The detrimental effect of iron on OA chondrocytes: Importance of pro‐inflammatory cytokines induced iron influx and oxidative stress

Abstract Iron overload is common in elderly people which is implicated in the disease progression of osteoarthritis (OA), however, how iron homeostasis is regulated during the onset and progression of OA and how it contributes to the pathological transition of articular chondrocytes remain unknown. In the present study, we developed an in vitro approach to investigate the roles of iron homeostasis and iron overload mediated oxidative stress in chondrocytes under an inflammatory environment. We found that pro‐inflammatory cytokines could disrupt chondrocytes iron homeostasis via upregulating iron influx transporter TfR1 and downregulating iron efflux transporter FPN, thus leading to chondrocytes iron overload. Iron overload would promote the expression of chondrocytes catabolic markers, MMP3 and MMP13 expression. In addition, we found that oxidative stress and mitochondrial dysfunction played important roles in iron overload‐induced cartilage degeneration, reducing iron concentration using iron chelator or antioxidant drugs could inhibit iron overload‐induced OA‐related catabolic markers and mitochondrial dysfunction. Our results suggest that pro‐inflammatory cytokines could disrupt chondrocytes iron homeostasis and promote iron influx, iron overload‐induced oxidative stress and mitochondrial dysfunction play important roles in iron overload‐induced cartilage degeneration.

joint injuries and gender are major risk factors for the disease, the intrinsic physiological and molecular mechanisms of OA still remain poorly understood. 1 Iron overload is common in tissues of elderly people and has been implicated to be responsible for many diseases or pathological conditions. 2 Abnormal iron metabolism has recently been implicated in the disease progression of OA. Multiple independent clinical studies on OA in the elderly people found that people with high serum ferritin levels have a 4-fold increased risk of OA, and that serum ferritin levels were positively correlated with radiographic severity. 3 OA is a common complication of many diseases with abnormal cartilage iron overload, including haemophilic arthropathy, hereditary hemochromatosis and rheumatoid arthritis. 4 Iron deposition in joint synovium and cartilage is an important pathological process in OA development, elevated iron concentration in synovial fluid and iron crystal deposition was observed in osteoarthritis and traumatic cartilage damage. 5 Hereditary hemochromatosis (HH) is a chronic systemic iron overload disease that is caused by mutations in the HFE gene. Recently, Camacho A et al demonstrated that HFE-KO mice developed more severe knee OA than wild-type (wt) mice after joint destabilization induced by partial meniscectomy. 6 However, the underlying mechanism of iron takes parts in OA development remains unknown.
Iron plays important roles in normal cellular processes including oxygen storage and transport, energy metabolism and DNA, RNA synthesize. Delicate regulation of iron homeostasis is required for maintaining normal cellular function, while excessive iron would damage cells by the increasing production of reactive oxygen species (ROS). Because of Fenton reaction, excess iron could generate highly toxic hydroxyl radicals and result in the peroxidation of membrane lipids, mitochondrial dysfunction, cellular proteins and nucleus acids damage and ultimately ferroptosis. 7 Oxidative stress and mitochondrial dysfunction were demonstrated to be an important contributor to joint OA development. 8,9 Mitochondria is reported to be the main cellular organ for iron metabolism and ROS production.
Iron could participate in mitochondrial oxidative respiratory chain by exchanging a single electron with a number of substrates which can lead to the generation of ROS. 10 Increased ROS production could damage mitochondria as well as other structures, leading to cell death. Damaged mitochondria could in turn produce excess ROS and result in decreased collagen production and increased matrixdegrading enzyme excretion. 9 Mitochondrial fission and fusion process could remove damaged mitochondria and restore mitochondrial morphology and function. During fission, Dynamin-related protein 1 (Drp1) is recruited from the cytosol to the mitochondria to form helixes around mitochondria and sever both the outer mitochondrial membranes (OMM) and inner mitochondrial membranes (IMM) by constriction. Other mitochondrial fission proteins that regulate mitochondrial fission included fission 1 homologue protein (FIS1) and mitochondrial fission factor (MFF). The damaged mitochondria are subsequently degraded by mitophagy. Mitophagy is a mitochondrial autophagy process that selectively removes damaged mitochondria. 11 Because of its double-edged sword effect in the body, iron homeostasis is delicately regulated. Cellular iron homeostasis is achieved by modulating the expression of proteins involved in iron uptake, storage, and export. Iron regulator proteins (IRPs) sense the concentration of active iron in the mitochondria and regulate the expression of iron uptake associated proteins transferrin receptor 1(TfR1) and divalent metal transporter 1 (DMT1). 12 IRP could also bind to the IRE of the 5' untranslated region of ferroportin (FPN) and inhibit the iron release from the cell. 13 Disruption of iron homeostasis is reported to take parts in many diseases, such as Parkinson disease, Alzheimer disease, cardiovascular diseases and osteoporosis. 14 Recent researches indicated that many risk factors, including ageing, mechanical overload, inflammation would disrupt cellular homeostasis and thus lead to iron overload. 15 The association between iron overload and OA pathogenesis is broadly appreciated regarding its effect on ROS production and oxidative stress. 16 OA is characterized by synovial tissue inflammation and cartilage degeneration. However, how iron homeostasis is regulated in chondrocytes under an inflammatory environment and how it contributes to the pathological transition of articular chondrocytes remain unknown. In the present study, we developed an in vitro approach using pro-inflammatory cytokines to mimic OA pathological conditions. We sought to investigate how iron homeostasis is regulated in chondrocytes under an inflammatory environment, and the roles of iron overload and iron overload mediated oxidative stress in cartilage degeneration.

