1. Immunology and Inflammation

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

  1. Zihao Yao
  2. Weizhong Qi
  3. Hongbo Zhang
  4. Zhicheng Zhang
  5. Liangliang Liu
  6. Yan Shao
  7. Hua Zeng
  8. Jianbin Yin
  9. Haoyan Pan
  10. Xiongtian Guo
  11. Anling Liu
  12. Daozhang Cai  Is a corresponding author
  13. Xiaochun Bai  Is a corresponding author
  14. Haiyan Zhang  Is a corresponding author
  1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, China
  2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, China
  3. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southern Medical University, China
https://doi.org/10.7554/eLife.83069
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Cite this article
  1. Zihao Yao
  2. Weizhong Qi
  3. Hongbo Zhang
  4. Zhicheng Zhang
  5. Liangliang Liu
  6. Yan Shao
  7. Hua Zeng
  8. Jianbin Yin
  9. Haoyan Pan
  10. Xiongtian Guo
  11. Anling Liu
  12. Daozhang Cai
  13. Xiaochun Bai
  14. Haiyan Zhang
(2023)
Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis
eLife 12:e83069.
https://doi.org/10.7554/eLife.83069
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Abstract

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.

Editor's evaluation

In this study, the authors demonstrated that patients with obese-OA and mice with ApoE deficiency showed phenotypes of synovitis and enhanced macrophage infiltration in synovial tissues. GAS6 secretion is decreased during M1 macrophage polarization during obese-OA, leading to impaired macrophage efferocytosis in synovial apoptotic cells. Intra-articular injection of GAS6 restored the phagocytic capacity of macrophages, decreased synovial cell apoptosis, and prevented OA progression in obese-OA mice.

https://doi.org/10.7554/eLife.83069.sa0

Introduction

Osteoarthritis (OA) is a common, chronic, degenerative joint disease and a significant cause of joint pain and even disability (Hunter and Bierma-Zeinstra, 2019). Epidemiological investigations have documented that obesity is one of the significant risk factors for OA (Martel-Pelletier et al., 2016; Teichtahl et al., 2015). A meta-analysis of joint replacements in obese patients in 2010 has shown that the risk of knee OA was five times higher in obese patients than in healthy individuals (Clement and Deehan, 2020). At the same time, being ‘overweight’ (i.e., obesity) doubled the proportion of joint replacement treatments required later in life (Franklin et al., 2009). At present, the incidence of obesity continues to increase (Caballero, 2019). It is estimated that by 2025, the global incidence of obesity will reach 18% in men and 21% in women (NCD Risk Factor Collaboration, 2016). Thus, elucidating the mechanisms by which obesity promotes OA development is essential for OA prevention and treatment.

It was initially believed that obesity affects OA by changing certain mechanical factors. However, the progression of OA continues in non-weight-bearing areas, even after the line of the force is corrected (Yusuf et al., 2010). Moreover, these obesity-related mechanical factors cannot be justified in the development of OA in non-weight-bearing joints such as the hands (Pottie et al., 2006). Scientists have paid specific attention to the effects and roles of various pro-inflammatory cytokines and adipokines in obesity (Neumann et al., 2016). Clinical data have confirmed that obese OA patients are often associated with severe chronic synovitis, which plays an essential role in the pathogenesis and progression of OA (Sellam and Berenbaum, 2010; Mathiessen and Conaghan, 2017). Our previous findings have indicated that synovial macrophage polarization is significantly correlated with synovitis in OA progression (Koski et al., 2006; Daghestani et al., 2015; Zhang et al., 2018). When synovitis occurs, macrophages are stimulated by various cytokines and consequently release inflammatory mediators (Sun et al., 2020). At the same time, excess energy caused by obesity leads to changes in various cell functions, including angiogenesis and inflammatory cell infiltration (Collins et al., 2021). However, the effect of obesity on synovial hyperplasia and macrophage polarization in OA development has not been reported yet. We speculate that changes in tissues and organs caused by obesity may also affect synovitis, which in turn affects the process of OA.

GAS6 is a secreted glycoprotein widely expressed throughout the body. It is well known for its vital role in bridging phosphatidylserine on the surface of apoptotic cells (ACs) with its receptors Tyro3, Axl, and Mer, triggering the engulfment of ACs in an inflammatory environment (Bellan et al., 2019). This macrophage-related phagocytic process is also known as ‘efferocytosis’, which is beneficial for resolving inflammation (Doran et al., 2020). Prior studies have shown that impaired efferocytosis weakens the ability to clear ACs, inducing the release of inflammatory factors and ultimately causing synovitis (Nepal et al., 2019). Nevertheless, obesity-related macrophage polarization and the effect of obesity on efferocytosis remain unclear.

The present study found that obese OA patients and obese Apoe−/− mice are more prone to M1 macrophage infiltration in synovial tissue. Obese OA mice had more severe cartilage destruction and increased synovial ACs than OA mice in the control group. Down-regulation of GAS6 by M1 macrophages resulted in impaired efferocytosis for synovial ACs, causing synovial hyperplasia and obesity-associated OA development. These data suggested that targeting macrophage phagocytosis and polarization in obese patients with OA may be a potential therapeutic strategy.

Results

Synovial tissues are highly hyperplastic in obese OA patients and infiltrated with more polarized M1 macrophages than non-obese OA patients

To investigate the role of obesity in synovial tissue in OA patients, levels of total cholesterol, triglycerides, and body mass index were examined in different patients. All subjects were divided into the following four groups based on the obtained values for these three factors: (1) normal individuals, (2) OA patients without obesity, (3) obese individuals, and (4) OA patients with obesity (Table 1). Consistent with our previous study, highly hyperplastic synovial tissues and abundant inflammatory cell infiltration were observed in human OA synovial tissue, combined with a significantly higher synovitis score than normal controls. Interestingly, the synovium tended to be hyperplastic in obese patients and reached a maximum in obese OA patients among the four groups (Figure 1A, C).

Figure 1
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Synovial hyperplasia and macrophage polarization in obese OA patients.

(A) Safranin O and Fast Green staining (top) of human articular cartilage, hematoxylin and eosin (H&E) staining (lower) of synovial tissue from normal individuals, OA patients without obesity, obese individuals, and OA patients with obesity. Scale bar: 200 µm, 50 µm. (B) Immunofluorescence of F4/80, inducible nitric oxide synthase (iNOS), and CD206 in normal and OA synovial tissues from normal and obese patients. F4/80: green; iNOS: red; DNA: blue. Scale bar: 50 µm. (C) Quantification of synovitis score in normal individuals, OA patients without obesity, obese individuals, and OA patients with obesity (n = 6 per group). (D) Quantification of F4/80, iNOS, and CD206 positive macrophages as a proportion of total lining cell population in (B). *p < 0.05, **p < 0.01, ns = not significant. One-way analysis of variance (ANOVA) was performed. Data are shown as mean ± standard deviation (SD).

Table 1
Blood lipids in obesity-related patients and general patients.
Normal individualsOA patients without obesityObese individualsOA patients with obesity
Total cholesterol (TC) (3.0–6.0)4.81 ± 0.74.7 ± 0.546.34 ± 0.796.2 ± 0.86
Triglyceride (TG) (0.56–1.7)1.34 ± 0.321.45 ± 0.214.3 ± 0.253.19 ± 0.48
BMI index22.9 ± 0.3523.5 ± 0.2228.6 ± 0.1728.4 ± 0.21

We further investigated the polarization level of macrophages in synovial tissues by staining with F4/80 (macrophage markers), inducible nitric oxide synthase (iNOS; M1 macrophage marker), and CD206 (M2 macrophage marker). As a result, the number of M1 macrophages in the synovial tissue of the OA group increased significantly compared to the control group. Moreover, synovial tissue in obese OA patients was infiltrated with more M1 macrophages than that in non-obese OA patients (Figure 1B, D). These results indicate that synovial tissues were highly hyperplastic in obese OA patients and infiltrated with more polarized M1 macrophages than in non-obese OA patients.

