Insulin Exacerbates Inflammation in Fibroblast-Like Synoviocytes

OA synovial tissues were macerated, chopped, and then incubated with type II collagenase (1 mg/ml, Sigma) in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Thermo Scientific) for 6 h at 37 °C and 5% CO₂ (Thermo Scientific). The tissues were treated with 0.25% trypsin (Solabio) diluted in a phosphate-buffered saline (PBS) solution at a volume equivalent to that of DMEM. The cells were filtered; incubated overnight in DMEM supplemented with 10% fetal bovine serum (FBS, HyClone, Thermo Scientific), penicillin (100 IU/ml), and streptomycin (100 μg/ml, Gibco); and passaged three times. FLSs at passages 4–6 were used for our study.

FLSs were plated on 6-well plates ( 3-5× 10⁵ cells/well) or 24-well plates (8× 10⁴ cells/well) in high glucose DMEM containing 10% or 2% FBS. Insulin (0, 100, 200, or 500 nM; Sigma-Aldrich, St. Louis, MO, USA; I0516) or inflammatory stimulators, including IL-1β, IL-6, and TNF-α (0, 0.01, 0.1, 1, 10, or 50 ng/ml), were added and then incubated for the indicated times. PBS was used as a control. PDTC (an NF-ĸB inhibitor; MCE, USA), LY294002 (a PI3K/Akt inhibitor; MCE, USA), and rapamycin (an mTOR inhibitor; MCE, USA) were used to block the signaling pathways, and an equal amount of inhibitor solvent (0.1% DMSO, 99.9% corn oil) was used as a control.

Cell suspensions were seeded on a 96-well plate at 1000 cells/well. Next, the cells were shaken, incubated for 4 to 6 h in a 37 °C incubator until they adhered, and incubated with insulin (0, 100, 200, or 500 nM) and with or without each of the inhibitors (LY294002 or rapamycin [10 μM]). The control group received the same amount of solvent. After 24 h or 48 h, 100 μl of CCK-8 reagent was added to each well, shaken, and subsequently incubated for 2 h. Afterward, the absorbance at 450 nm was measured with a microplate reader.

According to the source of the supernatant in the lower chamber, the experiments were divided into four groups: the control (N) group, the insulin (ISN)-mediated group (INS group), the fibroblast-like synoviocyte (FLS)-mediated group (FLS group), and the INS- and FLS-mediated group (INS+FLS group). When the FLSs in the 24-well plate reached 80% confluence, the DMEM was replaced with fresh DMEM or with DMEM containing a high INS concentration (500 nM). After 24 h, 1000 μl of the supernatant from each FLS-plated well under different stimulation conditions (DMEM with or without 500 nM INS) was aspirated and mixed well, and then 600 μl of it was added to a lower Transwell chamber (FLS and INS+FLS groups). The supernatant for the control group was 600 μl of DMEM, and the supernatant for the INS group was 600 μl of DMEM with 500 nM INS; these supernatants were placed in other lower Transwell chambers (N and INS groups) to start the experiments. When FLSs in a 24-well plate reached 80% confluence, insulin (500 nM) was added for 24 h. Then, 600 μl of the supernatant was aspirated and added to the lower Transwell chamber. After macrophage digestion, a cell suspension was prepared with DMEM/F12 medium, and 200 μl was added to the upper Transwell chamber. Next, the cells were incubated overnight, and the cells that did not migrate were wiped away with a cotton swab. Then, the cells in the lower chamber were stained with hematoxylin for 10 min at ambient temperature. The number of migrated cells was examined using a cell counter with microscopic imaging system.

FLSs were plated in 24-well plates and cultured in the presence of insulin (0, 100, 200, or 500 nM), the inflammatory stimulators IL-1β, IL-6, and TNF-α (0, 0.01, 0.1, 1, 10, or 50 ng/ml), or different signaling inhibitors (PDTC, LY294002, or rapamycin); then, the cells were analyzed 6, 12, or 24 h after treatment. Total RNA was extracted from FLSs using TRIzol reagent (Invitrogen) and reverse-transcribed using a ReverTra Ace qPCR RT kit (Toyobo, Japan) following the manufacturer’s protocol. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using a LightCycler 480 (Roche, Basel, Switzerland) according to the following protocol: denaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 1 min, and extension at 72 °C for 1 s. The primers for RT-qPCR were designed according to the consensus sequences by Oligo. GAPDH was used as an internal loading control. The forward and reverse primers are listed in Table 1, and all primers were created by BGI (Beijing, China). To measure the relative messenger RNA (mRNA) levels, the 2-ΔΔ cycle threshold (2-ΔΔCT) method was used.

