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
  13. Xiaochun Bai
  14. Haiyan Zhang

  • Curated by eLife

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    The authors demonstrate that patients with obesity-associated osteoarthritis and mice with the ApoE gene deficiency showed phenotypes of synovitis and enhanced macrophage infiltration in synovial tissues. Overall, this potentially important study is well-designed and carefully executed, although additional evidence is needed to fully support the authors' conclusions.

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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.

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  1. Version published to 10.7554/elife.83069 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    Understanding the molecular mechanism of obesity-associated OA is highly in clinical demand. Overall, the current study is well-designed and illustrated that down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis. 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. But the following major questions should be addressed.

    Major:

    1. Animal model: Ten-week-old animals received DMM surgery and were fed a standard/HFD diet for 4 or 8 weeks prior to specimen harvest. Since Wang J and other studies have shown that male ApoE(-/-) and C57BL/6J wild-type (WT) mice fed with a high-fat diet for 12 or 24 weeks, and the ApoE(-/-) mice gained less body weight and had less fat mass and lower triglyceride levels with better insulin sensitivity and lower levels of inflammatory markers in skeletal muscle than WT (Wang J, et al. Atherosclerosis. 2012 Aug;223(2):342-9. PMID: 22770993; Hofmann SM, et al. Diabetes. 2008 Jan;57(1):5-12. PMID: 17914034; Kypreos KE et al. J Biomed Res. 2017 Nov 1;32(3):183-90. PMID: 29770778). Thus, it is very important to provide the data on the final body weight gained in your groups and provide a relative background of the animal model chosen in the introduction or discussion. Please explain why ApoE-/- mouse model, and how this animal model is clinically relevant. Does a high-fat diet induced obsess OA available in C57BL/6 WT?

    Thank you for your valuable comment. We have added the body weight change data for each group of mice in Revised Figure 2-figure supplement 3. We also provided a relative background of the animal model in paragraph 2 of the Discussion section, which reads, “ApoE plays an important role in maintaining the normal levels of cholesterol and triglycerides in serum by transporting lipids in the blood. Mice lacking ApoE function develop hypercholesterolemia, increased very low-density lipoprotein (VLDL) and decreased high-density lipoprotein (HDL), exhibiting chronic inflammation in vascular disease and nonalcoholic steatohepatitis.”.

    Epidemiological study results suggest obesity is an independent risk factor for OA pathological progression. Gierman et al. found that increased plasma cholesterol levels play a vital role in the development of OA1,2. Mice deficient in ApoE-/- showed naturally high levels of LDL-cholesterol independent of gender and age, which could additionally be increased by a cholesterol-rich diet3,4. Moreover, recent studies found that ApoE-/- mice feeding with HFD gained more body weight than those feeding standard chow-diet groups5–7. We have re-analyzed the body weight statistics and found that ApoE-/- fed with HFD (19.81±1.33g) gained more body weight than the control (16.89±0.75g). These manuscripts indicated that feeding HFD to ApoE-/- mice for a short period could accelerate the increase in LDL cholesterol levels and cause more body weight gain. ApoE-/- mice may be partially clinically relevant to pathological progression in obese osteoarthritis patients with elevated plasma LDL cholesterol levels. As Reviewer #2 mentioned, an HFD induced obesity is available in C57BL/6 WT according to our weight gain data. However, the effect of obesity on OA progression in these two kinds of animals deserves further study.

    References：

    1. Gierman LM, Kühnast S, Koudijs A, et al. Osteoarthritis development is induced by increased dietary cholesterol and can be inhibited by atorvastatin in APOE*3Leiden.CETP mice—a translational model for atherosclerosis. Ann Rheum Dis. 2014;73(5):921-927.

    2. Gierman LM, van der Ham F, Koudijs A, et al. Metabolic stress-induced inflammation plays a major role in the development of osteoarthritis in mice. Arthritis Rheum. 2012;64(4):1172-1181.

    3. Wu D, Sharan C, Yang H, et al. Apolipoprotein E-deficient lipoproteins induce foam cell formation by downregulation of lysosomal hydrolases in macrophages. J Lipid Res. 2007;48(12):2571-2578.

    4. Naura AS, Hans CP, Zerfaoui M, et al. induces lung remodeling in ApoE-deficient mice: an association with an increase in circulatory and lung inflammatory factors. Lab Invest. 2009;89(11):1243-1251.

    5. Tung MC, Lan YW, Li HH, et al. Kefir peptides alleviate high-fat diet-induced atherosclerosis by attenuating macrophage accumulation and oxidative stress in ApoE knockout mice. Sci Rep. 2020;10(1):8802.

