TNF‑α increases inflammatory factor expression in synovial fibroblasts through the toll‑like receptor‑3‑mediated ERK/AKT signaling pathway in a mouse model of rheumatoid arthritis
- Authors:
- Published online on: April 19, 2018 https://doi.org/10.3892/mmr.2018.8897
- Pages: 8475-8483
Abstract
Introduction
Knee osteoarthritis is a disease with the degenerative changes in the articular hyaline cartilage (1). The incidence of knee osteoarthritis is increasing due to prevalence in aging societies worldwide, which is characterized by degenerative disease that mainly caused by inflammation and dysfunction of synovial cells (2–4). A systematic review has given the overview of factors (age, body mass index, level of physical function and level of physical activity) related to patients with hip or knee osteoarthritis (5). The pathological reasons of osteoarthritis are complex in classification system manner and the cellular pathogenesis in joints (6). Previous study has indicated that inflammation is a variable feature of knee osteoarthritis, and is associated with joint symptoms and progression of this disease (7). A report has showed that decreasing of tumor necrosis factor (TNF)-α, interleukin (IL)-1 and IL-6 contents in joint fluid markedly improved symptoms in the rabbit with knee osteoarthritis (8).
A previous study has investigated the association between systemic and local inflammation and incident and progressive radiographic secondary osteoarthritis and results found that TNF-α inhibitor infliximab therapy is effective against hand osteoarthritis (9). Qin et al reported that TNF/TNFR signal transduction pathway-mediated anti-apoptosis and anti-inflammatory effects of sodium ferulate on IL-1b-induced rat osteoarthritis chondrocytes (10). Study also identified that decreasing of circulating levels of TNF-α inhibited the development of osteoarthritis (11). Systemic blockade of STAT-3 can alleviate medial meniscus-induced osteoarthritis in mice (12). However, the associations between TNF-α and STAT3 pathway has not reported.
Currently, toll-like receptor-3 (TLR-3) can regulate the STAT3 pathway in rheumatoid arthritis fibroblast-like synoviocytes (13). Evidences have demonstrated that TLR-3 is overexpressed in synovial tissue in patients with early rheumatoid, suggesting that TLR-3 signal pathway may associate with the persistent inflammation and joint destruction in this disease process (14). Zhu et al have found that pristane-induced arthritis rats presented higher TLR3 expression levels in the synovium, and increased the activity of the TLR3 signal pathway (15). Domagala et al have reported that inhibition of IL-1β-induced activation of extracellular signal-regulated kinase (ERK) signal pathway showed benefits for the treatment of osteoarthritis (16). Klosowska et al have found that targeting of ERK/protein kinase B (AKT) signal pathway reduced Fractalkine-induced osteoarthritis fibroblast migration via alterations of cytoskeletal structure in the pathologic processes of osteoarthritis (17). However, the relationships between ERK/AKT signal pathway and osteoarthritis have not well investigated, yet.
In the present study, we investigated the role of TNF-α-mediated TLR-3/ERK/AKT pathways in synovial fibroblasts in osteoarthritis model. We also analyzed the in vivo effects of inhibition of TNF-α activity in monosodium iodoacetate-induced osteoarthritis mice model.
Materials and methods
Ethics statement
This study was approved by Ethics Committee of the 309th Hospital of People's Liberation Army. All surgeries were performed under intravenous injection of sodium pentobarbital anesthesia (35 mg/kg).
Animals study
A total of 40 male C57BL/6 mice (25–32 g, 5–6 months of age) were purchased from Shanghai SLAC Laboratory Animals Co., Ltd (Shanghai, China). All mice were housed under controlled temperatures in a 12 h light/dark cycle with easy access to food and water. All mice were identified by ear punching and used to establish osteoarthritis mice model induced by monosodium iodoacetate (0.2 mg per mice; Sigma-Aldrich Co., St. Louis, MO, USA) as described previously (18). On day 10 after monosodium iodoacetate administration, mice were randomly assigned to two groups vehicle group (n=20) and TNF-α inhibitor group (n=20). Mice were received subcutaneous injection of vehicle (0.2 mg/kg/day) or TNF-α inhibitor (0.2 mg/kg/day; Lenalidomide, Sigma-Aldrich) for a total of 32 days. All mice were sacrificed on day 40 for histological analysis. Body weights of experimental mice were measured prior and post treatments on day 40. The osteonecrosis mice were anesthetized under IV pentobarbital anesthesia (35 mg/kg) and sacrificed by cervical dislocation on day 40 for further analysis.