Obesity promotes synovial M1 macrophage accumulation, synovitis, and OA development in mice

The Apoe−/− mouse model was established to further explore the role of obesity in OA development, as it is considered an ideal model for investigating obesity. Previous studies have shown that feeding high-fat diets to Apoe−/− mice for a short period accelerate the increase in LDL cholesterol levels and induce an inflammatory state (Tung et al., 2020; Cao et al., 2020). Apoe−/− mice may be clinically relevant to pathological progression in obese OA patients characterized by elevated plasma LDL cholesterol levels (Gierman et al., 2014; Gierman et al., 2012). The body weight and plasma lipid levels were markedly elevated in Apoe−/− mice after administering a high-fat and high-energy diet (Tables 2–4). There were no significant differences in knee OA OARSI scores between Apoe−/− and C57BL/6 mice 4 weeks post-surgery (Figure 2—figure supplement 1). However, the OARSI score was significantly elevated in Apoe−/− mice 8 weeks post-surgery (Figure 2A, B), accompanied by a higher synovitis score and more infiltrated inflammatory cells (Figure 2A, C), indicating that obesity may promote OA development in mice. In addition, Apoe−/− OA mice expressed less aggrecan on cartilage and more MMP13 on cartilage and synovium than C57BL/6 mice (Figure 2D, E). Notably, the percentage of M1-like macrophages was increased with OA progression and reached a maximum in obese Apoe−/− OA mice 8 weeks post-surgery. However, the proportion of positive cells for M2-like macrophages in the OA synovium showed no significant change at both 4 and 8 weeks post-surgery (Figure 2F, G, Figure 2—figure supplement 2). These findings suggest that obesity exacerbates synovitis and M1-polarized macrophage accumulation during OA progression in mice.

Table 2
Comparison of specifications and energy of ordinary feed and high-fat feed.
Ordinary feedHigh-fat feed
Compositiong (%)kcal (%)g (%)kcal (%)
Protein19.2202420
Carbohydrate67.3704135
Fat4.3102445
Total100100
kcal/g3.854.73
Table 3
Lipid status of APOE−/− obese mice and C57BL/6 mice.
Apoe−/− miceC57BL/6 mice
Total cholesterol (TC) (3.0–6.0)17.82 ± 3.32.77 ± 0.62
Triglyceride (TG)
(0.56–1.7)		2.56 ± 0.430.92 ± 0.31
Table 4
Weight gain after feeding for 8 weeks (g).
C57BL/6 miceApoe−/− mice
Standard diet7.35 ± 1.229.13 ± 0.78
High-fat diet16.89 ± 0.7519.81 ± 1.33
Figure 2 with 2 supplements see all
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Cartilage loss, synovial hyperplasia, and macrophage polarization in Apoe−/− OA.

(A) Safranin O and Fast Green (first line) and hematoxylin and eosin (H&E; second line) staining of controls and destabilization of medial meniscus (DMM) knee cartilage or synovial membrane from normal and Apoe−/− mice. Scale bar: 200 µm, 50 µm. (B) Quantitative analysis of Osteoarthritis Research Society International (OARSI) scale in A (second line), n = 6 per group. (C) Synovitis score for joints described in (A) (third line), n = 6 per group. (D) Immunohistochemical staining for aggrecan (first line) and MMP-13 (middle and bottom) in controls and DMM knee cartilage from normal and Apoe−/− mice. Scale bar: 50 µm. (E) Quantification of MMP13-positive cells from cartilage or synovium in (D), n = 6 per group. (F) Immunofluorescence staining for inducible nitric oxide synthase (iNOS; first line) and CD206 (second line) in controls and DMM synovial tissues from normal and Apoe−/− mice. Scale bar: 50 µm; (G) Quantification of iNOS- and CD206-positive cells as a proportion of lining cell population in (F), n = 6 per group. *p < 0.05, **p < 0.01, ns = not significant. One-way analysis of variance (ANOVA) was performed. Data are shown as mean ± standard deviation (SD).

GAS6 expression is inhibited in synovial macrophages during obesity-associated OA development

The link between M1 macrophages and synovial hyperplasia during obesity-associated OA progression was further explored. GAS6, a member of the vitamin K-dependent protein family, has been previously found to be down-regulated in Lipopolysaccharide (LPS)-induced bone marrow-derived macrophages (BMDMs) compared to the controls (GSE53986, Figure 3—figure supplement 1; Noubade et al., 2014). GAS6 was investigated as a critical factor regulating cell proliferation and apoptosis by binding to its receptor Axl. The GAS6/Axl effects on OA remain unclear. The present study explored the association between synovial macrophage polarization types and the GAS6/Axl pathway in obesity-associated OA. Immunofluorescence (IF) staining analysis indicated that macrophage release of GAS6 expressed in both human and mouse normal synovial tissues tended to be diminished, especially in obesity-associated OA (Figure 3A–D). Moreover, enzyme-linked immunosorbent assay (ELISA) revealed a significant decrease in GAS6 levels in the synovial fluid from obese OA patients than in non-obese individuals (Figure 3E). An in vitro study in polarized M1 macrophages enhanced by LPS confirmed the M1 macrophage-associated reduction of GAS6 and increases in IL-1, IL-6, and TNF-α (Figure 3F, G, Figure 3—figure supplement 2, Figure 3—figure supplement 2—source data 1). Interestingly, F4/80-Axl double staining revealed no significant difference in the number of Axl-positive cells in the synovium of obese OA patients, obese Apoe−/− OA mice, and control subjects (Figure 3—figure supplement 3). These results indicate a potential role of M1 macrophage-mediated GAS6 in obesity-associated OA development.

Figure 3 with 3 supplements see all
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Loss of GAS6 expression in synovium of obese OA patients and Apoe−/− OA mice.

(A) Immunofluorescence staining for F4/80 (red) and GAS6 (green) in synovial tissue from normal individuals, OA patients without obesity, obese individuals, and OA patients with obesity. Scale bar: 50 µm. (B) Quantification of F4/80-GAS6-positive macrophages as a proportion of total lining cell population in (A), n = 6 per group. (C) Immunofluorescence staining (first line) for F4/80 (red) and GAS6 (green) in synovial tissue of controls and destabilization of medial meniscus (DMM) from C57BL/6 and Apoe−/− mice. Scale bar: 50 µm. (D) Quantification of F4/80-GAS6-positive macrophages (yellow) as a proportion of total F4/80-positive cells in (C) (first line). Quantification of GAS6-positive cells in (C) (second line), n = 6 per group. (E) Enzyme-linked immunosorbent assay (ELISA) for GAS6 in synovial fluid of non-obese and obese OA patients, n = 13 per group. (F) Immunofluorescence staining for F4/80(red) and GAS6 (green) in RAW264.7 cells treated with LPS for 24 and 48 hr. Scale bar: 50 µm. (G) Quantification of F4/80-GAS6-positive macrophages (yellow) as a proportion of total F4/80-positive cells (red), n = 6 per group. *p < 0.05, **p < 0.01, NS = not significant. One-way analysis of variance (ANOVA) was performed. Data are shown as mean ± standard deviation (SD).

