NOD2 attenuates osteoarthritis via reprogramming the activation of synovial macrophages.

OBJECTIVE: Synovial inflammation, which precedes other pathological changes in osteoarthritis (OA), is primarily initiated by activation and M1 polarization of macrophages. While macrophages play a pivotal role in the inflammatory process of OA, the mechanisms underlying their activation and polarization remain incompletely elucidated. This study aims to investigate the role of NOD2 as a reciprocal modulator of HMGB1/TLR4 signaling in macrophage activation and polarization during OA pathogenesis. DESIGN: We examined NOD2 expression in the synovium and determined the impact of NOD2 on macrophage activation and polarization by knockdown and overexpression models in vitro. Paracrine effect of macrophages on fibroblast-like synoviocytes (FLS) and chondrocytes was evaluated under conditions of NOD2 overexpression. Additionally, the in vivo effect of NOD2 was assessed using collagenase VII induced OA model in mice. RESULTS: Expression of NOD2 was elevated in osteoarthritic synovium. In vitro experiments demonstrated that NOD2 serves as a negative regulator of HMGB1/TLR4 signaling pathway. Furthermore, NOD2 overexpression hampered the inflammatory paracrine effect of macrophages on FLS and chondrocytes. In vivo experiments revealed that NOD2 overexpression mitigated OA in mice. CONCLUSIONS: Supported by convincing evidence on the inhibitory role of NOD2 in modulating the activation and M1 polarization of synovial macrophages, this study provided novel insights into the involvement of innate immunity in OA pathogenesis and highlighted NOD2 as a potential target for the prevention and treatment of OA.

STING/TBK1 Regulates Inflammation in Macrophages and Titanium Particles-Induced Osteolysis.

Inflammatory response in macrophages on account of prostheses-derived wear particles is the leading cause of artificial joint failure. However, the mechanism by which wear particles initiate macrophage inflammation has not been fully elucidated. Previous research studies have identified TANK-binding kinase 1 (TBK1) and stimulator of interferon genes (STING) as potential factors in inflammation and autoimmune diseases. Here, we found that both TBK1 and STING were increased in synovium from aseptic loosening (AL) patients and were activated in titanium particles (TiPs)-stimulated macrophages. Lentivirus-mediated knockdown of TBK or STING significantly inhibited the inflammatory effects of macrophages, while overexpression of TBK or STING exerted opposite results. In concrete, STING/TBK1 promoted the activation of NF-κB and IRF3 pathways and macrophage M1 polarization. For further validation, a mice cranial osteolysis model was constructed for in vivo assays, and we found that STING-overexpressed lentivirus injection exacerbated osteolysis and inflammation, which was counteracted by TBK1-knockdown injection. In conclusion, STING/TBK1 enhanced TiP-induced macrophage inflammation and osteolysis via orchestrating the activation of NF-κB and IRF3 pathways and M1 polarization, which suggested STING/TBK1 as potential therapeutic targets for preventing AL of prostheses.

HUCMSC-derived Exosomes Suppress the Titanium Particles-induced Osteolysis in Mice through Inhibiting CCL2 and CCL3.

OBJECTIVE: Wear particles induce inflammation and the further osteolysis around the prosthesis, has been proven to be the main cause of aseptic hip joint loosening. In this research, we aimed to clarify whether human umbilical cord mesenchymal stem cells (HUCMSCs) could inhibit the titanium particles-induced osteolysis and shed light upon its mechanism. METHODS: The expression of chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 3 (CCL3) and chemokine (C-C motif) ligand 5 (CCL5) were examinjed in clinical specimens of aseptic hip prosthesis loosening patients. Local injection of lentivirus that knocked down CCL2 or CCL3 in a cranial osteolysis mice model were used to exam the effect of CCL2 and CCL3 on titanium particles-induced osteolysis in vivo. Transwell assay was used to examine the effect of CCL2 and CCL3 on titanium particles-induced activation of macrophage in vitro. Furthermore, the therapeutic effect of HUCMSCs, and exosomes from HUCMSCs were also examed in vivo and vitro. Immunohistochemical and real-time PCR were used to examine the expression of relative pathways. Analysis of variance (ANOVA) and Student-Newman-Keuls post hoc t test were used to analyze the results and determine the statistical significance of the differences. RESULTS: Results showed that titanium particles caused the osteolysis at the mice cranial in vivo and a large number of macrophages that migrated, while local injection of HUCMSCs and exosomes did inhibit the cranial osteolysis and migration. An exosome inhibitor GW4869 significantly increased the osteolysis area in the mice cranium osteolysis model, and increased the number of migrated macrophages. Immunohistochemical results suggested that the expression of CCL2, CCL3 and CD68 in the cranial in Titanium particles mice increased significantly, but was significantly reduced by HUCMSCs or exosomes. HUCMSC and exosomes down-regulate the expression of CCL3 in vitro and in vivo. CONCLUSION: HUCMSCs and HUCMSC-derived exosomes could suppress the titanium particles-induced osteolysis in mice through inhibiting chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 3.

