IL-1β receptor antagonist (IL-1Ra) combined with autophagy inducer (TAT-Beclin1) is an effective alternative for attenuating extracellular matrix degradation in rat and human osteoarthritis chondrocytes

Background Autophagy induction is an effective approach for OA therapy. IL-1β is one of the major inflammatory cytokines linked to OA pathological progression, and its receptor blockade interrupts OA cartilage destruction. The objective of this study was to decipher the link between autophagy and regulatory mechanism of IL-1β and to investigate the effect of IL-1β receptor blockade by IL-1 receptor antagonist (IL-1Ra) combined with or without an autophagy inducer (TAT-Beclin1) on extracellular matrix (ECM) in OA chondrocytes in vitro and in vivo. Methods IL-1β-treated rat and human OA chondrocytes were cultured in response to IL-1Ra. The expression and distribution of signal molecules regulating ECM synthesis and autophagy were investigated via western blotting, immunoprecipitation, real-time PCR, immunofluorescence, and transmission electron microscope technique. Furthermore, after intra-articular injection of IL-1Ra, TAT-Beclin1, and a combination of both in a rat OA model established by anterior cruciate ligament transection and medial meniscus resection, the morphological changes of cartilage and related signal molecule expression levels were monitored using H.E., Safranin O-Fast green, and immunohistochemistry staining. Results Reduced autophagy by IL-1β contributed to ECM degradation, and blockade of IL-1β by IL-1Ra restored autophagy and attenuated ECM degradation in rat and human OA chondrocytes, as well as in a rat OA model. Akt/mTOR/ULK1, Akt/mTOR/NF-κB, and LC3B deacetylation were involved in autophagy regulated by IL-1β. Intra-articular injection of IL-1Ra combined with TAT-Beclin1 was more effective than IL-1Ra alone. Conclusions IL-1Ra restored autophagy and attenuated ECM degradation, with an implication that blocking IL-1β combined with enhancing autophagy might be a potential therapeutic strategy for OA. Electronic supplementary material The online version of this article (10.1186/s13075-019-1952-5) contains supplementary material, which is available to authorized users.


Background
Osteoarthritis (OA) pathological progression is linked to some cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor alpha. IL-1β is produced not only by activated synoviocytes and mononuclear cells, but also by articular cartilage chondrocytes [1]. It could significantly upregulate metalloproteinase (MMP) gene expression, resulting in extracellular matrix (ECM) degradation [2]. Blocking IL-1β, including recombinant IL-1 receptor antagonists (IL-1Ra) or soluble IL-1 receptor proteins, could modify OA progression [2]. Especially, elevated reproductively IL-1Ra to block IL-1β is a crucial element to promote cartilage regeneration in Orthokine®, which is an effective and well-tolerated alternative to currently predominant treatments of OA [3]. However, the mechanism of blocking IL-1β by IL-1Ra to promote cartilage regeneration is not fully understood [4].
As a highly conserved homeostatic process, autophagy could sequester and degrade cytosolic macromolecules, excess, or damaged organelles, and some pathogens to maintain cellular homeostasis in a healthy condition. Autophagy induction could interrupt OA pathological progression, including chondrocyte survival and ECM metabolism, and be an effective approach for OA therapy [5][6][7][8][9]. For instance, autophagy is a protective mechanism in normal cartilage [6]. The enhancement of autophagy by intra-articular injection of mTOR inhibitor, Rapamycin or Torin1, reduces degeneration of articular cartilage in an animal OA model [7][8][9]. However, whether and how IL-1β in OA progression linked to autophagy is not carefully elucidated. It is previously reported that autophagy regulates the secretion of IL-1β in macrophages [10][11][12]. Autophagy controls the production of IL-1β in macrophage through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome [10]. The inhibition of autophagy in macrophages by knockdown of autophagy-related gene (Atg)7 or Atg16L1 promotes the secretion of IL-1β in response to LPS [11]. On the other hand, IL-1β could modify autophagy in macrophage. IL-1 receptor blockade restores autophagy of macrophage and reduces inflammation in chronic granulomatous disease in mice and in humans [12]. Based on these studies, we hypothesize that the regulatory effect of IL-1β in OA progression might be associated with autophagy.
In this study, it was investigated the relationship between ECM synthesis and autophagy in IL-1βtreated rat chondrocytes (mimicking OA pathological condition, hereafter referred to as OA chondrocyte) and human OA chondrocytes in response to IL-1Ra. Furthermore, the effects of IL-1Ra on articular cartilage were monitored in a rat OA model. Our results demonstrated that reduced autophagy contributed to ECM degradation by IL-1β and blockade of IL-1β by IL-1Ra restored autophagy and attenuated ECM degradation in rat and human OA chondrocytes, as well as in a rat OA model. Akt/mTOR/ULK1, Akt/ mTOR/NF-κB, and LC3B deacetylation were involved in autophagy regulated by IL-1β. Intra-articular injection of IL-1Ra combined with autophagy inducer (TAT-Beclin1) is an effective alternative for attenuating extracellular matrix degradation in a rat OA model. Chondrocyte isolation, culture, and treatment with IL-1β