Cells isolation and culture
Synovial fibroblasts were isolated from experimental mice as described (19) and cultured in DMEM medium with 10% fetal bovine serum (FBS; both Sigma-Aldrich) at 37°C and 5% CO2 humidified atmosphere. Synovial fibroblasts (1×106 cells/well) were seeded into six-well plates and treated by TNF-α (2 mg/ml), TNF-α inhibitor (2 mg/ml) or PBS (all Sigma-Aldrich) for 24 h at 37°C for RT-qPCR and western blot analysis.
Enzyme-linked immunosorbent assay (ELISA)
Concentrations of TNF-α (MTA00B, Bio-Rad), IL-1β (MLB00C), IL-4 (M4000B) and IL-6 (DY406; all Bio-Rad, Berkeley, CA, USA) concentrations in serum from experimental mice were analyzed using ELISA. TNF-α, IL-1β, IL-4 and IL-6 were measured by using ELISA kits (Bio-Rad) according to the manufacturer's instructions. The results were performed by ELISA reader system (1775×Mark™; Bio-rad).
Knockdown of TLR-3
Small interference RNAs (siRNA) for TLR-3 (siRTLR-3) were synthesized by Ribobio Co., Ltd. (Guangzhou, China). siRTLR-3 sense, 5′-CCUGAGCUGUCAAGCCACUACCUUU-3′ and antisense, 5′-AAAGGUAGUGGCUUGACAGCUCAGG-3′; siRcontrol sense, 5′-CCUGUCGAACUACCGCAUCCAGUUU-3′ and antisense, 5′-AAACUGGAUGCGGUAGUUCGACAGG-3′. Synovial fibroblasts (1×106 cells/well) were seeded onto 6-well plates and transiently transfected with 120 nmol siRTLR-3 with negative control (NC) siRNA as control using RNAi MAX (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturers' instructions. All experiments were performed in triplicate and further analysis was performed after a 48-h transfection. After a 48-h transfection, cells were then treated with TNF-α inhibitor (2 mg/ml), or PBS for 24 h at 37°C for further analysis.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays
Total RNA was extracted from synovial fibroblasts using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturers' instructions. Extracted mRNA (1 µg) was transcribed into cDNA at 42°C for 2 h using a reverse transcription kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. The cDNA (10 ng) was used for qPCR using the SYBR-Green Master Mix system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's instrument. All primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) (Table I). After 120 sec incubation at 95°C, PCR was performed using 40 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec and elongation at 72°C for 30 sec. Relative gene expression levels were calculated using the 2−ΔΔCq method (20). The results were presented as the n-fold change compared with β-actin using Quantiscan 2.1 (software Demo of AB QuantStudio™ 12K Flex System; Thermo Fisher Scientific, Inc.).
Western blot analysis
Western blotting was used to evaluate protein levels in this study as previously described (21). Briefly, a total of 1×107 synovial fibroblasts were lysed in a lysis buffer containing 1% phenylmethanesulfonyl fluoride (Sigma-Aldrich) for 3 times of freezing-thawing and centrifuged at 8,000 × g for 10 min at 4°C. Concentration of proteins was measured by BCA kit (no. 23225; Thermo Fisher Scientific, Inc.). Proteins (20 µg) were separated using the 12% sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE). Then proteins were transferred onto proteins to polyvinylidene fluoride (PVDF) membrane (Millipore, Massachusetts, MA, USA). Then the nitrocellulose membrane was blocked with 5% (w/v) nonfat dry milk dissolved in tris-buffered saline plus Tween-20 (TBST) solution for 2 h at 37°C and followed by incubation with primary rabbit anti-mouse antibodies: TNF-α (1:1,000; ab6671), IL-1β (1:1,000; ab200478), IL-4 (1:1,000; ab9728), IL-6 (1:500; ab7737), PI3K (1:2,000; ab1678), ERK (1:2,000; ab196883), pERK (1:2,000; ab214362), AKT (1:1,000; ab8805), pAKT (1:1,000; ab133458), matrix metalloproteinase (MMP)-3 (1:1,000; ab53015) and MMP-9 (1:1,000; ab38898), receptor activator of NF-κB ligand (RANKL; 1:1,000; ab216484), NF-κBp65 (1:1,000; ab32536) and β-actin (1:2,000, ab8226; all Abcam, Cambridge, UK) for 12 h at 4°C. HRP-conjugated goat anti-rabbit IgG mAb (1:5,000; PV-6001; ZSGB-BIO, Beijing, China) was added for 24 h at 4°C. A Ventana Benchmark automated staining system was used for analyzing protein expression (Olympus BX51; Olympus, Tokyo, Japan). Protein expression signal was analyzed by scanning densitometry using a Microtek ScanMaker 8700 (Zhongjing Technology Co. Ltd, Beijing, China) with ScanWizard 5 software (Informer Technologies, Inc., Walnut, CA, USA).