GAS6 is involved in obesity-mediated inhibition of macrophage efferocytosis

Efferocytosis is an indispensable process through which dead and dying cells are removed by phagocytic cells. MER were widely used to label macrophages undergoing efferocytosis (Dransfield et al., 2015; Felton et al., 2018; Cai et al., 2017). IF staining analysis indicated the expression of MER in OA synovial macrophages decreased significantly, especially in obese Apoe−/− OA and obese OA patients (Figure 4—figure supplement 1). Immunochemical TUNEL and caspase-3 staining in the present study revealed that the number of ACs was increased in synovial tissues with OA progression, which increased significantly in obese Apoe−/− OA and obese OA patients (Figure 4A–D). Previous studies have shown that efferocytosis of ACs induced by macrophages is impaired in inflammatory diseases. Still, its role in obesity-associated OA and understanding of its mechanism are lacking. In addition, GAS6 has been described as a crucial bridging protein for macrophages to recognize and engulf ACs. Therefore, it was hypothesized that the high percentage of observed ACs might be due to ineffective macrophage efferocytosis caused by GAS6 suppression in OA. Primary BMDMs from Apoe−/− mice and controls were fed carboxyfluorescein succinimidyl ester (CFSE)-labeled ACs to determine the potency of efferocytosis induced by macrophages. The clearance of these fluorescent cells was quantified using flow cytometry. As expected, macrophage efferocytosis was impaired in the obesity microenvironment with reduced capacity for clearing fluorescent ACs in BMDMs from Apoe−/− mice compared to the controls (Figure 4E, F). Moreover, stimulation with GAS6 enhanced the phagocytotic activity of RAW264.7 cells, while inhibition of the Axl receptor by adding R428 diminished this effect (Figure 4H–K), which were further verified in BMDM primary cells (Figure 4—figure supplement 2). Adding recombinant mouse GAS6 (rmGAS6) significantly restored the up-regulation of inflammatory factors IL-1β, IL-6, and TNF-α induced by LPS in RAW264.7 cells (Figure 3—figure supplement 2). Nevertheless, rmGAS6 had no obvious effect on the polarization of macrophages, while R428 up-regulated CD86 and iNOS in BMDMs (Figure 4G, Figure 4—figure supplement 3). These data indicate that M1 macrophage-associated reduction of GAS6 in obesity-associated OA mice promotes the accumulation of ACs by decreasing macrophage efferocytosis.

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Accumulation of apoptotic cells in OA and impaired phagocytic ability of M1-polarized macrophages.

(A) Immunofluorescence staining for caspase-3 (top) and TUNEL (lower) in normal and OA synovial tissue from non-obese and obese patients. Scale bar: 50 µm. (B) Quantification of caspase-3- or TUNEL-positive cells as a proportion of total lining cell population in (A), n = 6 per group. (C) Immunofluorescence staining for caspase-3 (top) and TUNEL (lower) in controls and destabilization of medial meniscus (DMM) synovial tissue from C57BL/6 and Apoe−/− mice. Scale bar: 50 µm. (D) Quantification of caspase-3- or TUNEL-positive cells as a proportion of lining cell population in (C), n = 6 per group. (E) Immunofluorescence staining for F4/80 (red) in bone marrow-derived macrophages (BMDMs) extracted from Apoe−/− and C57BL/6 mice. Carboxyfluorescein succinimidyl ester (CFSE; green) in apoptotic thymocytes of C57BL/6 mice after 2 hr phagocytosis. (F) Quantification of positive BMDMs engulfing apoptotic thymocytes as a proportion of total F4/80-positive cells, n = 5 per group. (G) mRNA expression levels of inducible nitric oxide synthase (iNOS) or Arg1 after LPS, rmGAS6, or IL-4 stimulation for 24 hr. (H) Immunofluorescence staining for F4/80 (red) in RAW264.7 cells and CFSE (green) in apoptotic thymocytes after phagocytosis for 2 hr. Scale bar: 50 µm. (I) Flow cytometry analysis of CFSE-positive cells in total macrophages is shown as fluorescence‐intensity distribution plots. (J) Quantification of positive RAW264.7 cells engulfing apoptotic thymocytes as a proportion of total F4/80-positive cells, n = 6 per group. (K) Efferocytotic index was calculated as percentage of CFSE‐positive cells divided by percentage of total cells, n = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001, NS = not significant. One-way analysis of variance (ANOVA) was performed. Data are shown as mean ± standard deviation (SD).

Suppression of GAS6/Axl axis promotes synovial hyperplasia, synovitis, and obesity-associated OA development

To further investigate the role of GAS6/Axl signaling in the development of OA in vivo, an intra-articular injection intervention was performed in OA mice. As a result, 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 macrophages (Figure 2A–C and 3C, D). Therefore, rmGAS6 was injected into Apoe−/− obese OA mice to protect against synovial hyperplasia and cartilage damage induced by GAS6/Axl pathway suppression. On the other hand, the inhibitor R428 was injected into the joint cavity of C57BL/6 OA mice to stimulate OA progression. Interestingly, intra-articular injection of GAS6 significantly delayed synovial inflammation and cartilage destruction compared to vehicle-treated obese OA mice, manifested as lower synovitis and OARSI scores. In contrast, inhibition of Axl by R428 in C57BL/6 OA mice promoted synovial hyperplasia and cartilage destruction and enhanced synovitis and OARSI scores (Figure 5A, B). Moreover, intra-articular injection of GAS6 recombinant factor decreased the number of ACs stimulated by synovitis in synovial tissues (Figure 5C, D). To further explore the effect of inflammatory factors released by the stimulation of ACs on chondrocyte homeostasis dysfunction, ACs were co-cultured with BMDMs. The culture supernatant was collected after stimulation for 24 hr. The expression of MMP13 was increased after adding co-culture supernatant stimulation, together with senescence hallmarks such as p16 and p21, while COL2 were down-regulated after stimulation. However, the effect of supernatant promoting chondrocyte homeostasis dysfunction was partially alleviated when adding ACs with rmGAS6 to BMDMs, which were diminished by adding R428 (Figure 5—figure supplement 1, Figure 5—figure supplement 1—source data 1). Nevertheless, no apparent proteoglycan loss or increase was found in recombinant human GAS6 (rhGAS6)-treated cartilage explants (Figure 5—figure supplement 2). Moreover, western blotting showed no significant differences in the expression of COL2, MMP13, p16, and p21 in primary chondrocytes after stimulation with rhGAS6 (Figure 5—figure supplement 3, Figure 5—figure supplement 3—source data 1). Therefore, these data indicated that the GAS6/Axl axis might alleviate synovial hyperplasia and protect against obesity-associated OA development.

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GAS6 restored osteoarthritis (OA) cartilage loss and decreased apoptotic cell accumulation.

(A) Safranin O and Fast Green staining (top and middle) of knee cartilage, hematoxylin and eosin (H&E) staining of synovial tissues from destabilization of medial meniscus (DMM) mice and DMM mice treated with R428, and Apoe−/− mice treated with recombinant mouse (rmGAS6) 8 weeks after surgery. Scale bar: 200 µm, 50 µm. (B) Quantitative analysis of Osteoarthritis Research Society International (OARSI) scale and synovitis score in (A), n = 6 per group. (C) Immunofluorescence staining of caspase-3 or TUNEL in synovial tissue from DMM mice, DMM mice treated with R428, and Apoe−/− mice treated with recombinant mouse (rmGAS6) 8 weeks after surgery. Scale bar: 50 µm. (D) Quantification of caspase-3- or TUNEL-positive cells as a proportion of lining cell population in (C), n = 6 per group. *p < 0.05, **p < 0.01. One-way analysis of variance (ANOVA) was performed. Data are shown as mean ± standard deviation (SD).

Model for obesity-associated synovitis and OA development

Obesity stimulates synovial macrophage infiltration and M1 polarization, which suppress the secretion of GAS6. GAS6 binds to the Axl receptor on macrophages, while GAS6/Axl inhibition promotes the accumulation of ACs by decreasing macrophage efferocytosis to induce chondrocyte degradation and aggravate OA development (Figure 6).

Figure 6
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Model of GAS6 secreted by macrophages in modulating clearance of apoptotic cells and macrophage polarization during osteoarthritis (OA).

Macrophage polarization induced by obesity decreased the secretion of GAS6 and impaired the phagocytosis of apoptotic cells. The accumulation of apoptotic cell debris leads to the persistence of local inflammation and synovial hyperplasia, which aggravates the pathological process of OA.

Discussion

The present study revealed for the first time that M1-polarized macrophage infiltration in OA synovial tissue of obese patients is increased, accompanied by markedly down-regulated secretion of GAS6 and impaired macrophage-dependent efferocytosis for cleaning ACs. The intracellular contents released by accumulated ACs further trigger an immune response and lead to a release of inflammatory factors, such as TNF-α, IL-1β, and IL-6, which induce the dysfunction of chondrocyte homeostasis in obesity-associated OA. Therefore, targeting macrophage-associated efferocytosis or intra-articular injection of GAS6 is a potential therapeutic strategy for obesity-associated OA.