MiR-92a/KLF4/p110δ regulates titanium particles-induced macrophages inflammation and osteolysis.

As total joint replacement is widely applied for severe arthropathy, peri-prosthetic aseptic loosening as one of the main causes of implant failure has drawn wide attention. Wear particles such as titanium particles (TiPs) derived from prosthesis can initiate macrophages inflammation and sequentially activate osteoclasts, which results in bone resorption and osteolysis for long-term. Therefore, inhibiting wear particles induced macrophages inflammation is considered as a promising therapy for AL. In this research, we found that the inhibition of p110δ, a member of class IA PI3Ks family, could significantly dampen the TiPs-induced secretion of TNFα and IL-6. By the transfection of siRNA targeting p110δ, we confirmed that p110δ was responsible for TNFα and IL-6 trafficking out of Golgi complex without affecting their expression in TiPs-treated macrophages. As the upstream transcription-repressor of p110δ, Krüppel-like factor 4 (KLF4), targeted by miR-92a, could also attenuate TiPs-induced inflammation by mediating NF-κB pathway and M1/M2 polarization. To further ascertain the roles of KLF4/p110δ, TiPs-induced mice cranial osteolysis model was established and vivo experiments validated that KLF4-knockdown could exacerbate TiPs-induced osteolysis, which was strikingly ameliorated by knockdown of p110δ. In summary, our study suggests the key role of miR-92a/KLF4/p110δ signal in TiPs-induced macrophages inflammation and osteolysis.

LncRNA Neat1 promotes the macrophage inflammatory response and acts as a therapeutic target in titanium particle-induced osteolysis.

Aseptic loosening (AL), secondary to particle-caused periprosthetic osteolysis, is one of the main reasons of artificial joint failure. Suppressing the macrophage inflammatory response caused by wear particles extends the life of prosthesis, and the long noncoding RNAs (lncRNAs) may play a predominant part in it. Here, titanium particles' (TiPs') stimulation increases both the cytoplasmic and nuclear levels of lncRNA Neat1 in bone marrow derived macrophages (BMDMs), which further induces the inflammatory response. Mechanically, Neat1 facilitates Bruton's tyrosine kinase (BTK) transcription by reducing the transcriptional factor KLF4, which further activates the NF-κB pathway, NLRP3 inflammation, and M1 polarization in BMDMs. Cytoplasmic Neat1 also works as an miRNA sponge in miR-188-5p-regulated BTK expression in the post-transcriptional stage. In vivo, Neat1 downregulation can reduce the TiP-induced pro-inflammatory factors and reverse the osteolysis induced by BTK overexpression. In addition, the PLGA-based microparticles loaded with si-Neat1 are developed for the treatment of the mouse calvarial osteolysis model via local injection, presenting satisfactory anti-osteolysis efficacy. These findings indicate that Neat1 is a key regulator of AL. STATEMENT OF SIGNIFICANCE: Due to released particles, aseptic loosening (AL) is the most common reason for prosthesis failure and surgical revision and represents a substantial economic burden worldwide. Herein, we reported that lncRNA Neat1 is a key regulator in regulating wear particles-induced osteolysis by activating NF-κB pathway, NLRP3 inflammation and M1 polarization via BTK, and the underlying mechanisms of Neat1-BTK interaction were further portrayed. For potential clinical application, the microparticles are developed for effective si-Neat1 delivery, leading to a dramatically enhanced effect for the treatment of osteolysis, which might be a novel strategy to extend the life of the implant.