Tissue preparation and histopathologic analysis
The osteonecrosis mice were sacrificed on day 40 and the joints and articular cartilages were separated and fixed in 10% formalin. Paraffin-embedded joints and articular cartilages were cut into at a 4-µm thickness sections. Tissue sections were stained with 5% hematoxylin and eosin (H&E) for histological evaluation. Safranin O-fast green and Toluidine blue staining was used to evaluate proteoglycans in the cartilage matrix. Tissue sections were also fixed in 10% formalin at 37°C for 24 h and decalcificated using Gooding and Stewart's fluid (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and subsequently embedded in paraffin. 4-µm thick sections were stained with 0.05% toluidine blue (pH 4.1) and the severity of the degree of osteonecrosis was evaluated by the modified Mankin scoring system (22). The Mankin scoring system was scored as follows: 0, normal; 1, irregular surface; 2, pannus; 3, absence of superficial cartilage layers; 4, slight disorganization; 5, fissure into the calcified cartilage layer, and 6, disorganization. Histopathological evaluation was performed by two independent blinded observers.
Immunohistochemistry
The paraffinized joint tissue sections were heated in an oven at 65°C for 24 h, dewaxed to water and rinsed with PBS three times. The washed sections were placed in EDTA buffer (Beinuo Bioscience Inc., Shanghai, China), and then boiled at a low heat following an interval of 10 min at 65°C for a total of three intervals. Following natural cooling, the sections were washed with PBS three times, and were placed into 3% hydrogen peroxide solution (Beina Bioscience Inc.), for incubation at 37°C for 10 min, to block endogenous peroxidase. Free-floating sections were rinsed with PBS and placed in a solution containing primary mouse monoclonal antibodies directed against TNF-α (1:1,000; ab6671, Abcam), IL-1β (1:1,000, ab200478), IL-4 (1:1,000, ab9728), IL-6 (1: 500; ab7737), PI3K (1:2,000; ab1678), ERK (1:2,000; ab196883), pERK (1:2,000; ab214362), AKT (1:1,000; ab8805), pAKT (1:1,000; ab133458; Abcam) at 4°C for 12 h. After rinsing, sections were incubated for 1 h at 37°C with the avidin-biotin-peroxidase complex (1:5,000 dilution; PV-6001; ZSGB-BIO). The sections were then washed with PBS and observed by fluorescent video microscopy (BZ-9000; Keyence Corp., Osaka, Japan).
Statistical analysis
Statistical analysis was completed using SPSS 19.0 statistical software (IBM SPSS, Armonk, NY, USA) with the assistance of Microsoft Excel (Windows 2010; Microsoft Corporation, Redmond, WA, USA). All data are expressed as the mean ± standard deviation and experiments were performed three times. Statistical analyses were performed using one-way ANOVA followed by Tukey's multiple comparison post hoc tests using Graph Pad Prism 5 software. P<0.05 was considered to indicate a statistically significant difference.
Results
Inflammatory cytokines expression in iodoacetate-induced osteoarthritis mice
Inflammatory cytokines were investigated in osteoarthritis mice model. As shown in Fig. 1A, serum levels of TNF-α, IL-1β, IL-4 and IL-6 were significantly upregulated in osteoarthritis mice compared to healthy mice (P<0.01). Results demonstrated that TNF-α, IL-1β, IL-4 and IL-6 mRNA and protein levels were significantly upregulated in synovial fibroblasts in osteoarthritis mice compared to healthy mice (P<0.01, Fig. 1B and C). We observed that MMP-3, MMP-9, RANKL and NF-κB mRNA and protein levels were significantly upregulated in synovial fibroblasts in osteoarthritis mice compared to healthy mice (P<0.01, Fig. 1D and E). These results indicate that inflammatory cytokines are markedly upregulated in serum and synovial fibroblasts in osteoarthritis mice model.
