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

Obesity has always been considered a significant risk factor in 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 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 apoptotic cells. Intracellular contents released by accumulated apoptotic cells 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 apoptotic cells, and decreased the levels of TUNEL- and caspase-3-positive cells, preserving cartilage thickness and preventing the progression of obesity-associated OA. Therefore, blocking M1 macrophage polarization or intra-articular injection of GAS6 is a potential therapeutic strategy for obesity-associated OA.


Introduction 45
Osteoarthritis (OA) is a common, chronic, degenerative joint disease and a 46 significant cause of joint pain and even disability. 1 Epidemiological investigations have 47 documented that obesity is one of the significant risk factors for OA. 2,3 A meta-analysis 48 of joint replacements in obese patients in 2010 has shown that the risk of knee OA was 49 five times higher in obese patients than in healthy individuals. 4 At the same time, being 50 "overweight" (i.e., obesity) doubled the proportion of joint replacement treatments 51 required later in life. 5 At present, the incidence of obesity continues to increase. 6 It is 52 estimated that by 2025, the global incidence of obesity will reach 18% in men and 21% 53 in women. 7 Thus, elucidating the mechanisms by which obesity promotes OA 54 development is essential for OA prevention and treatment. 55 It was initially believed that obesity affects OA by changing certain mechanical 56 factors. However, the progression of OA continues in non-weight-bearing areas, even 57 after the line of the force is corrected. 8,9 Moreover, these obesity-related mechanical 58 factors cannot be justified in the development of OA in non-weight-bearing joints such 59 as the hands. 10,11 Scientists have paid specific attention to the effects and roles of various 60 pro-inflammatory cytokines and adipokines in obesity. [12][13][14] Clinical data have 61 confirmed that obese OA patients are often associated with severe chronic synovitis, 62 which plays an essential role in the pathogenesis and progression of OA. 15,16 Our 63 previous findings have indicated that synovial macrophage polarization is significantly 64 correlated with synovitis in OA progression. [17][18][19] When synovitis occurs, macrophages Results 91 1. Synovial tissues are highly hyperplastic in obese OA patients and infiltrated 92 with more polarized M1 macrophages than non-obese OA patients. 93 To investigate the role of obesity in synovial tissue in OA patients, levels of total 94 cholesterol, triglycerides, and body mass index (BMI) were examined in different 95 patients. All subjects were divided into the following four groups based on the obtained 96 values for these three factors: normal healthy individuals, normal OA patients, obese 97 patients, and obese OA patients (Supplementary Table 1). Consistent with our previous 98 study, highly hyperplastic synovial tissues and abundant inflammatory cell infiltration 99 were observed in human OA synovial tissue, combined with a significantly higher 100 synovitis score than normal controls. Interestingly, the synovium tended to be

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The ApoE -/mouse model was established to further explore the role of obesity in 116 OA development, as it is considered an ideal model for investigating obesity. The body 117 weight and plasma lipid levels were markedly elevated in ApoE -/mice after 118 administering a high-fat and high-energy diet (Supplementary Tables 2 and Table 3). 119 There were no significant differences in knee OA OARSI scores between ApoE -/and 120 C57BL/6 mice four weeks post-surgery (Supplementary Fig. S1A and B). However, the 121 OARSI score was significantly elevated in ApoE -/mice eight weeks post-surgery (Fig. 122 2A and B), accompanied by a higher synovitis score and more infiltrated inflammatory 123 cells ( Fig. 2A and C), indicating that obesity may promote OA development in mice. In 124 addition, ApoE -/-OA mice expressed less aggrecan on cartilage and more MMP13 on 125 cartilage and synovium than C57BL/6 mice ( Fig. 3D and E). Notably, the percentage 126 of M1-like macrophages was increased with OA progression and reached a maximum