Obesity has always been considered a significant risk factor in the progression of OA (Kulkarni et al., 2016), leading to more severe OA manifestations, including cartilage loss, subchondral bone sclerosis, and synovial inflammation (Wang and He, 2018). Researchers have accepted the role of synovial inflammation in the pathological progression of OA (Mathiessen and Conaghan, 2017; Felson et al., 2016). However, the underlying mechanism of obesity-related inflammation in the development of synovitis in OA remains unclear. Apoe plays an important role in maintaining the normal levels of cholesterol and triglycerides in serum by transporting lipids in the blood (Hatters et al., 2006). Mice lacking Apoe function develop hypercholesterolemia, increased very low-density lipoprotein and decreased high-density lipoprotein, leading chronic inflammation in vascular disease and nonalcoholic steatohepatitis (Liu et al., 2023). Apoe−/− mice may partially reflect clinical characteristics of obese OA patients with elevated plasma LDL cholesterol levels. The present study revealed that obese patients and Apoe−/− mice showed a more severe cartilage destruction with enhanced OARSI scores (Glasson et al., 2010). Furthermore, the lining layer of synovial tissue was more prone to hyperplasia. The number of macrophages increased significantly during the pathological development of OA in obese patients, which manifested as enhanced synovitis scores (Krenn et al., 2006) and increased numbers of F4/80-positive cells. These results suggest that obesity plays an essential mediating role in OA development.

Recent studies have reported that the imbalance in M1/M2 macrophage polarization plays a vital role in developing OA inflammation (Zhang et al., 2018). Classically, macrophages are divided into inflammatory M1 and anti-inflammatory M2 macrophages, though they can be interchanged or transformed into each other during various inflammatory reactions and thus function differently (Funes et al., 2018). Lumeng et al., 2007 have found that adipose tissue macrophages (ATMs) prefer to express TNF-α, iNOS, and other M1 macrophage markers, while ATMs in non-obese individuals highly express M2 macrophage markers. However, the effect of obesity on inducing macrophage polarization during OA development remains unclear. Many bursas and adipose tissue around the knee joint are typically characterized as the infrapatellar fat pad. Some researchers believe that the transformation of macrophages is triggered by lipids released by fat cells at the onset of obesity (Chatterjee et al., 2013). Thus, we investigated whether macrophage abnormalities in obesity-related knee OA affect the synovial membrane in the knee joint. We found that M1 macrophage infiltration increased in the OA synovial tissue of obese patients and Apoe−/− mice compared to non-obese patients and C57BL/6 mice, accompanied by increased secretion of TNF-α, IL-1, and IL-6. These results suggest that obesity may be a crucial factor affecting the functional status of macrophages, and targeting the polarization of macrophages in obese patients may alleviate the synovitis in OA.

The balance of progression and regression that controls the state of local inflammation has recently been in the spotlight (Oishi and Manabe, 2018; Feehan and Gilroy, 2019). The maintenance of efferocytosis dampens pro-inflammatory cytokine production and initiates inflammation resolution (Boada-Romero et al., 2020). Emerging studies have mentioned the effect of macrophage polarization on phagocytic ability. Yurdagul et al. have shown that M2 macrophages retain efferocytosis properties, which are crucial for resolving inflammation (Yurdagul et al., 2020). Another study demonstrated that DEL-1 contributes to the resolution of inflammation by promoting apoptotic neutrophil efferocytosis through macrophages and the emergence of an M2 macrophage phenotype (Kourtzelis et al., 2019). Although efferocytosis has been widely studied in multiple disease models, its role in synovial inflammation and OA development has not been reported. We found that the expression of MER decreased significantly in OA synovial macrophages, especially in obesity-associated OA. Our in vitro experiments demonstrated that macrophages extracted from the bone marrow of obese mice had decreased phagocytic capacity, and the phagocytic ability of macrophages for apoptotic thymocytes was reduced after stimulation with LPS (500 ng/ml). These results showed that the proportion of ACs was significantly increased in the synovium of Apoe−/− mice. This is partly due to the decreased phagocytic ability caused by the enhanced M1 macrophage polarization. We have further revealed that macrophages stimulated by ACs leading chondrocytes homeostasis dysfunction, yet administration of rmGAS6 partially alleviated the up-regulation of p16, p21, and MMP13. Intra-articular injection of GAS6 restored the phagocytic capacity of macrophages. It 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. These findings suggest that targeting the efferocytosis of macrophages in local inflammation of obesity-associated synovitis may maintain the homeostasis of the cartilage cavity and alleviate the obesity-related OA.

GAS6 and its receptor Axl are known to regulate apoptotic-related inflammation and may be implicated in lupus pathogenesis (Zhen et al., 2018). The remaining ACs are a source of autoantigens and can drive autoimmunity development (Szondy et al., 2014). The expression of GAS6 in the hyperplasia synovial tissues from obesity-associated OA in the present study was down-regulated and accompanied by an increase in M1 macrophage polarization. Moreover, the in vitro experiments demonstrated a decreased secretion of GAS6 protein and impaired phagocytic ability in macrophages after LPS stimulation. However, the proportion of macrophages that engulfed ACs was increased after incubation with recombinant GAS6 protein, while the phagocytic ability was significantly down-regulated after blocking the Axl receptor by adding R428. Exogenous cultured rmGAS6 with macrophages after stimulation of LPS can also decrease the levels of inflammatory cytokines such as IL-1, IL-6, and TNF-α. Nevertheless, there was no significant difference in the expression of its specific receptor Axl, which may be explained by the fact that GAS6 has three receptors (Axl, Mer, and Tyro3) with different affinities. These findings suggest that GAS6 may relieve local synovial inflammation by restoring the phagocytic ability of macrophages for ACs and decrease the induction of inflammatory chemokines, which alleviate the pathological progression of OA.

