IDN5706 Inhibits Synovial Inflammation via Inducing M2 Polarization of Synovial Macrophages in Osteoarthritis Rats.

IDN5706 is a tetrahydro derivative of hyperforin. In this study, we aimed to explore the effect of IDN5706 on synovial macrophages in osteoarthritis (OA) rats and the underlying mechanisms. OA rats were employed for the in vivo experiments, and RAW264.7 cells were employed for the in vitro experiments. Histopathological changes in synovium were examined using hematoxylin-eosin staining. Cell apoptosis in synovium was assessed by TUNEL staining. Macrophage polarization was determined by immunohistochemical analysis and flow cytometry. The mRNA expression and protein level of genes were detected by qRT-PCR and Western blot. The efferocytosis of macrophages was assessed by flow cytometry. IDN5706 reversed the increased CD86-positive cells (M1 macrophages) and decreased CD206-positive cells (M2 macrophages), both in synovium and synovial fluid of OA rats. The in vitro experiments further confirmed the promotion effect of IDN5706 on M2 macrophages, accompanied by the elevated Arg-1 and reduced iNOS. Also, the upregulated p-mTOR in synovium and synovial fluid of OA rats were reversed by IDN5706, and the decreased M1 macrophages and increased M2 macrophages induced by IDN5706 were reversed by the mTOR activator. IDN5706 enhanced the efferocytosis of IL-4-treated RAW264.7 cells, and the animal experiments further revealed the involvement of efferocytosis in the improvement of OA by IDN5706. IDN5706 enhanced the efferocytosis of synovial macrophages by inducing M2 polarization via inhibiting p-mTOR, thus suppressing synovial inflammation and OA development, providing a theoretical basis for IDN5706 as a clinical drug for inflammatory diseases.

Zhang J ，Cheng F ，Rong G ，Tang Z ，Gui B ... -  《-》

Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis.

Obesity has always been considered a significant risk factor in osteoarthritis (OA) progression, but the underlying mechanism of obesity-related inflammation in OA synovitis remains unclear. The present study found that synovial macrophages infiltrated and polarized in the obesity microenvironment and identified the essential role of M1 macrophages in impaired macrophage efferocytosis using pathology analysis of obesity-associated OA. The present study revealed that obese OA patients and Apoe-/- mice showed a more pronounced synovitis and enhanced macrophage infiltration in synovial tissue, accompanied by dominant M1 macrophage polarization. Obese OA mice had a more severe cartilage destruction and increased levels of synovial apoptotic cells (ACs) than OA mice in the control group. Enhanced M1-polarized macrophages in obese synovium decreased growth arrest-specific 6 (GAS6) secretion, resulting in impaired macrophage efferocytosis in synovial ACs. Intracellular contents released by accumulated ACs further triggered an immune response and lead to a release of inflammatory factors, such as TNF-α, IL-1β, and IL-6, which induce chondrocyte homeostasis dysfunction in obese OA patients. Intra-articular injection of GAS6 restored the phagocytic capacity of macrophages, reduced the accumulation of local ACs, and decreased the levels of TUNEL and Caspase-3 positive cells, preserving cartilage thickness and preventing the progression of obesity-associated OA. Therefore, targeting macrophage-associated efferocytosis or intra-articular injection of GAS6 is a potential therapeutic strategy for obesity-associated OA.

Yao Z ，Qi W ，Zhang H ，Zhang Z ，Liu L ，Shao Y ，Zeng H ，Yin J ，Pan H ，Guo X ，Liu A ，Cai D ，Bai X ，Zhang H ... -  《eLife》

Alpha defensin-1 attenuates surgically induced osteoarthritis in association with promoting M1 to M2 macrophage polarization.

Macrophages play an important part in the pathogenesis of osteoarthritis (OA). Our objective was to determine the effects of α-defensin-1 on macrophage polarization and consequently OA. OA synovial tissue and synovial fluid were assessed for the presence of M1 (CD68+CD16+CD206-) and M2 (CD68+CD206+CD16-) macrophages by flow cytometry. M0, M1, and M2 macrophages were co-cultured with OA chondrocytes to determine their influence on chondrogenic phenotype. Polarization of THP-1 activated monocytes from M1 to M2 in response to α-defensin-1 was evaluated by flow cytometry, RT-PCR and RNA sequencing. Effects of intra-articular α-defensin-1 in vivo were evaluated in a rat meniscal/ligamentous injury (MLI) model. The quantity of M1 exceeded M2 polarized macrophages in human OA synovial tissue (mean difference 26.1% [13.6-38.6%], P < 0.001) and fluid (mean difference 10.5% [5.0-16.1%], P = 0.003). M1 to M2 polarization in vitro was most effectively promoted with 10 ng/mL α-defensin-1. Compared with untreated macrophages, the α-defensin-1 polarized macrophages modified co-cultured OA chondrocytes from a pro-catabolic state to a pro-anabolic (regenerative-like) state based on expression of COL2A1, ACN, MMP3, MMP13 and ADAMTS5. Intra-articular α-defensin-1 decreased severity of cartilage damage and synovitis in the MLI rat model. RNAseq analyses suggested insulin and Toll-like receptor signaling pathways in the chondroprotective α-defensin-1 mechanism of action. α-defensin-1 promotes M1 to M2 macrophage polarization in vitro, has beneficial effects on chondrocytes indirectly via M2 macrophage polarization, and attenuates the severity of OA in vivo, suggesting it might be a candidate treatment for OA.