Increased expression of osteopontin in subchondral bone promotes bone turnover and remodeling, and accelerates the progression of OA in a mouse model.

Osteopontin (OPN) has been proved to be closely related to the pathogenesis of osteoarthritis (OA), but the role of OPN in the pathogenesis of OA has not been fully clarified. Current studies on OPN in OA mostly focus on articular cartilage, synovial membrane and articular fluid, while ignoring its role in OA subchondral bone turnover and remodeling. In this study, we used a destabilization OA mouse model to investigate the role of OPN in OA subchondral bone changes. Our results indicate that increased expression of OPN accelerates the turnover and remodeling of OA subchondral bone, promotes the formation of h-type vessels in subchondral bone, and mediates articular cartilage degeneration induced by subchondral bone metabolism. In addition, our results confirmed that inhibition of PI3K/AKT signaling pathway inhibits OPN-mediated OA subchondral bone remodeling and cartilage degeneration. This study revealed the role and mechanism of OPN in OA subchondral bone, which is of great significance for exploring specific biological indicators for early diagnosis of OA and monitoring disease progression, as well as for developing drugs to regulate the metabolism and turnover of subchondral bone and alleviate the subchondral bone sclerosis of OA.

Carnosol suppresses RANKL-induced osteoclastogenesis and attenuates titanium particles-induced osteolysis.

Osteolysis is a common medical condition characterized by excessive activity of osteoclasts and bone resorption, leading to severe poor quality of life. It is essential to identify the medications that can effectively suppress the excessive differentiation and function of osteoclasts to prevent and reduce the osteolytic conditions. It has been reported that Carnosol (Car), isolated from rosemary and salvia, has anti-inflammatory, antioxidative, and anticancer effects, but its activity on osteolysis has not been determined. In this study, we found that Car has a strong inhibitory effect on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation dose-dependently without any observable cytotoxicity. Moreover, Car can inhibit the RANKL-induced osteoclastogenesis and resorptive function via suppressing NFATc1, which is a result of affecting MAPK, NF-κB and Ca2+ signaling pathways. Moreover, the particle-induced osteolysis mouse model confirmed that Car could be effective for the treatment of bone loss in vivo. Taken together, by suppressing the formation and function of RANKL-induced osteoclast, Car, may be a therapeutic supplementary in the prevention or the treatment of osteolysis.

Spermidine activates RIP1 deubiquitination to inhibit TNF-α-induced NF-κB/p65 signaling pathway in osteoarthritis.

Spermidine has been known to inhibit the production of pro-inflammatory cytokines. However, there are no reports about anti-inflammatory effects of spermidine on osteoarthritis (OA). Herein, we examined whether OA progression could be delayed by intraperitoneal injection (i.p.) of spermidine in the anterior cruciate ligament transection (ACLT) and TNF-α induced arthritis (TIA) mouse models. During the process, human FLS cells (H-FLS) were used to investigate the potential ubiquitination mechanism of spermidine-mediated RIP1 in TNF-α-induced NF-κB/p65 signaling. We found that spermidine attenuated synovitis, cartilage degeneration and osteophyte formation, resulting in substantially lower OARSI scores and TNF-α scores in spermidine-treated ACLT and TIA mice. In terms of the mechanism, 9 µM spermidine did not affect the viability, proliferation, cell cycle and apoptosis of H-FLS, and exerted inhibitory effects by activating CYLD-mediated RIP1 deubiquitination on TNF-α-induced NF-κB/p65 signaling in H-FLS. From these data, we can conclude that spermidine attenuates OA progression by the inhibition of TNF-α-induced NF-κB pathway via the deubiquitination of RIP1 in FLS. Therefore, intake of spermidine could be a potential therapy for preventing OA.

ZBTB20-mediated titanium particle-induced peri-implant osteolysis by promoting macrophage inflammatory responses.