To conclude, the present study found that obese OA patients and Apoe−/− obese mice showed a more pronounced synovitis and enhanced macrophage infiltration in synovial tissue, accompanied by dominant M1 macrophage polarization. Obese OA mice had more severe cartilage destruction than OA mice in the control group. Enhanced M1-polarized macrophages in obese synovium decreased GAS6 secretion, impairing efferocytosis for synovial ACs and causing synovial hyperplasia and obesity-associated OA development. Therefore, these findings reveal that targeting GAS6-mediated macrophage polarization and phagocytosis in obese patients with OA may be a potential therapeutic strategy.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Mus musculus)RAW264.7PricellaHC2022083021Cell line has been authenticated by STR profiling and it did not be contaminated by mycoplasma
Antibodyanti-F4/80 (Mouse monoclonal)Santa Cruz BiotechnologyCat #: sc377009IF (1:100)
Antibodyanti-iNOS (Mouse monoclonal)Santa Cruz BiotechnologyCat #: sc-7271IF (1:100)
Antibodyanti-Aggrecan (Rabbit polyclonal)ProteintechCat #: 13880-1-APIF (1:200)
Antibodyanti-MMP13 (Rabbit polyclonal)ProteintechCat #: 18165-1-APIF (1:400)
WB (1:1000)
Antibodyanti-CD206 (Rabbit polyclonal)ProteintechCat #: 18704-1-APIF (1:100)
Antibodyanti-AXL (Rabbit polyclonal)AbcloneCat #: A20548IF (1:100)
Antibodyanti-CASPASE-3 (Rabbit polyclonal)ProteintechCat #: 19677-1-APIF (1:200)
Antibodyanti-GAS6 (Rabbit polyclonal)AbcloneCat #: A8545IF (1:100)
AntibodyPeroxidase AffiniPure Goat Anti-Rabb (Goat polyclonal)Jackson Immuno Research LaboratoriesCat #: 11-035-003IHC (1:200)
WB (1:3000)
AntibodyGoat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluo 488 (Goat polyclonal)InvitrogenCat #: A-11008IF (1:400)
AntibodyGoat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Goat polyclonal)InvitrogenCat #: A-11005IF (1:400)
AntibodyAnti-Collagen II antibody (Rabbit polyclonal)AbcamCat #: ab188570WB (1:1000)
Sequence-based reagentGas6_FThis paperPCR primersCCGCGCCTACCAAGTCTTC
Sequence-based reagentGas6_RThis paperPCR primersCGGGGTCGTTCTCGAACAC
Sequence-based reagentGapdh _FThis paperPCR primersAAATGGTGAAGGTCGGTGTGAAC
Sequence-based reagentGapdh _RThis paperPCR primersCAACAATCTCCACTTTGCCACTG
Sequence-based reagentIl-1β_FThis paperPCR primersGCAACTGTTCCTGAACTCAACT
Sequence-based reagentIl-1β_RThis paperPCR primersATCTTTTGGGGTCCGTCAACT
Sequence-based reagentIl-6_FThis paperPCR primersACAACCACGGCCTTCCCTACTT
Sequence-based reagentIl-6_RThis paperPCR primersCAGGATTTCCCAGCGAACATGTG
Sequence-based reagentTnf-α_FThis paperPCR primersCCTCCCTCTCATCAGTTCTA
Sequence-based reagentTnf-α_RThis paperPCR primersACTTGGTTTGCTACGAC
Commercial assay or kitTUNEL Apoptosis Detection Kit (Alexa Fluor 488)YeasenCat #: 40307ES20-
Commercial assay or kitHuman Gas6 DuoSet ELISAR&DCat #: DY885B-
Commercial assay or kitRNAiso Plus (Trizol)Takara Bio IncCat #: T9108-
Commercial assay or kit5× HiScript II qRT SuperMix IIVazyme BiotechCat #: R223-01-
Commercial assay or kit2× ChamQ SYBR qPCR Master MixVazyme BiotechCat #: Q311-02-
Chemical compound, drugDAPISigma-AldrichCat #: F6057-20ML-
Chemical compound, drugCarboxyfluorescein succinimidyl ester (CFSE)TopscienceCat #: T6802-
Peptide, recombinant proteinR428TopscienceCat #: 1037624-75-1-
Peptide, recombinant proteinLipopolysaccharideMed Chem ExpressCat #: HY-D1056-
Peptide, recombinant proteinIL-4 Protein, Mouse (CHO)Med Chem ExpressCat #: HY-D1056-
Peptide, recombinant proteinRecombinant GAS6 Protein (Mouse)Sino BiologicalCat #: 58026-M08H-
Peptide, recombinant proteinRecombinant GAS6 Protein (Human)NovoproteinCat #: C01W-
Software, algorithmSPSSSPSSSPSS 25.0-

Human synovial and cartilage tissue

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Synovial tissue and synovial fluid samples of normal individuals (n = 6, age 34 ± 8.15 years, three males, three females) or obese individuals (n = 6, age 35 ± 7.36 years, four males, two females) were obtained from patients who received arthroscopic treatment for acute anterior cruciate ligament rupture or meniscus injury Other joint diseases were excluded from the study. OA synovial tissues, and synovial fluid samples were obtained from obese patients (n = 6, age 64 ± 5.75 years, two males, four females) or patients without obesity (n = 6, age 65 ± 4.26 years, three males, three females) who underwent total knee arthroplasty. OA cartilage tissues were obtained from patients who underwent total knee arthroplasty, the tibial plateau cartilage was carefully separated and cut into 1 mm3, cultured in vitro. Informed consent was obtained from all recruited patients and was identified by the ethics committee of the Third Affiliated Hospital of Southern Medical University.

Destabilization of medial meniscus animal model

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Ten-week-old male C57BL/6 mice and Apoe-deficient (Apoe−/−) male mice were purchased from the Experimental Animal Center of Guangdong Province, China. All animals were housed in cages without pathogens at a temperature of 24 ± 5°C and with a relative humidity of 40%. C57BL/6 mice were fed a standard diet, and Apoe−/− mice were fed a high-fat diet. The feed specifications are shown in Table 2. This study was performed in strict accordance with the recommendations in Chinese Laboratory animal-Guideline for ethical review of animal welfare (GB/T 35892-2018). The protocol was approved by the Southern Medical University Animal Care and Use Review Board (Permit Number: 2021-Ethical review-053). All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.

In the destabilization of medial meniscus-OA model (Kamekura et al., 2005), mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital and the skin was cut along the medial collateral ligament. The joint capsule was cut open and the femoral condyle was exposed. The connection between the medial meniscus and the tibial plateau was cut to release the medial meniscus. The joint capsule and skin were sutured after the operation.

Animal treatment and specimen preparation

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After the surgery, 50 ng/g (about 5 µl as total volume) of recombinant mouse GAS6 (rmGAS6, Sino Biological, China, #58026-M08H) was administered into the articular once per week. The right legs were harvested 4–8 weeks post-surgery (n = 6 in each group). Knee joints from mice in different experimental groups were fixed in 4% paraformaldehyde for 48 hr and decalcified for 21 days. The specimens were embedded in paraffin, and 4 μm serial sections were cut from the sagittal portion through the inner side of the knee. The Southern Medical University Animal Care and Use Committee approved all procedures involving mice.

Histology and immunohistochemical/IF staining

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Histology sections were stained with Safranin O-fast green/hematoxylin and eosin for morphological analysis. Immunohistochemical (IHC) and IF staining was performed on the 4-μm-thick tissue sections. Slides were deparaffinized, rehydrated, and washed in phosphate-buffered saline (PBS) three times for 5 min each time. Antigen retrieval was performed by soaking slides in citric acid overnight in a 60°C water bath. After washing three times in PBS, slides were quenched in 3% hydrogen peroxide for 10 min at room temperature and washed with PBS three more times. Then, slides were blocked with 10% normal bovine serum for 1 hr at room temperature (IHC staining). Slides were then incubated with primary antibodies at 4°C overnight. A secondary antibody for IHC or fluorescent secondary antibody for IF was applied for 1 hr at room temperature. Then, IHC slides were stained with diaminobenzidine and hematoxylin, dehydrated, and mounted. IF slides were processed with 4,6-diamidino-2-phenylindole (DAPI) staining solution and mounted with cover glass. Antibodies used for IHC/IF staining were as follows: rabbit anti-Caspase-3, rabbit anti-GAS6, rabbit anti-Axl, rabbit anti-MMP13, rabbit anti-Aggrecan, mouse anti-F4/80, mouse anti-iNOS, rabbit anti-CD206, species-matched horseradish peroxidase-conjugated secondary antibodies, and species-matched Alexa-488- or 594-labeled secondary antibody.

Cartilage and synovium structure grading

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Histology sections of the knee joints were graded based on the Osteoarthritis Research Society International (OARSI) scoring system developed by Glasson et al., 2010 by two observers blinded to the experimental conditions. Generally, sections were assigned a grade of 0–6: 0, normal cartilage; 0.5, slight loss of Safranin O staining without structural changes; 1, small fibrillations without loss of cartilage; 2, vertical clefts down to the layer below the superficial layer; 3–6, vertical clefts or erosion to the calcified cartilage affecting <25% (grade 3), 25–50% (grade 4), 50–75% (grade 5), and >75% (grade 6) of the articular surface. Synovitis severity was estimated based on the synovial lining cell layer enlargement, resident cell density, and inflammatory infiltration. A nine-point scale was used, where low scores indicated moderate synovitis and high scores represented severe synovitis (Krenn et al., 2006).

TUNEL

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The TUNEL assay was performed according to the manufacturer’s instructions (TUNEL Apoptosis Detection Kit [Alexa Fluor 488], Yeasen, #40307ES20) to detect cell death in the synovial membrane. The assay used the green channel at 488 nm. DAPI was applied as a nuclear counterstain in the blue channel at 461 nm. Images were taken with an Olympus BX43 fluorescent microscope and Olympus DP73 digital camera at ×400 magnification with cellSens software (Olympus). Exposure settings were adjusted to minimize oversaturation.

Apoptosis induction and thymocyte IF staining

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Murine thymocytes were isolated from C57BL/6 mice and then stimulated with 25 μmol/l dexamethasones for 3 hr to induce apoptosis, followed by washing twice and resuspension with phagocyte culture medium to a concentration of 1 × 107 cells/ml. Then, thymocytes were labeled with CFSE dye following the manufacturer’s instructions.