Reagents and antibodies
After the approval of the Committee on the Ethics of Animal Experiments of Medical School, Xiamen University, rat chondrocytes of knee cartilage were isolated from neonatal male Sprague-Dawley (SD) rats (within 24-72 h after birth) [13,14]. Briefly, rats were killed and articular cartilages were removed under sterile conditions. Thin slices of cartilage were sequentially digested with 0.25% Trypsin 37°C for 5 min, followed with 0.1% type I collagenase (Sigma-Aldrich in Shanghai, China) in humidified incubator (37°C and 5% CO 2 ) overnight. Type I collagenase was prepared with Dulbecco's modified Eagle's medium (DMEM)/F12 (Invitrogen, Carlsbad, CA, USA) containing 10% FBS supplemented with antibiotics: penicillin (100 UI/ml, Sigma) and erythromycin (100 μg/ml). Primary chondrocytes were cultured in DMEM containing 10% fetal bovine serum to 80% confluence and plated in 60-mm cell culture dishes (1 × 10 6 /ml). Passage 1-2 chondrocytes obtained from the same rat in each experiment were pretreated by IL-1β (20 ng/ml) for 36 h to mimic OA pathological condition for the subsequent experiments [14].
Human OA cartilage was obtained from 27 patients (Table 1) with advanced OA who were undergoing total knee replacement surgery without protreatment of arthroscopy, after receiving all patient consent and in accordance with the ethical guidelines approved by the Ethics Committee of Medical School, Xiamen University, China. Thin slices of cartilage were sequentially digested with 0.25% trypsin 37°C for 30 min and 0.15% type II collagenase (Sigma-Aldrich in Shanghai, China) in a humidified incubator (37°C and 5% CO 2 ) for 8-12 h. Type II collagenase preparation is the same as type I collagenase. Primary chondrocytes were cultured in DMEM containing 10% fetal bovine serum to 80% confluence and plated in 60-mm cell culture dishes (1 × 10 6 /ml). Passage 1-2 chondrocytes were pretreated by IL-1β (10 ng/ml) for 24 h to maintain OA pathological condition for the subsequent experiments as described [15][16][17][18].

Establishment of a rat experimental model of OA
The protocol was approved by the Committee on the Ethics of Animal Experiments of Medical School, Xiamen University. Four-to five-week-old male SD rats (120-150 g) purchased from Slaccas.com (Shanghai, China) were acclimatized to the laboratory environment for one week before the experiments and were randomly divided into three parts, normal (n = 12 rats), Sham (n = 12 rats), and OA (n = 60 rats). Sixty rats underwent anterior cruciate ligament transection and medial menisci resection [15,17] and were divided into 5 groups, OA (n = 12 rats), injection with normal saline (NS, 0.9% NaCl) (n = 12 rats, a total volume of 40 μl per injection), injection with IL-1Ra (n = 12 rats, 500 μg/ml in a total volume of 40 μl per injection), TAT (n = 12 rats, 1.5 mg/kg in a total volume of 40 μl per injection), and IL-1Ra+TAT (n = 12 rats, IL-1Ra: 1000 μg/ml in a total volume of 20 μl; TAT: 1.5 mg/kg in a total volume of 20 μl per injection). As described previously [20,21], different reagents or NS were injected intra-articularly once every three days for two weeks after three days post-surgery. Rats were not sacrificed until three and five weeks after post-operation, respectively. Animal hepatic tissue was collected to detect the side effect of different reagents and NS injected intra-articularly.