Aseptic loosening (AL) caused by wear particles released from implant surfaces is one of the main causes for the failure of artificial joints, which is initiated by macrophage inflammatory responses. Emerging evidence suggests that the member of a broad-complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) family as well as zinc finger and BTB domain-containing protein 20 (ZBTB20) can inhibit IκBα gene transcription, promote NF-κB activation, and initiate innate immune responses. The molecular mechanism(s) by which ZBTB20 contributes to titanium particle (TiP)-induced macrophage inflammatory responses and osteolysis has not been fully elucidated. Here, we showed that ZBTB20 increased either in the AL group's synovial membranes or in TiP-stimulated bone-marrow-derived macrophages (BMDMs) as compared to that in the control groups. Moreover, the knockdown of ZBTB20 led to the inhibition of proinflammatory factors induced by TiPs in BMDMs, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-ß (IFN-ß). Here, we also reported that the knockdown of ZBTB20 suppressed TiP-induced NF-κB activation and M1 polarization as well as stabilized the trans Golgi network (TGN) in BMDMs. The dual-luciferase reporter assay identified the binding between the IκBα promoter and ZBTB20, and IκBα knockdown could rescue the antiinflammatory effects induced by the ZBTB20 knockdown in BMDMs. Finally, we found that sh-ZBTB20 lentivirus injection could reduce TiP-induced osteolysis in mouse calvaria, inhibiting TiP-induced proinflammatory factors and loss of bone volume/total volume (BV/TV) as well as bone mineral density (BMD). These results suggest that ZBTB20 positively regulated NF-κB activation and M1 polarization as well as the production of TGN-derived tubular carriers in BMDMs, playing a positive role in macrophage activation and mouse cranial osteolysis induced by TiPs. It may be a potential therapeutic target for the prevention of aseptic loosening of prostheses.

The USP14-NLRC5 pathway inhibits titanium particle-induced osteolysis in mice by suppressing NF-κB and PI3K/AKT activities.

Total hip arthroplasty (THA) is a widely-used surgical intervention for treating patients with end-stage degenerative and inflammatory osteoarthropathy. However, wear particles from the artificial titanium joint can induce osteolysis, limiting the long-term survivorship of THA. Monocyte/macrophage lineage cells are the key players in the response to wear particles, and the proinflammatory NF-κB and phosphoinositide 3-kinase (PI3K)-AKT Ser/Thr kinase (AKT)-signaling pathways have been shown to be the most important contributors to wear particle-induced osteolysis. In contrast, ubiquitin-specific protease 14 (USP14) specifically removes the polyubiquitin chains from the nucleotide-binding and oligomerization domain (NOD)-like receptor family Caspase recruitment domain (CARD)-containing 5 (NLRC5) and thereby enhances the NLRC5-mediated inhibition of NF-κB signaling. In this study, we aimed to clarify the role of the USP14-NLRC5 pathway in wear particle-induced osteolysis in vitro and in vivo We found that NLRC5 or USP14 overexpression inhibits titanium particle-induced proinflammatory tumor necrosis factor α (TNFα) production and NF-κB pathway activation, and it also decreases M1 macrophage polarization and PI3K/AKT pathway activation. Of note, NLRC5 and USP14 overexpression attenuated titanium particle-induced cranial osteolysis in mice. In conclusion, the findings of our study indicate that the USP14-NLRC5 pathway inhibits titanium particle-induced osteolysis by suppressing the NF-κB and PI3K/AKT pathways both in vitro and in vivo.

Efficiency of Zoledronic Acid in Inhibiting Accelerated Periprosthetic Bone Loss After Cementless Total Hip Arthroplasty in Osteoporotic Patients: A Prospective, Cohort Study.

OBJECTIVE: To investigate the influence of preoperative osteopenia/osteoporosis on periprosthetic bone loss after total hip arthroplasty (THA) and the efficiency of zoledronate (ZOL) treatment in periprosthetic bone preservation. METHODS: This multicenter, prospective cohort study was conducted in four centers between April 2015 and October 2017. Patients were assigned to Normal BMD, Osteopenia, and Osteoporosis+ZOL groups. Patients with osteopenia received daily oral calcium (600 mg/d) and vitamin D (0.5 µg/d), while patients in the Osteoporosis+ZOL group received additional ZOL annually (5 mg/year). Periprosthetic bone mineral density (BMD) in seven Gruen zones, radiographic parameters, Harris hip score, EuroQol 5-Dimensions (EQ-5D) score, and BMD in hip and spine were measured within 7 days, 3 months, 12 months postoperation and annually thereafter. RESULTS: A total of 266 patients were enrolled, while 81 patients that completed the first year follow-up were involved in the statistical analysis. The mean follow-up time was 1.3 years. There were significant decreases of mean BMD in total Gruen zones (-4.55%, P < 0.05) and Gruen zone 1 (-10.22%, P < 0.01) in patients with osteopenia during the first postoperative year. Patients in the Osteoporosis+ZOL group experienced a marked increase in BMD in Gruen zone 1 (+16%) at the first postoperative year, which had a significant difference when compared with the Normal BMD group (P < 0.05) and the Osteopenia Group (P < 0.001). Low preoperative BMD in hip and spine was predictive of bone loss in Gruen zone 1 at 12 months after THA in patients with normal BMD (R2 = 0.40, P < 0.05). CONCLUSIONS: Patients with osteopenia are prone to higher bone loss in the proximal femur after cementless total hip arthroplasty (THA). ZOL, not solely calcium and vitamin D, could prevent the accelerated periprosthetic bone loss after THA in patients with osteopenia and osteoporosis.

