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.

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.