Efferocytosis assay

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An efferocytosis assay was performed as previously described (Heo et al., 2014). Briefly, RAW264.7 or BMDM cells were plated in a 6-well plate (1 × 106 cells/well) with Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and cultured overnight. RAW264.7 cell line was purchased from Pricell Life Science & Technology. The cells were authenticated by STR profiling and were not contaminated by mycoplasma. The cells were then treated with LPS, rmGAS6, or R428 for 24 or 48 hr. CFSE‐labeled apoptotic thymocytes were then added at a ratio of 10:1 and incubated for an additional 120 min. The cells were extensively washed three times with PBS to remove unengulfed thymocytes. The ability of macrophages to engulf apoptotic thymocytes was quantified by flow cytometry or visualized using IF microscopy. The efferocytotic index was calculated using the following formula: (number of macrophages containing apoptotic bodies)/(total macrophages) × 100% and then normalized using the control group as 100%.

Real‐time polymerase chain reaction

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Total RNA was isolated from RAW264.7 cells and ground cartilage from human tibial plateaus using TRIzol reagent. For mRNA quantification, 1 mg of total RNA was purified with gDNA remover and reverse transcribed using 5× HiScript II qRT SuperMix II. Each PCR reaction consisted of 10 µl of 2× ChamQ SYBR qPCR Master Mix, 10 µM of forward and reverse primers, and 500 ng of cDNA. For miRNA quantification, 1 mg of total RNA was purified with gDNA wiper mix and then reverse transcribed using Hiscript II Enzyme Mix, 10× RT Mix, and specific stem-loop primers. Template DNA was mixed with 2× miRNA Universal SYBR qPCR Master Mix, specific primers, and mQ primer R. All reactions were run in triplicate. Mouse primer sequences are listed in the Key Resources Table.

Enzyme-linked immunosorbent assay

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Human synovial fluid samples were collected as described above. All samples were spun down at 4500 × g for 15 min. Human GAS6 Quantikine Kit (R&D Systems) was used to measure the concentration of GAS6 in the synovial fluid.

Statistical analyses

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Data were represented as the mean ± standard deviation. An unpaired Student’s t-test was performed for experiments comparing two groups of data. A one-way analysis of variance was performed for data involving multiple groups, followed by Tukey’s post hoc test. p values of <0.05 were considered statistically significant.

Data availability

Sequencing data have been deposited in GEO under accession code GSE53986. Source Data has been uploaded in Dryad, which was named after 'Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis' (https://doi.org/10.5061/dryad.d2547d86d).

The following data sets were generated
    1. Zihao Y
    (2023) Dryad Digital Repository
    Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis.
    https://doi.org/10.5061/dryad.d2547d86d
The following previously published data sets were used
    1. Noubade R
    (2014) NCBI Gene Expression Omnibus
    ID GSE53986. NRROS negatively regulates ROS in phagocytes during host defense and autoimmunity.
    https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE53986

References

Decision letter

  1. Di Chen
    Reviewing Editor; Chinese Academy of Sciences, China
  2. Mone Zaidi
    Senior Editor; Icahn School of Medicine at Mount Sinai, United States
  3. Chao Xie
    Reviewer; University of Rochester Medical Center, United States

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Mone Zaidi as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Chao Xie (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

In this study, the authors demonstrated that patients with obesity-associated osteoarthritis and mice with the ApoE gene deficiency showed phenotypes of synovitis and enhanced macrophage infiltration in synovial tissues.

GAS6 is a glycoprotein and its secretion is decreased during M1 macrophage polarization during obesity-associated osteoarthritis, leading to impaired macrophage efferocytosis in synovial apoptotic cells.

Intra-articular injection of GAS6 restored the phagocytic capacity of macrophages, decreased synovial cell apoptosis, and prevented osteoarthritis progression in mice with obesity-associated osteoarthritis.

Essential Revisions:

1) The authors need to carefully explain the rationale behind their design of the experimental groups.

2) The authors also need to analyze changes in macrophage efferocytosis maker F4/80 in OA synovitis.

3) If the administration of GAS6 can also reverse OA-like feasure in human cartilage explants or human articular chondrocytes.

Reviewer #1 (Recommendations for the authors):

The authors need to address the comments below.

1) If the administration of GAS6 can also reverse OA-like feasure in human cartilage explants or human articular chondrocytes.

2) To determine that GAS6/Axl signaling is indeed involved in OA development, the authors may need to provide evidence about the role of Axl in OA development.

Reviewer #2 (Recommendations for the authors):

The manuscript entitled "Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis" by Dr. Yao et al. observed that M1 macrophage infiltration significantly increased in the synovial tissue of obesity-associated OA. Overall, this is a well-designed, highly clinical demand and potentially translational study. Based on the patient's sample, the data indicated Synovial tissues are highly hyperplastic in obese OA patients and infiltrated with more polarized M1 macrophages than in non-obese OA patients.

Further authors proved that obesity promotes synovial M1 macrophage accumulation and GAS6 was inhibited in synovitis during OA development in mice models. The sample size, data collection, and quality of the IHC and immunofluorescent histological sections are outstanding. The results were well presented with appropriate interpretation.

Reviewer #3 (Recommendations for the authors):

The main strengths of the paper are the discovery of the underlying mechanism of obesity-associated osteoarthritis. However, some claims and conclusions were not well supported by their data. There are some issues that need to be carefully clarified and studied:

1. The design of experimental groups was defective, all C57BL/6 mice were fed a standard diet, while ApoE-/- mice were fed a high-fat diet. Both C57BL/6 and ApoE-/- mice should be fed the standard and high-fat diet respectively.

2. The source of cartilage and synovial tissue of obese OA patients described in Figure 1 should be added to the Materials and methods.

3. The authors deemed that the expression of GAS6 in chondrocytes was decreased in the obese ApoE-/- OA mice, however, in Figure 3C, its expression seemed to be increased.

4. The authors declared that GAS6 expression was inhibited in synovial macrophages, whether GAS6 is mainly expressed in synovial macrophages? Why was the expression level decreased in chondrocytes?

5. In Figure 1B, the F4/80 labeled macrophages were increased in synovial tissues of obese OA patients, however, in Figure 3A-D, the expression of F4/80 in synovial tissues of obese OA seemed to be decreased.

6. In Figure 4 H-J, the authors should add a group that is stimulated with GAS6 and R428 in RAW264.7 cells to prove that inhibition of the Axl receptor by R428 diminished the enhanced phagocytotic activity.

7. The authors declared that blocking M1 macrophage polarization could be a potential therapeutic strategy for obesity-associated OA, however, the authors did not investigate the effect of blocking M1 macrophage polarization.

8. The authors declared that enhanced M1-polarized macrophages in obese synovium decreased GAS6 secretion, the evidence for this conclusion was weak, and the decreased GAS6 secretion maybe not be due to the polarization of macrophages.

9. To study macrophage efferocytosis, the markers F4/80 should be used, but not the M1 macrophage marker iNOS. Besides, the primary macrophage such as BMDM is better than 264.7 cells.

10. The authors thought that accumulated apoptotic cells lead to the release of inflammatory factors that induced chondrocyte homeostasis dysfunction in obese OA patients, however, additional evidence is needed to support this conclusion.

https://doi.org/10.7554/eLife.83069.sa1

Author response

Essential Revisions:

1) The authors need to carefully explain the rationale behind their design of the experimental groups.

We greatly appreciate the careful review and helpful suggestions for improving manuscript. The experimental group was divided into four groups according to disease model and genotype: C57BL/6 control group, C57BL/6 OA group, ApoE-/- control group and ApoE-/- OA group.