Effect of TNF-α on inflammatory cytokines expression levels in synovial fibroblasts
In vitro assay showed that TNF-α increased TNF-α, IL-1β, IL-4 and IL-6 mRNA and protein levels were significantly upregulated in synovial fibroblasts (Fig. 2A and B). TNF-α inhibitor treatment downregulated TNF-α, IL-1, IL-4 and IL-6 mRNA and protein levels in synovial fibroblasts (Fig. 2C and D). These results indicate that TNF-α inhibitor can lead to downregulation of inflammatory cytokines expression levels in synovial fibroblasts in vitro.
Effect of TNF-α on expression of pro-inflammation factors in synovial fibroblasts
We investigated the effects of TNF-α on pro-inflammation factors in synovial fibroblasts. We showed that TNF-α treatment stimulated mRNA and protein expression levels of MMP-3, MMP-9, RANKL and NF-κB in synovial fibroblasts (P<0.01, Fig. 3A and B). TNF-α inhibitor treatment significantly decreased mRNA and protein expression levels of MMP-3, MMP-9, RANKL and NF-KB in synovial fibroblasts (P<0.01, Fig. 3C and D). These results suggest that TNF-α is a target for inhibition of pro-inflammation factors in synovial fibroblasts.
Effect of TNF-α on TLR-3-medicated ERK/AKT signal pathway
We investigated the regulatory effects of TNF-α on TLR-3-medicated ERK/AKT signal pathway in synovial fibroblasts. Results demonstrated that TLR-3, ERK and AKT expression levels were significantly upregulated in synovial fibroblasts in osteoarthritis mice compared to healthy mice (P<0.01, Fig. 4A). TNF-α treatment increased TLR-3, ERK and AKT expression levels in synovial fibroblasts compared to control (P<0.01, Fig. 4B). Reversely, TNF-α inhibitor significantly decreased TLR-3, ERK and AKT expression levels in synovial fibroblasts compared to control (P<0.01, Fig. 4C). These results suggest that TNF-α can regulate TLR-3-medicated ERK/AKT signal pathway in synovial fibroblasts.
TNF-α regulates inflammatory factors expression via TLR-3-meidated ERK/AKT signal pathway
We further analyzed the possible mechanism medicated by TNF-α in synovial fibroblasts. Results revealed that knockdown of TLR-3 (siRTLR-3) canceled TNF-α inhibitor-decreased expression and phosphorylation levels of ERK and AKT in synovial fibroblasts (Fig. 5A). TNF-α inhibitor-decreased TNF-α, IL-1β, IL-4 and IL-6 expression levels were also abolished by siRTLR-3 in synovial fibroblasts (Fig. 5B). Also, TNF-α inhibitor-decreased MMP-3, MMP-9, RANKL and NF-κB expression levels were canceled by TLR-3 knockdown in synovial fibroblasts (Fig. 5C). These results indicate that TNF-α regulates inflammatory factors expression via TLR-3-meidated ERK/AKT signal pathway in synovial fibroblasts.
TNF-α inhibitor improves bone destruction in iodoacetate-induced osteoarthritis mice model
Finally, we further investigated the in vivo role of TNF-α inhibitor in the iodoacetate-induced osteoarthritis mice model. As shown in Fig. 6A, TNF-α inhibitor improved the osteoarthritis determined by osteoarthritis score. Results demonstrated that TNF-α inhibitor markedly downregulated bone destruction compared to control (Fig. 6B). TNF-α inhibitor also improved body weight for osteoarthritis mice compared to control (Fig. 6C). Inflammatory factors of TNF-α, IL-1, IL-4 and IL-6 expression levels were downregulated in cartilago articularis (Fig. 6D). TNF-α inhibitor decreased TLR-3 and increased expression and phosphorylation levels of ERK and AKT in cartilago articularis (Fig. 6E). These results suggest that TNF-α inhibitor presents many benefits for the treatment of osteoarthritis mice model.