NOD2 negatively regulated titanium particle-induced osteolysis in mice.

For patients undergoing total joint replacement (TJR), one of the complications, aseptic loosening, could cause serious consequences, such as revision surgery. In early research, pattern recognition receptors (PRRs) were reported to play vital roles in recognizing wear particles from the prosthesis and initiating an inflammation response. In this research, we aimed to clarify the role of nucleotide-binding and oligomerization domain containing protein 2 (NOD2), one of the PRRs, in macrophage-induced aseptic loosening in vivo and in vitro. High expressions of NOD2 and TNFα were observed from twenty patients who underwent primary or revision total hip replacements (THR). The effect of NOD2 on the activation of inflammation pathways was observed in RAW264.7 cells and CRISPR-Cas9 NOD2-knockout mice. The expressions of NOD2, the NF-κB pathway, the MAPK pathway and proinflammatory cytokine TNF-α in macrophages stimulated by wear particles were up-regulated. Otherwise, inhibition of NOD2 further up-regulated the expressions of NOD2, the NF-κB pathway, the MAPK pathway and TNF-α. Knockdown of the NOD2 gene enhanced the cranial osteolysis induced by titanium particles in a mouse model. In conclusion, our study demonstrated that NOD2 plays a negative role in osteolysis induced by titanium particles in vitro and in vivo.

Study on the mechanism of platinum(ii)-catalyzed asymmetric ring-opening addition of oxabicyclic alkenes with arylboronic acids.

The mechanism of an asymmetric ring-opening (ARO) addition of oxabicyclic alkenes catalyzed by a platinum(ii) catalyst was investigated by M06-2X/6-311G(d,p) using density functional theory (DFT). All the structures were optimized in the solvent model density (SMD) solvation model (solvation = the mixture of H2O/CH2Cl2 1 : 10, v/v) for consistence with experimental conditions. The overall mechanism is considered as a four-step reaction including transmetalation, carboplatinum, ß-oxygen elimination, and hydrolysis. The transmetalation and carboplatinum steps are multi-step processes, and both the regioselectivity and the enantioselectivity lie in the carboplatinum process. Based on the natural population analysis (NPA) and the orbital composition analysis of oxabicyclic alkenes, the preferable coordination site with a platinum(ii) center is considered as the bridging oxygen atom by exo-coordination because of the less steric hindrance and the stronger electronic effect. This coordination is thought of as origin of the regioselectivity and the enantioselectivity, which is different from that proposed previously. The Gibbs free energy profiles show that the rate-determining step involves the migration of an aryl group from the platinum(ii) center to one of the closer enantiotopic carbon atoms in an alkene of the oxabicyclic alkenes. The theoretically predicted enantiomeric excess (ee) value of 82% for this reaction is very close to the experimental ee value of 80%. It was found that the hydrogen bonds between the oxabicyclic alkenes and water molecules promotes the platinum(ii) catalyst leaving the reaction system effortlessly and entering the next catalysis recycle. In the overall catalytic cycle, the highest free energy barrier is 30.1 kcal mol-1 and the process releases an energy of 26.3 kcal mol-1. The results confirm that the Pt(ii)-catalyzed ARO reactions take place at mild experimental conditions, which is consistent with the experiment observations. Thus, this study is important for understanding the catalytic behavior of the transition metal platinum(ii) in an asymmetric ring-opening reaction.