| Cell isolation and culture
Chondrocytes were obtained from five days old C57/BL6 male mice.
Briefly, bilateral knee joints were isolated and cartilage was minced into small pieces. After washed by cold PBS, cartilage pieces were digested with 0.25% trypsin-EDTA for 30 minutes at 37°C. Then trypsin-EDTA was removed and sample pieces were washed by PBS twice.
Samples were then re-suspended in 0.25% collagenase solution at 37°C for 6 hours. Finally, cells were cultured in DMEM/F12 medium containing 10% FBS and passaged when cells reached 80% confluence.
Chondrocytes at passage 1 and 2 were used in our study. All animal protocols were approved by the Institutional Animal Care of the Shandong Provincial Hospital affiliated to Shandong First Medical University.

| Reverse transcription and real-time polymerase chain reaction (RT-PCR) analysis
Total RNA isolation and quantitative RT-PCR amplification were performed as previously described. 17 Briefly, complementary DNA (cDNA) synthesis was performed by using a first Strand cDNA Synthesis Kit

| Measurement of intracellular iron levels
Iron-sensitive fluorescent Calcein-AM dye was used to evaluate the intracellular iron levels. Briefly, cells were pre-treated with 10 ng/mL IL-1β for 24 hours and then incubated with 0.5 μmol/L Calcein-AM for 30 minutes at 37°C. Chondrocytes were then washed with PBS for three times to wash out the excess calcein dye. 100 μmol/L FAC was added into medium and ferrous iron influx into chondrocytes was deter-

| Evaluation of intracellular ROS
Chondrocytes were treated with FAC (ferric ammonium citrate) with or without 100 μmol/L DFO (deferoxamine) or NAC (N-acetyl-L-cysteine) for 24 hours. After treatment, the intracellular ROS level determination was performed using the Reactive Oxygen Species Assay Kit (S0033, Beyotime, China) as previously described. 18 To quantify intracellular ROS level, chondrocytes were collected and the mean fluorescence intensity was evaluated with a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).

| Mitochondrial specific fluorescence staining
The morphological changes of mitochondria were assessed using

| Statistical analysis
The comparisons between multiple groups, such as RT-PCR, western blot and immunofluorescence analyses were performed using multiple comparisons by one-way ANOVA followed by Tukey's test.
For western blot and intracellular ROS evaluation data, Student's t test and one-way ANOVA with Dunnett's test were used for pairwise comparisons and multi-group comparison, respectively.
Results are represented as mean ± SD, P values <.05 were considered to be significant. All analyses were performed with GraphPad Prism software (Version 6.0).