    6. Bao M hua, Luo H qing, Chen L hua, et al. Impact of high fat diet on long non-coding RNAs and messenger RNAs expression in the aortas of ApoE(−/−) mice. Sci Rep. 2016;6(1):34161.

    7. Cao X, Guo Y, Wang Y, et al. Effects of high-fat diet and Apoe deficiency on retinal structure and function in mice. Sci Rep. 2020;10(1):18601.

    1. Control group: The DMM surgery was performed on the right leg, and the contralateral knee joint should be used as a baseline to show the level of M1 macrophage infiltration under the obsess microenvironment.

    Thank you for this insightful comment. The reason why we used the right lower limb as the control group in our experiment was mainly because we considered the impact of right knee surgery on the left lower limb. A book published in 2014 described a series of method for inducing mouse osteoarthritis model, authors noted that sham-operated left knee joints would develop OA-like symptoms after right knee joints received DMM. Thus, Lorenz et al. strongly recommend using a separate control group for sham surgeries.

    References：

    1. Lorenz, J., Grässel, S. (2014). Experimental Osteoarthritis Models in Mice. In: Singh, S., Coppola, V. (eds) Mouse Genetics. Methods in Molecular Biology, vol 1194. Humana Press, New York, NY.
  3. eLife

    eLife assessment

    The authors demonstrate that patients with obesity-associated osteoarthritis and mice with the ApoE gene deficiency showed phenotypes of synovitis and enhanced macrophage infiltration in synovial tissues. Overall, this potentially important study is well-designed and carefully executed, although additional evidence is needed to fully support the authors' conclusions.

  4. eLife

    Reviewer #1 (Public Review):

    Obesity is a risk factor for OA development and progression and its molecular mechanisms remain unknown. In this study, the authors demonstrated that obese OA patients and ApoE KO mice showed a pronounced synovitis and enhanced macrophage infiltration in synovial tissues. In addition, obese OA mice had severe cartilage degradation and increased apoptotic cells in synovial tissues than OA mice without obesity. GAS6 is a secreted glycoprotein and during M1 macrophage polarization, GAS6 secretion is decreased, leading to impaired macrophage efferocytosis in synovial apoptotic cells. Intra-articular injection of GAS6 restored the phagocytic capacity of macrophages and decreased the levels of TUNEL-positive cells, preserving cartilage thickness and preventing OA progression in obese OA mice. Overall speaking, this study is well-designed and carefully executed. The data presented are supportive of the conclusion that the authors made.

  5. eLife

    Reviewer #2 (Public Review):

    Understanding the molecular mechanism of obesity-associated OA is highly in clinical demand. Overall, the current study is well-designed and illustrated that down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis. 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. But the following major questions should be addressed.

    Major:
    1. Animal model: Ten-week-old animals received DMM surgery and were fed a standard/HFD diet for 4 or 8 weeks prior to specimen harvest. Since Wang J and other studies have shown that male ApoE(-/-) and C57BL/6J wild-type (WT) mice fed with a high-fat diet for 12 or 24 weeks, and the ApoE(-/-) mice gained less body weight and had less fat mass and lower triglyceride levels with better insulin sensitivity and lower levels of inflammatory markers in skeletal muscle than WT (Wang J, et al. Atherosclerosis. 2012 Aug;223(2):342-9. PMID: 22770993; Hofmann SM, et al. Diabetes. 2008 Jan;57(1):5-12. PMID: 17914034; Kypreos KE, et al. J Biomed Res. 2017 Nov 1;32(3):183-90. PMID: 29770778). Thus, it is very important to provide the data on the final body weight gained in your groups and provide a relative background of the animal model chosen in the introduction or discussion. Please explain why ApoE-/- mouse model, and how this animal model is clinically relevant. Does a high-fat diet induced obsess OA available in C57BL/6 WT?
    2. Control group: The DMM surgery was performed on the right leg, and the contralateral knee joint should be used as a baseline to show the level of M1 macrophage infiltration under the obsess microenvironment.

  6. eLife

    Reviewer #3 (Public Review):

    In this study, the authors studied the underlying mechanism of obesity-related inflammation in OA synovitis. They found more pronounced synovitis and enhanced macrophage infiltration accompanied by dominant M1 macrophage polarization in obese OA patients and ApoE-/- mice synovial tissues. Enhanced M1-polarized macrophages in obese synovium decreased growth arrest-specific 6 (GAS6) secretion, which resulted in impaired macrophage efferocytosis in synovial apoptotic cells. 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. 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.

  7. Version published to 10.1101/2022.09.20.508661v1 on bioRxiv