Xie JW ，Wang Y ，Xiao K ，Xu H ，Luo ZY ，Li L ，Pei FX ，Kraus VB ，Huang ZY ... -  《-》

Frugoside delays osteoarthritis progression via inhibiting miR-155-modulated synovial macrophage M1 polarization.

Direct inhibition of M1 polarization of synovial macrophages may be a useful therapeutic treatment for OA and OA-associated synovitis. Frugoside (FGS) is a cardiac glycoside compound isolated and extracted from Calotropis gigantea. Cardiac glycosides possess interesting anti-inflammatory potential. However, the corresponding activity of FGS has not been reported. Therefore, our aim was to find direct evidence of the effects of FGS on synovial macrophage M1 polarization and OA control. Collagenase was used to establish an experimental mouse OA model (CIOA) with considerable synovitis. Then, FGS was intra-articular administered. The mRNA and protein levels of iNOS were analysed by real-time PCR and Western blotting in vitro. Immunohistochemical and immunofluorescence staining were used to measure the expression of F4/80, iNOS, Col2α1 and MMP13 in vivo. The levels of pro-inflammatory cytokines in FGS-treated M1 macrophage culture supernatants were analysed by flow cytometry. FGS attenuates synovial inflammation and delays the development of OA in CIOA mice. Further results demonstrate that FGS inhibits macrophage M1 polarization in vitro and in vivo, which subsequently decreases the secretion of IL-6 and TNF-α, in turn delaying cartilage and extracellular matrix (ECM) degradation and chondrocyte hypertrophy. FGS inhibits macrophage M1 polarization by partially downregulating miR-155 levels. This study demonstrates that intra-articular injection of FGS is a potential strategy for OA prevention and treatment, even at an early stage of disease progression. This is a novel function of FGS and has promising future clinical applications.

Wang H ，Zhang H ，Fan K ，Zhang D ，Hu A ，Zeng X ，Liu YL ，Tan G ，Wang H ... -  《-》

Mechanical stress protects against chondrocyte pyroptosis through lipoxin A(4) via synovial macrophage M2 subtype polarization in an osteoarthritis model.

Our previous study found that lipoxin A4 (LXA4) exerts therapeutic effects on osteoarthritis (OA). In this study, we evaluated the effects of LXA4 via synovial macrophage M1/M2 subtype polarization on chondrocyte pyroptosis and corresponding mechanisms during mechanical stimulation. Synovial macrophages were subjected to various LXA4 concentrations and cyclic tensile strain (CTS) conditions to determine optimal co-culture conditions. The effects of LXA4 on chondrocyte pyroptosis, as represented by macrophage M1/M2 subtype polarization, were detected by western blot, immunofluorescence, and flow cytometry analyses. Forty male Sprague-Dawley rats were randomly divided into four groups (n = 10): control (CG), OA (OAG), OA with moderate-intensity treadmill exercise (OAE), and OAE + BOC-2 (an LXA4 antagonist). All rats were evaluated using histology, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and western blot analyses. We found that with increasing Kellgren-Lawrence grade, LXA4 expression was downregulated in articular fluid and that CD86 and Arg1 expression was upregulated in the synovium of patients. In vitro, CTS and LXA4 both promoted M2 subtype polarization of synovial macrophages, which inhibited the nuclear translocation of NF-κB p65 and formation of NLRP3 in chondrocytes. In vivo, the OAE treatment exerted protective effects on articular cartilage and facilitated M2 polarization of synovial macrophages. These effects were suppressed by BOC-2 treatment. We concluded that moderate CTS enhances therapeutic effects of LXA4 by inhibiting the nuclear translocation of NF-κB p65 and NLRP3. Furthermore, the therapeutic effects of LXA4 during treadmill exercise in monoiodoacetate-induced OA were driven by promotion of synovial macrophage M2 subtype polarization.

Shen P ，Jia S ，Wang Y ，Zhou X ，Zhang D ，Jin Z ，Wang Z ，Liu D ，Bai L ，Yang Y ... -  《-》