Discussion
In recent years, many reports have found that anti-TNF-α therapy has achieved the therapeutic effects for patients with osteoarthritis by targeting TNF-α in synovial fluid (9,23–25). In this study, we analyzed the relationships between TNF-α and TLR-3-mediatred ERK/AKT signal pathway in synovial fibroblasts in mice with osteoarthritis. Güler-Yüksel et al have indicated that treatment with TNF-α inhibitor infliximab could reduce hand osteoarthritis (9). Findings in this study indicate that TNF-α upregulates the inflammatory cytokines and decreases ERK/AKT signal pathway in synovial fibroblasts in mice with osteoarthritis. Notably, we found that TNF-α increased TLR-3 expression in synovial fibroblasts in mice with osteoarthritis. Here, we reported that TNF-α can increase regulate inflammation in synovial fibroblasts via regulation of TLR-3-mediated ERK/AKT signal pathway in mice with osteoarthritis both in vitro and in vivo.
Study has observed that osteoarthritis Marker YKL-39 is stimulated by IL-4 in differentiating macrophages (26). We reported that TNF-α increased Il-4 expression in synovial fibroblasts. Treatment with TNF-α inhibitor infliximab treatment significantly reduced hand osteoarthritis in patients with rheumatoid arthritis (9). In addition, suppressing the release of synovia IL-1β and TNF-α in knee osteoarthritis of rabbits may contribute to the treatment of osteoarthritis (27). Our results showed that TNF-α treatment upregulated IL-1 and IL-6 expression levels in synovial fibroblasts and in cartilago articularis in osteoarthritis mice model. Study has showed that TNF-α and MMP-3 correlated significantly with the swollen joint count (28). MMP-3 and MMP-9 expression levels were upregulated in osteoarthritis mice, which can be downregulated by TNF-α inhibitor (29). Evidences have showed that RANKL and NF-KB expression levels were increased in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy and osteoarthritis (30). We reported that TNF-α inhibitor significantly downregulated RANKL and NF-KB in synovial fibroblasts and in synovial tissue in osteoarthritis mice. These results indicate that TNF-α is a mediator for inflammation in the processes of osteoarthritis mice. We will evaluate the effects TNF-α on P-38, P-65 and IRF-3 expression in synovial fibroblasts in our future work.
Currently, TLR-3 SNP is associated with knee osteoarthritis and increasing of TLR-3 levels has been observed in a Chinese Han population (31). We found that TLR-3 was upregulated and TNF-α inhibitor decreased TLR-3 expression levels in synovial fibroblasts. Domagala et al have suggested that inhibition of IL-1β-induced activation of MEK/ERK pathway provided a potential mechanism for the treatment of osteoarthritis (16). Fu et al have showed that regulation of the PI3K/AKT signaling pathway could inhibit inflammation and chondrocyte apoptosis in a rat model of osteoarthritis (32). In this study, we found that TNF-α inhibitor downregulated TLR-3, ERK and AKT expression levels in synovial fibroblasts in mice model of osteoarthritis. Ballak et al have found that TLR-3 appears to play a redundant role in obesity-induced inflammation and insulin resistance (33). Additionally, TLR-3 mediated the synovial inflammation in rheumatoid arthritis (34). We reported that TNF-α regulates inflammation via TLR-3-mediated ERK/AKT signal pathway in synovial fibroblasts.
In conclusion, this study found that TNF-α inhibitor treatment not only decreased inflammatory factors, but also associated with osteoarthritis score in mice model. The pro-inflammatory cytokines in osteoarthritis, such as MMP-3, MMP-9, RANKL and NF-KB, which participate in the pathogenesis of osteoarthritis, are downregulated by treatment of TNF-α inhibitor. Importantly, findings have indicated that TNF-α can regulate inflammation expression via TLR-3-mediated ERK/AKT signal pathway in synovial fibroblasts both in vitro and in vivo. Therefore, ERK/AKT may provide a novel potential target for osteoarthritis therapy. However, further reports need to elucidate the possible mechanisms mediated by TNF-α in the pathogenesis of osteoarthritis.
Acknowledgements
Not applicable.
Funding
The present study was supported by National Science Foundation (grant no. 30801159), the 12th five-year plan of the military (grant no. 39770714) and Beijing municipal starting special (grant no. 2016-3-5071).
Availability of data and materials
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.
Authors' contributions
FYY, CQX, CLJ and JTS performed the experiments and analyzed the experimental data. XWH designed the study.
Ethics approval and consent to participate
The present study was approved by Ethics Committee of the 309th Hospital of People's Liberation Army.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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