| The pro-inflammatory cytokines disrupted iron homeostasis with increased TfR1 expression and decreased FPN expression
To elucidate the change of iron homeostasis and the associated iron regulators in chondrocytes under an inflammatory environment, F I G U R E 1 Pro-inflammatory cytokines IL-1β and TNFα disrupted iron homeostasis in chondrocytes via upregulating iron influx protein, downregulating iron efflux protein. A-D, Chondrocytes were treated with increasing concentrations of IL-1β or TNFα and iron regulators, IRP1/2, TfR1 and FPN proteins expression were determined by western blot analysis. The band density of IRP1/2, TfR1 and FPN were quantified and normalized to control. E, Chondrocytes were treated with 10 ng/mL IL-1β for 24 h and RT-PCR was conducted to evaluated TfR1 and FPN gene expression. F, Chondrocytes were treated with 10 ng/mL IL-1β with or without 100 μmol/L FAC for different time intervals and iron regulators, IRP1/2, TfR1 and FPN protein expression were determined by western blot analysis. G-J, The band density of IRP1/2, TfR1 and FPN were quantified and normalized to control. Data are presented as mean ± SD from three different experiments. *P < .05; **P < .01; ***P < .001 vs Ctrl; # P < .05; ## P < .01; ### P < .001vs FAC (100 μmol/L, 0 min) chondrocytes were treated with IL-1β and TNFα and western blot analysis was performed to detect the protein levels of IRP1/2, TfR1 and FPN. IL-1β and TNFα showed a similar trend that IRP1 and TfR1 were upregulated and FPN was downregulated after treatment of various concentrations of IL-1β and TNFα. While no significant difference was observed in IRP2 protein expression after both IL-1β and TNFα treatment ( Figure 1A-D). RT-PCR experiment obtained similar results that IL-1β significantly promoted iron influx mediators TfR1, inhibited iron efflux mediator FPN genes expression ( Figure 1E

| Iron overload promoted expression of chondrocyte matrix-degrading enzymes
To evaluate the detrimental effect of iron in chondrocytes, primary mouse chondrocytes were initially treated with 100 μmol/L FAC with or without 10 ng/mL IL-1β, fluorescence dye calcein was used to assess the ferrous iron uptake process in chondrocytes. As shown in Figure 2A (Figure 2C-D). These results indicate that FAC could promote mitochondrial fission. Overdose and prolonged FAC treatment would inhibit mitochondrial fission and fusion, which might inhibit mitophagy and further cause mitochondrial destruction.

| Oxidative stress and mitochondrial dysfunction mediates iron overload-induced chondrocytes apoptosis and matrix metalloproteinases expression
Because of Fenton reaction, excess iron could generate highly toxic hydroxyl radicals and result in mitochondrial dysfunction. We next examined whether excess iron-induced oxidative stress and mitochondrial dysfunction mediates the accelerated progression of OA.
Primary chondrocytes were treated with DFO to chelate iron ions, or NAC to inhibit iron overload-induced oxidative stress. As shown in Figure 4A, excess iron-induced ROS overproduction was significantly inhibited by DFO or NAC treatment. Collapse of mitochondrial membrane potential (MMP) represents mitochondrial dysfunction.
As shown in Figure 4B