From a critical perspective, the experimental grouping should be more rigorous in the manuscript. According to previous studies which revealed that feeding ApoE-/- mice with HFD accelerates the increase in LDL cholesterol levels and causes more body weight gain, compared with C57BL/6 mice fed with an HFD (references were provided in Answer 1# for Reviewer #2). We regard ApoE-/- mice fed with an HFD as the total variable associated with obesity, while C57BL/6 mice fed with a standard-chow diet as control. During revising the article, we reared mice and divided them into four groups, as Reviewer #3 suggested. Body weight gain was re-analyzed and provided in revised Table 4. Our statistical data also revealed a significant increase in body weight by feeding ApoE-/- mice with an HFD (19.81±1.33g) for 8 weeks compared with feeding C57BL/6 mice with an HFD (16.89±0.75g), which partly verified the effectiveness of inducing obesity by feeding ApoE-/- mice with an HFD for a short-term period. Accordingly, we selected feeding ApoE-/- mice with an HFD as the experimental group while feeding C57BL/6 mice with a standard-chow diet as control. Moreover, further studies were designed to explore the mechanism of obesity in the progression of OA.

2) The authors also need to analyze changes in macrophage efferocytosis maker F4/80 in OA synovitis.

Thanks for this valuable comment. Previous studies showed that MER is closely related to the maintenance of macrophages efferocytosis. Dransfield et al. identified Mer as a receptor uniquely capable of tethering ACs to the macrophage surface and driving their subsequent internalization1. Moreover, Felton and colleagues further revealed that knocking out Mer impaired phagocytosis of mouse eosinophils by macrophages, contributing to the persistence of airway inflammation2. Cai et al. found that higher macrophage MerTK improved efferocytosis and increased the ratio of pro-resolving versus proinflammatory lipid mediators, which attenuated plaque inflammation, fibrous cap thinning, and thrombosis in atherosclerosis3. These results suggest that MER is essential for the normal functioning of macrophage efferocytosis. In this manuscript, we found a decreased expression of MER in OA hyperplastic synovial tissue, especially in obesity-associated OA. These results indicated that the efferocytosis of macrophages is impaired in obesity-associated OA.

We have now added this part of the results in Revised Results 4, which reads “MER were widely used to label macrophages undergoing efferocytosis. Immunofluorescence staining analysis indicated the expression of MER in OA synovial macrophages decreased significantly, especially in obese ApoE-/- OA and obese OA patients.” We have now included these results in the revised Figure 4—figure supplement 1.

3) If the administration of GAS6 can also reverse OA-like feasure in human cartilage explants or human articular chondrocytes.

Thanks for this insightful comment. We have now used both human cartilage explants and primary articular chondrocytes obtained from patients undergoing total knee arthroplasty to confirm the effect of GAS6 on chondrocytes. Patient consent and the approval of the Ethics Committee of the Third Affiliated Hospital of Southern Medical University (Guangzhou, China) were obtained before the human tissue samples were harvested. No apparent proteoglycan loss or increase was found in rhGAS6 treated cartilage explant. Moreover, Western blotting showed no significant differences in the expression of COL2, MMP13, p16, and p21 in primary chondrocytes after stimulation with rhGAS6. These data are shown in revised Figure 5—figure supplement 2 and Figure 5—figure supplement 3

Reviewer #1 (Recommendations for the authors):

The authors need to address the comments below.

1) If the administration of GAS6 can also reverse OA-like feasure in human cartilage explants or human articular chondrocytes.

Thank you for this valuable comment, and thank the Reviewer very much for the opportunity to improve the manuscript. Please refer to Response to Essential Revisions comments Question 3.

2) To determine that GAS6/Axl signaling is indeed involved in OA development, the authors may need to provide evidence about the role of Axl in OA development.

Thank you for this insightful comment. As shown in our manuscript, F4/80-Axl double staining revealed no significant difference in the number of Axl-positive cells in the synovium of obese OA patients, obese ApoE-/- OA mice, and control subjects (previous Figure 3—figure supplement 3). Articular administration of R428 (Axl inhibitor) in C57BL/6 OA mice promoted synovial hyperplasia and cartilage destruction and enhanced synovitis and OARSI scores (previous Figure 5A and B). Moreover, in vitro experiments showed that the phagocytotic activity of RAW264.7 cells was decreased after administration of R428 (previous Figure 4H).

As the reviewer points out, more evidence is required to support the role of Axl in OA development. To demonstrate the direct role of Axl in macrophage, R428 was administrated to Raw264.7 cells to inhibit Axl. The results showed an increased expression of iNOS and CD86, together with up-regulated IL-1、IL-6, and TNF-α. These data are shown in revised Figure 3—figure supplement 2C and Figure 4—figure supplement 3.

Reviewer #3 (Recommendations for the authors):

The main strengths of the paper are the discovery of the underlying mechanism of obesity-associated osteoarthritis. However, some claims and conclusions were not well supported by their data. There are some issues that need to be carefully clarified and studied:

1. The design of experimental groups was defective, all C57BL/6 mice were fed a standard diet, while ApoE-/- mice were fed a high-fat diet. Both C57BL/6 and ApoE-/- mice should be fed the standard and high-fat diet respectively.

Thank you for this valuable comment, and thank the Reviewer very much for the opportunity to improve the manuscript. From a critical perspective, the experimental grouping should be more rigorous in the manuscript. According to previous studies which revealed that feeding ApoE-/- mice with HFD accelerates the increase in LDL cholesterol levels and causes more body weight gain, compared with C57BL/6 mice fed with an HFD (references were provided in Answer 1# for Reviewer #2). We regard ApoE-/- mice fed with an HFD as the total variable associated with obesity, while C57BL/6 mice fed with a standard-chow diet as control. During revising the article, we reared mice and divided them into four groups, as you suggested. Body weight gain was re-analyzed and provided in revised Table 4. Our statistical data also revealed a significant increase in body weight by feeding ApoE-/- mice with HFD (19.81±1.33g) for 8 weeks compared with feeding C57BL/6 mice with an HFD (16.89±0.75g), which partly verified the effectiveness of inducing obesity by feeding ApoE-/- mice with an HFD for a short-term period. Accordingly, we selected feeding ApoE-/- mice with an HFD as the experimental group while feeding C57BL/6 mice with a standard-chow diet as control. Moreover, further studies were designed to explore the mechanism of obesity in the progression of OA.

2. The source of cartilage and synovial tissue of obese OA patients described in Figure 1 should be added to the Materials and methods.

Thank you for your suggestions. We have added the source of cartilage and synovial tissue from obese OA patients in the first section of Materials and methods section. It reads “Synovial tissue and synovial fluid samples of normal individuals (n = 6, age 34 ± 8.15 years, three males, three females) or obese individuals (n = 6, age 35 ± 7.36 years, four males, two females) were obtained from patients who received arthroscopic treatment for acute anterior cruciate ligament rupture or meniscus injury Other joint diseases were excluded from the study. OA synovial tissues, and synovial fluid samples were obtained from obese patients (n = 6, age 64 ± 5.75 years, two males, four females) or patients without obesity (n = 6, age 65 ± 4.26 years, three males, three females) who underwent total knee arthroplasty. OA cartilage tissues were obtained from patients who underwent total knee arthroplasty, the tibial plateau cartilage was carefully separated and cut into 1mm3, cultured in vitro.”

3. The authors deemed that the expression of GAS6 in chondrocytes was decreased in the obese ApoE-/- OA mice, however, in Figure 3C, its expression seemed to be increased.

Thank you for your comments. We have replaced it with more representative images.

4. The authors declared that GAS6 expression was inhibited in synovial macrophages, whether GAS6 is mainly expressed in synovial macrophages? Why was the expression level decreased in chondrocytes?

We greatly appreciate your careful review and helpful suggestions. Our previous results showed that both macrophages and chondrocytes expressed GAS6 protein. However, we found a decrease in chondrocyte protein expression but did not investigate its functional impact. As suggested by Reviewer 1, rhGAS6 were used to stimulate OA cartilage explants or primary chondrocytes, we found that rhGas6 may not reverse the dysregulation of anabolic and catabolic homeostasis in OA articular cartilage explants, these data are shown in revised Figure 5—figure supplement 2 and Figure 5—figure supplement 3 (referring to answer for Essential Revisions 3). Since administration of GAS6 has no obvious effect on chondrocyte metabolism, we delete this part of figures. Nevertheless, the underlying mechanism of the decreased expression of GAS6 deserves further investigation.