Real-time PCR (RT-PCR)
After total RNA in chondrocytes was extracted using TRIzol (Invitrogen, CA, USA), cDNA was synthesized with 1 μg of total RNA at 37°C for 15 min using a Primescript RT Master Mix Kit (Takara, Dalian, China). Real-time PCR was then performed using an ABI StepOnePlus Sequence Detection System v2.1 (Applied Biosystems, Singapore) with a SYBR Premix Ex Taq II Kit (Takara, Dalian, China). The results were normalized to GAPDH and analyzed using SDS software v2.1 [22,23]. The primers used for quantitative PCR to measure gene expression levels are listed in Table 2.

Separation of cytoplasmic and nuclear fraction
Cells were suspended in 2 ml MS buffer (210 mmol/l mannitol, 70 mmol/l sucrose, 5 mmol/l Tris-HCl, pH 7.5, and 1 mmol/l EDTA, pH 7.5), containing a 1% protease inhibitor cocktail, and then homogenized using a Dounce homogenizer [24]. The homogenate was spun at 12,000×g for 30 s at 4°C to pellet the nuclei and unbroken cells. The supernatant was the cytoplasmic fraction.

Western blotting analysis and immunoprecipitation assay
Protein extracts were subjected to SDS-PAGE (6-12%) and transferred to a PVDF membrane (GE Healthcare, Hertfordshire, UK) as described [22,23]. The membrane was incubated with various antibodies as required at 4°C overnight, followed by the addition of the corresponding secondary antibodies at room temperature for 1 to 2 h. An enhanced chemiluminescence (ECL) detection kit was used to detect antibody reactivity (Pierce, Rockford, IL, USA).
As described previously [25,26], 400 μg of nuclear protein was mixed with 8 μl of Protein A&G Sepharose (Sigma-Aldrich, Shanghai, China) and 8 μl of anti-LC3B antibody or immunoglobulin (IgG) control for 3 h at 4°C. The protein-antibody complexes that were recovered on the beads were subjected to western blot analysis as above using anti-LC3B and antiacetylated-lysine antibodies.

Transmission electron microscopy
Cells were scraped and then pelleted by centrifugation at 2000×g for 15 min at 4°C, followed by fixation for 2 h at 4°C in 2.5% glutaraldehyde in 0.1 M PBS (PH7.4) as described [27]. After samples were dehydrated and embedded in Embed-812 resin, 70-nm sections were cut using an ultramicrotome (Leica EM UC7, LEICA, Shanghai, China) and stained with uranyl acetate and lead citrate. Autophagic vacuoles were observed under a transmission electron microscope (Tecnai G2 Spirit BioTWIN, FEI Company, Hillsboro, OR, USA).

Histopathological assay
Samples were fixed in 4% paraformaldehyde for 48 h followed with decalcification in 10% EDTA-2Na for three weeks, and then paraffin-embedded for further routine histological preparation. Three-micrometerthick sections were deparaffinized in xylene and rehydrated in graded alcohols and distilled water prior to H.E. and Safranin O-Fast green staining as described [15,17]. Histological sections were imaged using the Virtual Slide Microscope (VS120-S6-W, Olympus, Tokyo, Japan). The articular cartilage thickness of each femur condyle was measured using Image-Pro Plus 6.0 software. According to the Osteoarthritis Research Society International (OARSI) scoring system established for grading OA changes [28], semiquantitative histopathological grading was performed by two different blinded pathologists. Score 0 represents normal articular cartilage, and an increasing score indicates a more biologically cartilage degeneration (a maximum possible score of 24).

Immunohistochemistry staining
Sections were incubated overnight at 4°C with primary antibody: type II of collagen (Col II, 1:1000), Aggrecan (1:800), autophagy marker LC 3B (1:200), and Beclin1 (1:200) dilutions, respectively, prior to incubation with secondary antibodies, as described in the manufacturer's instructions (MAIXIN.BIO, Fuzhou, China). Diaminobenzidine was then used to visualize the immunohistochemical reaction followed by being counterstained with hematoxylin. Images were scanned using the Virtual Slide Microscope (VS120-S6-W, Olympus, Tokyo, Japan). Dark brown cells or area were considered to be positive. The positive chondrocytes were counted semi-automated using Image-Pro Plus 6.0 Software, and area was measured using ImageJ Software, followed by analysis with Graph-Pad Prism version 5 [15,17,29,30].

