Pro-resolving lipid mediator ameliorates obesity induced osteoarthritis by regulating synovial macrophage polarisation

Abstract

Non-resolved persistent macrophage-mediated synovial inflammation is considered as one of the main drivers of both the establishment and progression of obesity-associated osteoarthritis (OA). Herein, we used clodronate-loaded liposomes (CL) to locally deplete macrophages in the synovial joints to examine the role of macrophages in the progression of obesity-induced OA. Furthermore, resolvin D1 (RvD1), a unique family of pro-resolving lipid mediator derived from the omega-3 polyunsaturated fatty acid, have shown marked potency in changing the pro-inflammatory behaviour of the macrophages. We sought to determine whether RvD1 administration ameliorates obesity-induced OA by resolving macrophage-mediated synovitis. Therapeutic properties of RvD1 and macrophage depletion (CL) were tested for its ability to slow post-traumatic OA (PTOA) in obese mice models. PTOA was induced in C57Bl/6 mice fed with high-fat diet (HFD) by surgically destabilising the meniscus. Firstly, CL treatment showed beneficial effects in reducing synovitis and cartilage destruction in obese mice with PTOA. In vitro treatment with RvD1 decreased the levels of pro-inflammatory markers in CD14+ human macrophages. Furthermore, intra-articular treatment with RvD1 diminishes the progression of OA in the knee joint from mice as follows: (a) decreases macrophages infiltration in synovium, (b) reduces the number of pro-inflammatory macrophages in synovium and (c) improves the severity of synovitis and cartilage degradation. Thus, our results provide new evidence for the potential targeting of macrophages in the treatment of obesity-induced OA.

Figure 1

High-fat diet promotes weight gain and altered metabolic parameters. (A) Body weight of CD or HFD mice were monitored over 16 weeks. (B) Ventral view of the mice showing the changes in the total abdominal length caused by the two diets after 16 weeks. (C–J) Effect of HFD on metabolic parameters. Measurement of serum resistin (C), leptin (D), insulin (E), adiponectin (F), total cholesterol (G), LDL (H), HDL (I) and triglyceride (J). (K) Immunostained section of the infrapatellar fat pad (IFP) of mice fed a control or HFD diet. Bar = 100 µm. Graphs represent mean ± SD (n = 7). *p < 0.05. CD, control diet-fed mice; HFD, high fat diet-fed mice.

Figure 2

High-fat diet induces OA-like cartilage changes and accelerates surgically-induced OA. (A) Top panel: Representative Safranin O and fast green stained sagittal sections of sham or experimental OA knee regions in mice fed a CD or HFD. Scale bars, 100 µm. The inset boxes in upper re shown at higher resolution in lower panels. Scale bars, 100 µm. Bottom panel: Similar sections were stained with COL10, DIPEN, and NITEGE to determine if HFD resulted in OA-like cartilage molecular changes. Scale bars, 100 µm. (B) Severity of articular cartilage degradation was graded using Mankin scoring system. Graphs represent mean ± SD (n = 7). *p < 0.05. (C-E)The percentage of COL10 (C), DIPEN (D), and NITEGE (E) - positive cells per knee section were counted. Graphs represent mean ± SD (n = 6). *p < 0.05. Saf-O: Safranin O and fast green staining; MT: medial tibia.

Figure 3

Inflamed synovium expresses a dominant M1 signature during the challenge of High-fat diet. (A)Top panel: Representative Safranin O and fast green stained sagittal sections of sham or experimental OA knee regions in mice fed a CD or HFD diet. Scale bars, 100 µm. Bottom panel: Similar sections were stained with F4/80, iNOS, and CD206 to determine the phenotype of synovial macrophage in activated synovium from CD- or HFD-fed mice. Scale bars, 100 µm. Insets are enlarged images of stained sections. (B) Synovial inflammation was assessed using synovitis scoring based on degree of cell thickness in the synovial lining layer and cell density of the synovial stroma. Graphs represent mean ± SD (n = 7). *p < 0.05. (C–E)The percentage of F4/80 (C), iNOS (D), and CD206 (E) - positive cells per knee section were counted. Graphs represent mean ± SD (n = 5). *p < 0.05. (F) Schematic diagram showing the experimental procedure, from the isolation of synoviocytes from animals to analyse the phenotype of synovial macrophage in the synovium from multiple biopsies of CD- and HFD-fed mice. qPCR analysis of pro-inflammatory M1-like (G) or anti-inflammatory M2-like (H) genes in MACS isolated F4/80+ synovial macrophage from CD- or HFD-fed mice. (I–J) FACS analysis of synovial macrophage from CD- or HFD-fed mice. (K)Same biopsies were further stained with MHC II to analyse the population of M1 activated macrophage. Saf-O: Safranin O and fast green staining; MT: medial tibia.