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Efferocytosis is an indispensable process through which dead and dying cells are 161 removed by phagocytic cells. Immunochemical TUNEL and caspase-3 staining in the 162 present study revealed that the number of apoptotic cells was increased in synovial 163 tissues with OA progression, which increased significantly in obese ApoE -/-OA and 164 obese OA patients . Previous studies have shown that efferocytosis of 165 apoptotic cells induced by macrophages is impaired in inflammatory diseases. Still, its 166 role in obesity-associated OA and understanding of its mechanism are lacking. In 167 addition, GAS6 has been described as a crucial bridging protein for macrophages to 168 recognize and engulf apoptotic cells. Therefore, it was hypothesized that the high 169 percentage of observed apoptotic cells might be due to ineffective macrophage   To further investigate the role of GAS6/Axl signaling in the development of OA in 188 vivo, an intra-articular injection intervention was performed in OA mice. As a result, 189 the degree of synovial inflammation and cartilage degeneration in C57BL/6 mice was far lower than that in ApoE -/mice, with a lower level of GAS6 expression in 191 macrophages ( Fig. 2A-C). Therefore, GAS6 recombinant factor was injected into 192 ApoE -/obese OA mice to protect against synovial hyperplasia and cartilage damage 193 induced by GAS6/Axl pathway suppression. On the other hand, the inhibitor R428 was 194 injected into the joint cavity of C57BL/6 OA mice to stimulate OA progression.    abnormalities in obesity-related knee OA affect the synovial membrane in the knee joint. 249 We found that M1 macrophage infiltration increased in the OA synovial tissue of obese 250 patients and ApoE -/mice compared to non-obese patients and C57BL/6 mice, 251 accompanied by increased secretion of TNF-α, IL-1, and IL-6. These results suggest 252 that obesity may be a crucial factor affecting the functional status of macrophages, and  However, the proportion of macrophages that engulfed apoptotic cells was increased 285 after incubation with recombinant GAS6 protein, while the phagocytic ability was 286 significantly down-regulated after blocking the Axl receptor by adding R428.

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Exogenous cultured rmGAS6 with macrophages after stimulation of LPS can also 288 decrease the levels of inflammatory cytokines, such as IL-1, IL-6, and TNF-ɑ. 289 Nevertheless, there was no significant difference in the expression of its specific 290 receptor Axl, which may be explained by the fact that GAS6 has three receptors (Axl, 291 Mer, and Tyro3) with different affinities. These findings suggest that GAS6 may relieve To conclude, the present study found that obese OA patients and ApoE -/obese 296 mice showed a more pronounced synovitis and enhanced macrophage infiltration in 297 synovial tissue, accompanied by dominant M1 macrophage polarization. Obese OA 298 mice had more severe cartilage destruction than OA mice in the control group.

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Enhanced M1-polarized macrophages in obese synovium decreased GAS6 secretion, 300 impairing efferocytosis for synovial apoptotic cells and causing synovial hyperplasia 301 and obesity-associated OA development. Therefore, these findings reveal that targeting 302 GAS6-mediated macrophage polarization and phagocytosis in obese patients with OA 303 may be a potential therapeutic strategy.  Ten-week-old male C57BL/6 mice (body weight 23 ± 2 g) and ApoE-deficient 317 (ApoE -/-) male mice (body weight 33 ± 2 g) were purchased from the Experimental 318 Animal Center of Guangdong Province, China. All animals were housed in cages 319 without pathogens at a temperature of 24 ± 5°C and with a relative humidity of 40%. 320 C57BL/6 mice were fed a standard diet, and ApoE -/mice were fed a high-fat diet. The 321 feed specifications are shown in Table 2. The protocol was approved by the Southern 322 Medical University Animal Care and Use Review Board.

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In the DMM-OA model, mice were anesthetized by intraperitoneal injection of 5% 324 chloral hydrate and the skin was cut along the medial collateral ligament. The joint 325 capsule was cut open and the femoral condyle was exposed. The connection between 326 the medial meniscus and the tibial plateau was cut to release the medial meniscus. The 327 joint capsule and skin were sutured after the operation.                     UDP-N-ace diaminopim activity///co activity///co activity///cy glutamic ac activity///pr elongating/ initiating///r activity///tu activity///tu protein liga activity///ub