Cells were cultured and then stimulated as described above, and the supernatants were collected at 6 h, 12 h, or 24 h. The release of proinflammatory cytokines (IL-1β, IL-6, and TNF-α), matrix metalloproteinases (MMP-9 and MMP-13), and chemokines (CXCL12, CCL2/MCP-1, and CCL5/RANTES) was analyzed using enzyme-linked immunosorbent assay (ELISA) kits (Multi Sciences, Hang Zhou, China) following the manufacturer’s instructions.

Whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). Western blotting was performed using anti-LC3I (1:1000, Abcam, USA), anti-LC3II (1:1000, Abcam, USA), anti-PI3K (1:1000, CST), anti-phospho-PI3K (1:500, CST), anti-Akt (1:3000, CST), anti-phospho-Akt (1:1000, CST), anti-mTOR (1:3000, CST), anti-phospho-mTOR (1:1000, CST), anti-phospho-p50 NF-ĸB (1:1000, CST), p50 NF-ĸB (1:2000, CST), anti-phospho-p65 NF-ĸB (1:1000, CST), and anti-p65 NF-ĸB (1:2000, CST) antibodies. GAPDH (1:10000, Abcam, USA) was used as a loading control for proteins. The band intensities were analyzed using an ECL Plus detection system (Thermo Scientific, Pittsburgh, PA, USA).

FLSs were grown in 6-well plates with high insulin stimulation (500 nM) for 24 h. Preconditioned cells were washed slowly three times with PBS for 5 min each, fixed with 4% paraformaldehyde for 30 min, washed three times with PBS (5 min each), and then treated with 5% bovine serum albumin (BSA) for 1 h. The cells were then incubated with anti-p50 (1:100 dilution) and anti-p65 (1:150 dilution) antibodies at 4 °C overnight. After the cells were washed slowly three times with PBS for 5 min each, FITC- and TRITC-conjugated secondary antibodies were used to visualize the proteins under a fluorescence microscope (Olympus, Tokyo, Japan). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI).

Statistical analysis was conducted using the GraphPad Prism 5 software package (La Jolla, CA, USA). A t test was used to assess significant differences between two groups. The results of three different experiments are expressed as the mean ± SEM. Differences in the results with P < 0.05 were considered statistically significant.

A previous study suggested that FLSs are vital for osteoarthritis pathogenesis and that synoviocyte hyperplasia and abnormal synoviocyte proinflammatory cytokine production were both important pathological hallmarks of OA [2‐5]. CCK-8 assays were applied to assess the effects of insulin (0, 100, 200, or 500 nM) on the viability of FLS. In response to insulin (200 or 500 nM), cell viability was significantly enhanced at 24 h (Fig. 1a) and 48 h (Fig. 1b). Next, the regulatory effects of insulin on proinflammatory cytokines and MMP mechanisms in FLSs were investigated. To determine whether insulin can directly regulate proinflammatory cytokine expression, FLSs were treated with incremental concentrations of insulin (0, 100, 200, or 500 nM). The qRT-PCR results (Fig. 1c–g) showed that IL-1β, IL-6, TNF-α, MMP-9, and MMP-13 expression levels were increased in a concentration-dependent manner compared with those of the control group (P < 0.05) at 6 h, 12 h, and 24 h after treatment with insulin (0, 100, 200, or 500 nM), and these levels were increased most significantly at 24 h. A similar increase in FLS secretion in response to insulin stimulation was observed by ELISA (Fig. 1h–l).

Because macrophage infiltration is a significant pathological feature of OA, macrophages can also contribute to OA [4, 5]. Chemokines are the major drivers of leukocyte adhesion and cell migration in inflammatory disease development [32, 33]. Among the chemokines, CXCL12, CCL2/MCP-1, and CCL5/RANTES can induce macrophage chemotaxis and are closely involved in OA development [34‐36]. It is unclear whether insulin can regulate FLS-mediated macrophage infiltration and chemokine production. Transwell assays were employed to analyze the role of insulin in macrophage infiltration. The results suggest that the number of transmigrated macrophages was significantly increased at 24 h in the presence of FLSs treated with high insulin (500 nM). In addition, ELISA was used to detect CXCL12, CCL2/MCP-1, and CCL5/RANTES secretion by FLSs after 24 h. It was observed that insulin could independently attract macrophages in the absence of FLSs (Fig. 2a). Moreover, CXCL12, CCL2/MCP-1, and CCL5/RANTES secretion increased following insulin stimulation (500 nM, Fig. 2b–d).