Figure 4

Clodronate liposome attenuates cartilage damage and synovial inflammation induced by High-fat diet in post-traumatic OA model. (A) Top panel: Knee sections were stained with F4/80 to determine the effect of Clodronate liposome on synovial macrophages. Scale bars, 100 µm. Bottom panel: Representative safranin-O image showed treatment effects from Clodronate liposome in the synovium of HFD-fed mice with surgically-OA. Scale bars, 100 µm. (B) The percentage of F4/80-positive cells per knee section were counted. Graphs represent mean ± SD (n = 5). *p < 0.05. (C) Synovial inflammation was assessed using synovitis scoring based on degree of cell thickness in the synovial lining layer and cell density of the synovial stroma. Graphs represent mean ± SD (n = 7). *p < 0.05. (D) Top panel: Representative Safranin O and fast green stained sagittal sections of experimental OA knee regions in mice fed HFD diet after Clodronate liposome treatment. Scale bars, 100 µm. The inset boxes in upper re shown at higher resolution in lower panels. Scale bars, 100 µm. Bottom panel: Similar sections were stained with COL10, DIPEN, and NITEGE to determine if Clodronate liposome attenuated cartilage damage. Scale bars, 100 µm. (E) Severity of articular cartilage degradation was graded using Mankin scoring system. Graphs represent mean ± SD (n = 8 ). *p < 0.05. (F-H) The percentage of COL10 (F), DIPEN (G), and NITEGE (H) - positive cells per knee section were counted. Graphs represent mean ± SD (n = 6). *p < 0.05. PBSL: PBS liposome; CL: Clodronate liposome. Saf-O: Safranin O and fast green staining; MT: medial tibia.

Figure 5

Resolvin D1 treatment improves the resolution of synovial inflammation. (A) MACS isolated human CD14+ synovial macrophage were stimulated to M1 phenotype and then treated with RvD1 for 24 h, qPCR analysis of pro-inflammatory M1-like genes were performed in vitro. Graphs represent mean ± SD (n = 4). *p < 0.05. (B,C) FACS analysis of synovial macrophage from CD- or HFD-fed mice with or without OA after RvD1 treatment. (D) Same biopsies were further stained with MHC II to analyse the population of M1 activated macrophage. (E) Knee sections were stained with F4/80, iNOS, and CD206 to determine the phenotype of synovial macrophage in activated synovium from HFD-fed mice with or without OA after RvD1 treatment. Scale bars, 100 µm. RvD1: resolvin D1; Veh: placebo (1% ethanol in saline).

Figure 6

Resolvin D1 treatment reduces the severity of HFD in surgically induced OA. (A) Top panel: Representative Safranin O and fast green stained sagittal sections of sham or experimental OA knee regions in mice fed a HFD diet with or without RvD1 treatment. Scale bars, 100 µm. The inset boxes in upper re shown at higher resolution in lower panels. Scale bars, 100 µm. Bottom panel: Similar sections were stained with COL10, DIPEN, and NITEGE to determine the effect of RvD1 on cartilage. Scale bars, 100 µm. Representative safranin-O image show treatment effects of RvD1 in the synovium of HFD-fed mice with surgically-OA. Scale bars, 10 µm. (B) Severity of articular cartilage degradation was graded using Mankin scoring system. Graphs represent mean ± SD (n = 8). *p < 0.05. (C) Synovial inflammation was assessed using synovitis scoring based on degree of cell thickness in the synovial lining layer and cell density of the synovial stroma. Graphs represent mean ± SD (n = 8). *p < 0.05. (D–F)The percentage of COL10 (D), DIPEN (E), and NITEGE (F) - positive cells per knee section were counted. Graphs represent mean ± SD (n = 6). *p < 0.05. RvD1: resolvin D1; Veh: placebo (1% ethanol in saline). Saf-O: Safranin O and fast green staining; MT: medial tibia.