Statistical analysis
Data were expressed as the mean ± 95% confidence interval (CI) of three independent experiments in each cell experiment and six independent samples in each group of animal experiments. One-way analysis of variance (ANOVA) with Tukey's post hoc test was used to compare two or multiple groups by GraphPad Prism 5 software. The error bars of all figures represent 95% CI. A value of p < 0.05 was regarded as statistically significant.

IL-1 receptor blockade by IL-1Ra restored autophagy to attenuate ECM degradation in human OA chondrocytes
In human OA chondrocytes (Table 1), IL-1β reduced Col II, Aggrecan, Beclin1 protein levels, and the ratio of LC3B-II/I, while addition of IL-1Ra reversed the effect of IL-1β on them (Fig. 2a, *p < 0.05,****p < 0.0001, vs control or IL-1β-treated human OA chondrocytes). Increased autophagic vacuoles were obviously observed in IL-1β+IL-1Ra-treated human OA chondrocytes, compared with IL-1β-treated human chondrocytes (Fig. 2b). LC3Bpositive structures in IL-1β+IL-1Ra-treated human OA chondrocytes seemed to be more than that in IL-1βtreated human OA chondrocytes (Fig. 2c). Like rat OA chondrocytes, the differential distribution of LC3Bpositive structure in nucleus was observed in human OA chondrocytes between IL-1β-treated and IL-1β+IL-1Ra chondrocytes (Fig. 2c, Additional file 1: Figure S1). The results of the separation of cytoplasmic and nuclear fraction showed that IL-1β promoted the translocation of LC3B from the cytoplasm to nucleus in human OA chondrocytes, and the addition of IL-1Ra reversed the effect of IL-1β on the translocation of LC3B (Fig. 2d). Meanwhile, IL-1Ra led to the deacetylation of LC3B in IL-1β-treated human OA chondrocytes (Fig. 2e). Additionally, IL-1β upregulated the phosphorylation of mTOR, ULK1 and Akt, along with an increase in NF-κB (p65) protein level, and IL-1Ra reversed the effect of IL-1β on them (Fig. 2f, ****p < 0.0001, vs control or IL-1β-treated human OA chondrocytes). The effect of IL-1Ra, TAT, and a combination of both on articular cartilage degeneration in a rat OA model Western blotting analysis showed that the promoted effect of a combination of IL-1Ra with TAT-Beclin1 (autophagy inducer, TAT) on Col II and Aggrecan protein levels was more effective than IL-1Ra alone in IL-1βtreated rat and human OA chondrocytes (Fig. 3a, b). Hence, we investigated the effect of IL-1Ra, TAT, and a combination of both on cartilage degeneration and whether the "additive" or "synergistic" effect of IL-1Ra and TAT treatment exists in rat OA model. Each knee was cut through the intercondylar of femur, and femoral cartilage was then observed in the current study as tibial cartilage was not intact at some sections (Additional file 2 Figure S2 and Additional file 3: Figure S3). The intraarticular injection of IL-1Ra, TAT, or IL-1Ra+TAT in three weeks post-operative groups resulted in a significant decrease of OARSI score (Fig. 3c, ****p < 0.0001, vs OA+NS group). Similar results were observed in five weeks post-operative groups (Fig. 3d, **p < 0.01, ****p < 0.0001, vs OA+NS group). In either three weeks or five weeks post-operative groups, OARSI score of IL-1Ra+TAT group was lower than IL-1Ra or TAT alone (Fig. 3c,d, *p < 0.05,**p < 0.01), but closer to that of the normal group. Additionally, the intra-articular injection of IL-1Ra and TAT did not result in liver injury of rat OA model (Additional file 4: Figure S4).