The above-mentioned data suggest that insulin could increase the inflammatory effect of FLSs. The PI3K/Akt/mTOR signaling pathway is involved in the pathogenesis of inflammation, including OA [28‐31], and NF-ĸB is a known signaling pathway of OA inflammation [11, 12]. Thus, whether insulin can regulate PI3K/Akt/mTOR and NF-ĸB signaling pathway activation in FLSs was verified. Western blotting was used to evaluate the phosphorylation of PI3K/Akt/mTOR and p50/p65 (NF-ĸB subunits). According to the Western blotting results, the phosphorylation of PI3K/Akt/mTOR (Fig. 3a) and p50/p65 (Fig. 3b) was increased remarkably in FLSs treated with high insulin concentrations (100, 200, or 500 nM) for 24 h. These results verified PI3K/Akt/mTOR and NF-ĸB signaling pathway activation by high insulin conditions in FLSs. Immunofluorescence confirmed that a high insulin level (500 nM) could improve p50 and p65 (Fig. 3c, d) translocation into the nucleus in FLSs.

Autophagy is a critical cellular process that maintains homeostasis, and autophagy inhibition is closely associated with OA [21‐25]. To evaluate how insulin regulates autophagy activation in FLSs, Western blotting was employed to detect the expression of LC3II, a major autophagy effector. LC3II expression and the LC3II/LC3I ratio were decreased with concomitantly increased PI3K/Akt/mTOR phosphorylation levels (Fig. 3a).

According to the above-mentioned data, insulin could activate the PI3K/Akt/mTOR and NF-ĸB signaling pathways in FLSs (Fig. 3a–c). In addition, the PI3K/Akt/mTOR pathway is a fundamental intracellular signaling pathway that is widely involved in autophagy regulation [28, 29]. To further verify the regulation of both signaling pathways by insulin, the effects of specific inhibitors on insulin-induced inflammatory response exacerbation and autophagy inhibition were also studied. The data revealed that inhibiting the PI3K/Akt/mTOR signaling pathways could eliminate the increased FLS viability following insulin treatment (0, 100, 200, or 500 nM) for 24 or 48 h (Fig. 4a, b). Furthermore, the decreases in LC3II expression and the LC3II/LC3I ratio caused by insulin (500 nM) could be restored by inhibitor treatment. The results demonstrated that the PI3K/Akt/mTOR pathway plays a role in autophagy regulation in FLSs (Fig. 4c).

Because NF-ĸB signaling could significcytokines (e.g., IL-1β, IL-6, and TNF-α) directly and MMPs indirectly [11‐14], we speculated that the overproduction of proinflammatory factors and chemokines following insulin treatment might result from NF-ĸB signaling pathway activation. To verify the mechanism of high insulin-induced proinflammatory factor and chemokine production, an inhibitor targeting the NF-ĸB (PDTC, 10 μM) signaling pathway was used. To evaluate the role of different pathways involved in OA progression, inhibitors targeting the PI3K/Akt/mTOR signaling pathway (LY294002, rapamycin) were employed. Subsequently, the inhibition of NF-ĸB signaling pathway could inhibit the CXCL12, CCL2/MCP-1, and CCL5/RANTES oversecretion induced by a high insulin concentration (500 nM) at 24 h (Fig. 4d–f). Similar results showing that inhibiting the PI3K/AKT/mTOR and NF-ĸB signaling pathways could reduce the overexpression (Fig. 4g–k) and secretion (Fig. 4l–p) of IL-1β, IL-6, TNF-α, MMP-9, and MMP-13 induced by a high insulin concentration (500 nM) at 24 h were also obtained. These experiments provide evidence that the proinflammatory effect of insulin on FLSs could be exerted through PI3K/mTOR/Akt and NF-ĸB pathway activation.