5. In Figure 1B, the F4/80 labeled macrophages were increased in synovial tissues of obese OA patients, however, in Figure 3A-D, the expression of F4/80 in synovial tissues of obese OA seemed to be decreased.

We greatly appreciate your careful review and helpful suggestions. We are sorry for the ambiguity caused by the quality of images and we have chosen more representative images in revised Figure 3A-D.

6. In Figure 4 H-J, the authors should add a group that is stimulated with GAS6 and R428 in RAW264.7 cells to prove that inhibition of the Axl receptor by R428 diminished the enhanced phagocytotic activity.

Thank you for your suggestions. We added the group treated with both GAS6 and R428. The phagocytotic activity was indeed diminished after stimulated with rmGAS6 and R428 compared administration with rmGAS6 alone. We have now included these results in revised Figure 4H-K.

7. The authors declared that blocking M1 macrophage polarization could be a potential therapeutic strategy for obesity-associated OA, however, the authors did not investigate the effect of blocking M1 macrophage polarization.

We greatly appreciate your careful review and helpful suggestions. We are sorry for the ambiguity caused by the expression. Our previous findings showed that blocking M1 polarization of synovial macrophages attenuated OA progression. In this manuscript, we revealed that M1-polarized macrophage infiltration in OA synovial tissue of obese patients is significantly increased. Nevertheless, evidence supporting the effect of blocking M1 polarization could attenuate OA development is insufficient. We have deleted such ambiguous expressions and corrected the expression in the manuscript, which reads “targeting macrophage associated efferocytosis or intra-articular injection of GAS6 is a potential therapeutic strategy for obesity-associated OA.”

8. The authors declared that enhanced M1-polarized macrophages in obese synovium decreased GAS6 secretion, the evidence for this conclusion was weak, and the decreased GAS6 secretion maybe not be due to the polarization of macrophages.

We greatly appreciate your careful review and helpful suggestions. In order to explore the effect of M1 macrophage polarization on GAS6 secretion, BMDMs were treated with LPS or IL-4 for 36 hours. As is shown in revised Figure 3—figure supplement 2B, the expression of iNOS and CD86 were increased while GAS6 decreased significantly after treated BMDMs with LPS for 36 hours. These data indicated that the decreased GAS6 secretion was mainly due to the M1 polarization of macrophages.

9. To study macrophage efferocytosis, the markers F4/80 should be used, but not the M1 macrophage marker iNOS. Besides, the primary macrophage such as BMDM is better than 264.7 cells.

Thank you for your comment. In Figure 4E-J, marker F4/80 (red) and CFSE (green) were used to label macrophages and apoptotic cells respectively. Moreover, as you proposed, we performed an experiment with BMDMs to illustrate the effect of rmGAS6 in regulating efferocytosis of macrophages, which were showed in Revised Figure 4—figure supplement 2.

10. The authors thought that accumulated apoptotic cells lead to the release of inflammatory factors that induced chondrocyte homeostasis dysfunction in obese OA patients, however, additional evidence is needed to support this conclusion.

Thank you for your comment. To further explore the effect of inflammatory factors released by the stimulation of apoptotic cells (ACs) on chondrocyte homeostasis dysfunction, ACs were co-cultured with BMDMs, and the culture supernatant was collected after stimulation for 24 hours. The expression of MMP13 was increased after adding co-culture supernatant stimulation, together with senescence hallmarks such as p16 and p21, while COL2 were down-regulated after stimulation. These data partly indicated that macrophages stimulated by apoptotic cells secreted chemokines leading chondrocyte homeostasis dysfunction. However, the effect of supernatant promoting chondrocyte homeostasis dysfunction was partially alleviated when adding ACs with rmGAS6 to BMDMs for 24 hours, which were diminished by adding R428. We adding these contexts in Results 5, which reads “To further explore the effect of inflammatory factors released by the stimulation of apoptotic cells (ACs) on chondrocyte homeostasis dysfunction, ACs were co-cultured with BMDMs and the culture supernatant was collected after stimulation for 24 hours. The expression of MMP13 were increased after adding co-culture supernatant stimulation, together with senescence hallmarks such as p16 and p21, while COL2 were down-regulated after stimulation. However, the effect of supernatant promoting chondrocyte homeostasis dysfunction was partially alleviated when adding ACs with rmGAS6 to BMDMs, which were diminished by adding R428.” These data were showed in revised Figure 5—figure supplement 1.

https://doi.org/10.7554/eLife.83069.sa2

Article and author information

Author details

  1. Zihao Yao

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Data curation, Investigation, Writing - original draft
    Contributed equally with
    Weizhong Qi and Hongbo Zhang
    Competing interests
    No competing interests declared

  2. Weizhong Qi

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Data curation, Software, Formal analysis
    Contributed equally with
    Zihao Yao and Hongbo Zhang
    Competing interests
    No competing interests declared

  3. Hongbo Zhang

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Conceptualization, Software, Methodology
    Contributed equally with
    Zihao Yao and Weizhong Qi
    Competing interests
    No competing interests declared

  4. Zhicheng Zhang

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Validation, Investigation
    Competing interests
    No competing interests declared

  5. Liangliang Liu

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Resources, Validation, Investigation
    Competing interests
    No competing interests declared

  6. Yan Shao

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Software, Methodology
    Competing interests
    No competing interests declared

  7. Hua Zeng

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Resources, Software
    Competing interests
    No competing interests declared

  8. Jianbin Yin

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Supervision, Methodology
    Competing interests
    No competing interests declared

  9. Haoyan Pan

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Supervision, Visualization, Methodology
    Competing interests
    No competing interests declared

  10. Xiongtian Guo

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Software, Validation
    Competing interests
    No competing interests declared

  11. Anling Liu

    Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
    Contribution
    Supervision, Funding acquisition
    Competing interests
    No competing interests declared

  12. Daozhang Cai

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Conceptualization, Writing - review and editing
    For correspondence
    cdz@smu.edu.cn
    Competing interests
    No competing interests declared

  13. Xiaochun Bai

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Software, Validation, Methodology
    For correspondence
    baixc15@smu.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9631-4781

  14. Haiyan Zhang

    1. Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    2. Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China
    Contribution
    Conceptualization, Formal analysis, Funding acquisition
    For correspondence
    zhhy0704@126.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9361-3134

Funding

National Natural Science Foundation of China (81902229)

  • Haiyan Zhang

National Natural Science Foundation of China (81871745)

  • Anling Liu

Natural Science Foundation of Guangdong Province (2020A1515011062)

  • Haiyan Zhang

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (grant numbers: 81902229 and 81871745) and Natural Science Foundation of Guangdong Province (2020A1515011062).

Ethics

Informed consent was obtained from all recruited patients and was identified by the ethics committee of the Third Affiliated Hospital of Southern Medical University.

This study was performed in strict accordance with the recommendations in Chinese Laboratory animal-Guideline for ethical review of animal welfare (GB/T 35892-2018). The protocol was approved by the Southern Medical University Animal Care and Use Review Board (Permit Number: 2021-Ethical review-053). All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.

Senior Editor

  1. Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States

Reviewing Editor

  1. Di Chen, Chinese Academy of Sciences, China

Reviewer

  1. Chao Xie, University of Rochester Medical Center, United States

Version history

  1. Received: August 30, 2022
  2. Preprint posted: September 21, 2022 (view preprint)
  3. Accepted: May 4, 2023
  4. Accepted Manuscript published: May 5, 2023 (version 1)
  5. Version of Record published: May 17, 2023 (version 2)

Copyright

© 2023, Yao, Qi, Zhang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Zihao Yao
  2. Weizhong Qi
  3. Hongbo Zhang
  4. Zhicheng Zhang
  5. Liangliang Liu
  6. Yan Shao
  7. Hua Zeng
  8. Jianbin Yin
  9. Haoyan Pan
  10. Xiongtian Guo
  11. Anling Liu
  12. Daozhang Cai
  13. Xiaochun Bai
  14. Haiyan Zhang
(2023)
Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis
eLife 12:e83069.
https://doi.org/10.7554/eLife.83069

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