| D ISCUSS I ON
Osteoarthritis is characterized by overproduction of ROS in afflicted joints. 8 Iron overload, due to its effect in the production of ROS and crystal deposition in the joints, is implicated in the disease progression of OA. 19 Recent clinical studies indicated the link between iron overload and the incidence and progression of OA. 20 However, no evidence available to date clearly shows the involvement of iron homeostasis in the OA pathogenesis. In the present study, we demonstrated that pro-inflammatory cytokines disrupted chondrocytes iron homeostasis via upregulating iron influx mediators TfR1, downregulating iron efflux mediator FPN expression. Excess iron could promote OA-related catabolic markers MMP3 and MMP13 expression in chondrocytes. Moreover, we found that oxidative stress and mitochondrial dysfunction play important roles in iron overloadinduced chondrocytes OA-related catabolic markers expression.
Although many researches and clinical evidences indicated that iron might be involved in the development of OA, there are still many problems that need to be further clarified. Epidemiological evidence showed that iron overload is a common physiological and pathological state in the elderly due to the lack of an effective way to excrete iron in the human body. 21 It is reported that the serum ferritin concentration in women increased rapidly after menopausal with 106 ng/mL which is more than twice that of premenopausal women.
The average serum ferritin concentration of middle-aged and elderly men is 121 ng/mL, which is 3-4 times that of adolescent. 22 But not all elderly people would eventually develop into OA, we guess this may be related to cellular iron metabolism regulation. Iron is a "double- Iron homeostasis is delicately regulated in the human body, but many risk factors, including age, mechanical load, inflammation could disturb iron homeostasis and cause iron accumulation in tissues. 24 Pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumour necrosis factor-a (TNF-a), are major components of OA F I G U R E 5 Iron chelator DFO or antioxidant NAC inhibited iron overload induced apoptosis, mitochondrial dysfunction and matrix metalloproteinases (MMPs) upregulation. A-B, Chondrocytes were treated with 100 μmol/L FAC with or without 100 μmol/L NAC for 24 h and mitochondrial fission protein (DRP1, MFF, FIS1) were detected using western blot analysis. The band density were quantified and normalized to control. C-D, Chondrocytes were treated with 100 μmol/L FAC with 100 μmol/L NAC or DFO for 24 h, Annexin V-FITC/PI flow cytometric analysis was conducted to detect the apoptosis rate. Data are presented as mean ± SD. *P < .05 vs Ctrl group; # P < .05 vs FAC treatment group. E-F, Chondrocytes were treated with 100 μmol/L FAC with or without 100 μmol/L NAC for 24 h and matrix metalloproteinases (MMP3, MMP13) were detected using western blot analysis. The band density were quantified and normalized to control. Data are presented as mean ± SD from three different experiments. *P < .05; ***P < .001 inflammation that play key roles in OA primary cartilage damage. 25 In the present study, we found that IL-1β or TNFα increased ferrous iron influx and decreased iron efflux, with increased TfR1 expression and decreased FPN expression. What's more, the level of IRP1 was elevated in IL-1β or TNFα treated chondrocytes. IRPs could sense cellular iron status and regulate the iron homeostasis by binding to iron-responsive element (IRE). Upregulated IRP1 binding to IRE increased TfR1 mRNA stability and repressed FPN mRNA translation. 23,26 Our results were consistent with previous studies indicating that IRP1 was upregulated after pro-inflammatory cytokines stimulation in Parkinson's disease. 27 We next investigated the detrimental effect of iron influx induced by pro-inflammatory cytokines in chondrocytes. Our in vitro experiments showed that IL-1β promoted iron influx and lead to chondrocytes iron overload. Excess iron in turn accelerated IL-1β induced matrix-degrading enzymes MMPs expression. Recently, oxidative stress and mitochondrial dysfunction were reported to play pivotal roles in the progression of OA. 28,29 However, the role of mitochondrial dysfunction in iron overload-induced chondrocytes apoptosis and matrix-degrading enzyme expression remain to be elucidated. Iron overload is common in tissues of elderly people and has been implicated to be responsible for many diseases or pathological conditions. In iron overloaded status, the free Fe 2+ is increased and could catalyze the formation of free radicals, which could induce mitochondrial damage and reactive oxygen species generation, resulting in cell damage and eventually cell death. 15,30 Mitochondria are major cellular organelles that generate ROS, but excessive ROS production could in turn induce oxidative stress and mitochondrial dysfunction. 31 Therefore, we first examined whether there is a connection between iron overload and mitochondrial dysfunction. We found that FAC promoted chondrocytes ROS production and mitochondrial fission proteins expression in a dose-dependent manner. Our results indicated that excess iron could lead to mitochondrial dysfunction and morphology destruction. Furthermore, ROS production was significantly reduced by direct ROS scavenging with NAC as well as indirect ROS reduction through decreases in iron accumulation by iron chelation with DFO. Mitochondrial membrane potential decrease was also reversed after treatment with NAC or DFO. Our results indicated that iron accumulation in chondrocytes promoted ROS generation and caused mitochondrial dysfunction.
Our in vitro experiments also showed that FAC promoted chondrocytes apoptosis and matrix-degrading enzymes MMPs expression. While antioxidant NAC treatment partly reversed iron overload-induced mitochondrial dysfunction and MMPs production, NAC exhibited a similar protective effect with DFO, an iron chelator that could reduce cellular iron content, indicating oxidative stress and mitochondrial dysfunction play key roles in iron overloadinduced matrix-degrading enzymes expression.
In conclusion, our results indicated that pro-inflammatory cytokines could disturb cellular iron homeostasis via upregulating iron influx protein, TfR1, downregulating iron efflux protein FPN.
Excess iron in chondrocytes leads to oxidative stress via ROS overproduction and mitochondrial dysfunction. Oxidative stress then induces upregulation of the crucial effector matrix-degrading enzymes, MMP3 and MMP13. Our findings indicate that antioxidant treatment or local depletion of iron using an iron chelator would be effective therapeutic approaches for the treatment of iron overloadinduced cartilage degeneration.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.