NF-ĸB contributes to the production of proinflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α), and in turn, the proinflammatory effects of IL-1β, IL-6, and TNF-α are mediated through the activation of several signaling pathways, including NF-ĸB signaling [2, 11, 12]. Thus, a feedback mechanism exists among inflammatory cytokines and signaling pathways, which can activate each other and have a close relationship. Based on the fact that insulin is a positive regulator of proinflammatory cytokine production and was found to upregulate the downstream targets of PI3K/mTOR/Akt/NF-ĸB signaling in FLSs, we hypothesized that insulin might be involved in the interaction between IL-1β/IL-6/TNF-α and PI3K/mTOR/Akt/NF-ĸB signaling. Cytokines act as important mediators of intercellular communication through specific cytokine receptors on the cell surface and initiate a series of specific biochemical reactions. The receptors responsible for the proinflammatory effects of IL-1β/IL-6/TNF-α-activated intracellular signaling pathways include interleukin-1 receptor 1 (IL-1R1), interleukin-1 receptor 3 (IL-1R3, interleukin-1 receptor accessory chain), interleukin-6 receptor (IL-6R), glycoprotein 130 (GP130, interleukin-6 coreceptor), tumor necrosis factor receptor 1 (TNFR1 or p55), and tumor necrosis factor receptor 2 (TNFR2 or p75) [37‐39]. Thus, we measured the expression of these proinflammatory cytokine receptors in insulin-stimulated FLSs by qRT-PCR. The results showed that IL-1R1, IL-1R3, IL-6R, GP130, TNFR1, and TNFR2 expression levels were increased in a concentration-dependent manner compared with those of the control group (P < 0.05) at 24 h after treatment with insulin (0, 100, 200, or 500 nM); these receptors were upregulated most significantly at the 500 nM concentration (Fig. 5a–f). To explore the association of cytokine receptor upregulation with PI3K/mTOR/Akt/NF-ĸB signaling in FLSs, the effect of inhibitors targeting the PI3K/Akt (LY294002, 10 μM), mTOR (rapamycin, 10 μM), and NF-ĸB (PDTC, 10 μM) signaling pathways on insulin-induced cytokine receptor expression was studied. The data revealed that inhibiting the PI3K/Akt/mTOR and NF-ĸB signaling pathways could reduce the IL-1R1, IL-1R3, IL-6R, GP130, TNFR1, and TNFR2 overexpression induced by high insulin (500 nM) at 24 h (Fig. 5g–l). These experiments provide evidence that when FLSs are stimulated with insulin, the expression of proinflammatory cytokine (e.g., IL­1-β, IL­6, and TNF-α) receptors depends on PI3K/Akt/mTOR and NF-ĸB signaling pathway activation.

In synovial joint pathology, a major hallmark in response to increased levels of IL-1β, IL-6 and TNF-α is the overexpression of MMPs, including MMP-9 and MMP-13, which contributes to cartilage degradation [13, 14]. To determine whether insulin can directly regulate cytokine-mediated inflammatory reactions, we chose MMP-9 and MMP-13 expression as a functional readout for the effect of insulin. FLSs were treated with incremental concentrations of IL-1β, IL-6 and TNF-α (0, 0.01, 0.1, 1, 10, or 50 ng/ml) or were cotreated with high insulin (500 nM) and all three cytokines separately for comparison. The qRT-PCR results showed that the expression of MMP-9 and MMP-13 increased (P < 0.05) after treatment with IL-1β, IL-6 and TNF-α (0, 0.01, 0.1, 1, 10, or 50 ng/ml) at 24 h; the MMPs were increased most significantly (P < 0.01) at the 50 ng/ml concentration, and the response was further increased by the presence of insulin (Fig. 6a–f).

Finally, we sought to investigate whether insulin could affect IL-1β, IL-6 and TNF-α-mediated signaling activation in FLSs, as suggested by the increase in cytokine-induced MMP expression. FLSs were treated with inflammatory cytokines (IL-1β, IL-6 and TNF-α, 50 ng/ml) and insulin (500 nM) separately or cotreated with the two stimuli simultaneously. Western blotting was used to test the phosphorylation of PI3K/Akt/mTOR and p50/p65 in different groups. After treatment, the PI3K/Akt/mTOR and p50/p65 phosphorylation levels in the three groups increased significantly and were even higher in the cotreatment group than in the other two groups (Fig. 6g). These experiments showed that insulin sensitized cellular signaling transduction activation by inflammatory cytokines. Based on the above-mentioned research, we suggest that insulin is involved in a positive feedback loop between IL-1β/IL-6/TNF-α and PI3K/mTOR/Akt/NF-ĸB signaling in FLSs.