Discussion
Our findings demonstrated that IL-1β degraded ECM synthesis concomitant with reduced autophagy in IL-1β-treated rat and human OA chondrocytes. IL-1Ra reversed the effect of IL-1β by restoring autophagy, associated with Akt/mTOR/ULK1 and Akt/NF-κB signaling pathways, as well as the deacetylation of LC3B. Furthermore, intra-articular injection of IL-1Ra, TAT, and IL-1Ra+TAT, respectively, ameliorated cartilage degeneration in a rat OA model, in which the effect of a combination of IL-1Ra with TAT was significantly higher than IL-1Ra alone. Therefore, reduced autophagy could contribute to ECM degradation induced by IL-1β and IL-1 receptor blockade by IL-1Ra could restore autophagy to promote ECM synthesis in OA chondrocytes in vitro and in vivo. A combination of blocking IL-1β effect by IL-1 receptor antagonist with enhancing autophagy induction by Previous studies have shown that the autophagy induction suppresses IL-1β secretion and IL-1 receptor blockade could restore autophagy in macrophages [8][9][10]. Consistent with those studies, in the current study, IL-1β degraded ECM synthesis concomitant with reduced autophagy and IL-1Ra reversed its effect by enhancing autophagy induction in rat and human OA chondrocytes, while 3-MA reversed the effect of IL-1Ra. Furthermore, in rat OA model, intra-articular injection of IL-1Ra ameliorated cartilage degeneration, similar to an autophagy inducer TAT that could protect mammalian cells against damage by autophagy induction [34][35][36]. Hence, we suggested that reduced autophagy could contribute to ECM degradation induced by IL-1β and that IL-1 receptor blockade by IL-1Ra could restore autophagy to promote ECM synthesis in OA chondrocytes in vitro and in vivo. As for why Col II and Aggrecan predominantly were distributed in the intra-and pericellular region in this study, there might exist two possibilities. One might be associated with the duration and dose of injected reagents. Another might be due to the shortage of detecting effectiveness. This will be considered in the future work. mTOR, as a "classical" autophagy suppressor, acts by blocking the activity of the ULK1 complex [37]. Akt, an important signal molecule in cell events, has been reported to activate mTOR pathway to suppress autophagy [38,39]. For instance, microRNA-99 family modulates hepatitis B virus replication by promoting IGF-1R/PI3K/Akt/mTOR/ULK1 signalinginduced autophagy [38] PRKCD/Akt/mTOR/ULK1 signaling pathway is involved in autophagy suppression in cisplatin nephrotoxicity [39]. In the current study, IL-1Ra restored autophagy reduced by IL-1β and reversed the promoted effect of IL1β on the phosphorylation of Akt, mTOR, and ULK1, implying that Akt/mTOR/ULK1 pathway could be involved in OA chondrocyte autophagy restored by IL-1Ra, consistent with the abovementioned studies. In addition, in the current study, IL-1Ra led to the decrease of NF-κB, one of the downstreams of Akt, in rat and human OA chondrocytes. Li et al. have found that cerebral ischemia induced autophagy-like injury is regulated by the NF-κB pathway [40]. Huang et al. have recently reported that the inhibition of the mTOR/NF-κB signaling pathway potentiates HTEA against myocardial IR injury by autophagy and apoptosis in rats [41]. It is believable that the Akt/mTOR/ NF-κB pathway was also involved in IL-1Ra-restored autophagy in OA chondrocytes. Therefore, we suggested that IL-1Ra could restore autophagy via Akt/ mTOR/ULK1 and Akt/mTOR/NF-κB pathways in rat and human OA chondrocytes.
Differential acetylation of autophagy-related proteins participates in autophagic flux. For instance, LC3B-II deacetylation by HDAC6 is involved in serum-starvation-induced autophagic degradation of Hela cells [31]. Erythropoietin (EPO) alleviates hepatic steatosis by activating autophagy via SIRT1-dependent deacetylation of LC3 [32]. In response to nutrient depletion, activated Sirt1 interacts with and deacetylates nuclear LC3. Through binding to DOR, deacetylated LC3 is transported to the cytoplasm to carry out PE conjugation by sequential interaction with Atg7 and Atg3 [33]. Consistent with these studies, we also found that IL-1Ra led to the translocation of LC3B from nucleus to cytoplasm via its deacetylation, suggesting that LC3B deacetylation could contribute to the restoration of autophagy induction by IL-1Ra in rat and human OA chondrocytes. The regulatory mechanism of LC3B deacetylation in OA pathological progression is further to be studied.

Conclusions
IL-1Ra restored autophagy and attenuated ECM degradation, in which Akt/mTOR/ULK1, Akt/mTOR/NF-κB, and LC3B deacetylation were involved in rat and human OA chondrocytes, as well as a rat OA model. IL-1β receptor antagonist (IL-1Ra) combined with an autophagy inducer (TAT-Beclin1) is an effective alternative for attenuating extracellular matrix degradation of osteoarthritis in rats and humans.