameliorates cartilage degeneration via a p38 MAPK-dependent mechanism. Int J Mol Med 37(6):1542–1550

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Leptin augments the inflammatory effect of Interleukin 1 Beta on synoviocytes mainly through NF-kB and STAT3 prompting its possible implementation in OA pathogenesis

Status:

Version 1

Abstract

Background

Methods and results

Conclusions

Figures

1. Introduction

2. Method

2.1. Cell culture

2.2. Treatment conditions

2.3. Cell viability assay

2.4. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR)

2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

2.6. Western blot

2.7. Statistical analysis

3. Result

3.1. Leptin enhances the effect of IL-1β on increasing of pro-inflammatory cytokines production

3.2. Leptin enhances the effect of IL-1β via p65 and STAT3 signaling proteins

3.3. Pro-inflammatory cytokines induced by leptin and IL-1β are suppressed by signaling inhibitors, BAY11-7082 and ruxolitinib

3.5. DHA and EPA prevent inflammation of SW982 induced by combination of leptin with IL-1β

4. Discussion

Conclusions

Declarations

Competing Interests

Ethics declarations

Funding

Author Contribution

Acknowledgement

References

Additional Declarations

Status:

Version 1