Targeted knockdown of PGAM5 in synovial macrophages efficiently alleviates osteoarthritis

Abstract

Osteoarthritis (OA) is a common degenerative disease worldwide and new therapeutics that target inflammation and the crosstalk between immunocytes and chondrocytes are being developed to prevent and treat OA. These attempts involve repolarizing pro-inflammatory M1 macrophages into the anti-inflammatory M2 phenotype in synovium. In this study, we found that phosphoglycerate mutase 5 (PGAM5) significantly increased in macrophages in OA synovium compared to controls based on histology of human samples and single-cell RNA sequencing results of mice models. To address the role of PGAM5 in macrophages in OA, we found conditional knockout of PGAM5 in macrophages greatly alleviated OA symptoms and promoted anabolic metabolism of chondrocytes in vitro and in vivo. Mechanistically, we found that PGAM5 enhanced M1 polarization via AKT-mTOR/p38/ERK pathways, whereas inhibited M2 polarization via STAT6-PPARγ pathway in murine bone marrow-derived macrophages. Furthermore, we found that PGAM5 directly dephosphorylated Dishevelled Segment Polarity Protein 2 (DVL2) which resulted in the inhibition of β-catenin and repolarization of M2 macrophages into M1 macrophages. Conditional knockout of both PGAM5 and β-catenin in macrophages significantly exacerbated osteoarthritis compared to PGAM5-deficient mice. Motivated by these findings, we successfully designed mannose modified fluoropolymers combined with siPGAM5 to inhibit PGAM5 specifically in synovial macrophages via intra-articular injection, which possessed desired targeting abilities of synovial macrophages and greatly attenuated murine osteoarthritis. Collectively, these findings defined a key role for PGAM5 in orchestrating macrophage polarization and provides insights into novel macrophage-targeted strategy for treating OA.

References

1. Coryell, P. R., Diekman, B. O.& Loeser, R. F. Mechanisms and therapeutic implications of cellular senescence in osteoarthritis. Nat. Rev. Rheumatol. 17, 47-57 (2021).
2. Loeser, R. F., Collins, J. A.& Diekman, B. O. Ageing and the pathogenesis of osteoarthritis. Nat. Rev. Rheumatol. 12, 412-420 (2016).
3. Conaghan, P. G.et al.Therapeutic options for targeting inflammatory osteoarthritis pain. Nat. Rev. Rheumatol. 15, 355-363 (2019).
4. Robinson, W. H.et al.Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis. Nat. Rev. Rheumatol. 12, 580-592 (2016).
5. Zhang, H.et al.Synovial macrophage M1 polarisation exacerbates experimental osteoarthritis partially through R-spondin-2. Ann. Rheum. Dis. 77, 1524-1534 (2018).
6. Dai, M.et al. Cartilage repair in degenerative osteoarthritis mediated by squid type II collagen via immunomodulating activation of M2 macrophages, inhibiting apoptosis and hypertrophy of chondrocytes. Biomaterials 180, 91-103 (2018).
7. Ma, Y.et al.Artificial M2 macrophages for disease-modifying osteoarthritis therapeutics. Biomaterials 274, 120865 (2021).
8. Lu, W.et al.Genetic deficiency of the mitochondrial protein PGAM5 causes a Parkinson's-like movement disorder. Nat. Commun. 5, 4930(2014).
9. Wang, Z.et al. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell 148, 228-243 (2012).
10. Ma, K.et al.Dynamic PGAM5 multimers dephosphorylate BCL-xL or FUNDC1 to regulate mitochondrial and cellular fate. Cell Death Differ. 27, 1036-1051 (2020).
11. Yu, B.et al.Mitochondrial phosphatase PGAM5 modulates cellular senescence by regulating mitochondrial dynamics. Nat. Commun. 11, 2549(2020).
12. Moriwaki, K.et al.The mitochondrial phosphatase PGAM5 is dispensable for necroptosis but promotes inflammasome activation in macrophages. J. Immunol. 196, 407-415 (2016).
13. Knights, A. J.et al.Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 in osteoarthritis. Ann. Rheum. Dis. 82, 272-282 (2023).
14. Sica A.& Mantovani, A. Macrophage plasticity and polarization: in vivo veritas. J. Clin. Investig. 122, 787-795 (2012).
15. Schaefer, L.et al.The matrix component biglycan is proinflammatory and signals through Toll-like receptors 4 and 2 in macrophages. J. Clin. Investig. 115, 2223-2233 (2005).
16. Yang, H.et al. MTORC1 coordinates the autophagy and apoptosis signaling in articular chondrocytes in osteoarthritic temporomandibular joint. Autophagy 16, 271-288 (2020).
17. Olefsky J. M.& Glass, C. K. Macrophages, inflammation, and insulin resistance. Annu. Rev. Physiol. 72, 219-246 (2010).
18. Guo, B.et al.Antagonism of PPAR-γ signaling expands human hematopoietic stem and progenitor cells by enhancing glycolysis. Nat. Med. 24, 360-367 (2018).
19. Li, J.et al.STAT6 contributes to renal fibrosis by modulating PPARα-mediated tubular fatty acid oxidation. Cell Death Dis. 13, 66(2022).
20. Rauschenberger, V.et al. The phosphatase Pgam5 antagonizes Wnt/β-Catenin signaling in embryonic anterior-posterior axis patterning. Development 144, 2234-2247 (2017).
21. Feng, Y.et al.Wnt/β-catenin-promoted macrophage alternative activation contributes to kidney fibrosis. J. Am. Soc. Nephrol. 29, 182-193 (2018).
22. Tian, X.et al.Long noncoding RNA LINC00662 promotes M2 macrophage polarization and hepatocellular carcinoma progression via activating Wnt/ β-catenin signaling. Mol. Oncol. 14, 462-483 (2020).
23. Oguma, K.et al.Activated macrophages promote Wnt signalling through tumour necrosis factor-alpha in gastric tumour cells. EMBO J. 27, 1671-1681 (2008).
24. Lu, Y.et al.lncRNA MIR100HG-derived miR-100 and miR-125b mediate cetuximab resistance via Wnt/β-catenin signaling. Nat. Med. 23, 1331-1341 (2017).
25. Fire, A.et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811 (1998).
26. Evans C. H.Catering to chondrocytes. Sci. Transl. Med. 10, 469(2018).
27. Kazi, A.et al.GSK3 suppression upregulates β-catenin and c-Myc to abrogate KRas-dependent tumors. Nat. Commun. 9, 5154(2018).
28. Mahmoudian, A.et al.Early-stage symptomatic osteoarthritis of the knee - time for action. Nat. Rev. Rheumatol. 17, 621-632 (2021).
29. Wynn, T. A. & Vannella, K. M. Macrophages in tissue repair, regeneration,fibrosis. Immunity 44, 450-462 (2016).
30. Mantovani, A.et al.Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 23, 549-555 (2002).
31. Zhu, L.et al.TSC1 controls macrophage polarization to prevent inflammatory disease. Nat. Commun. 5, 4696(2014).
32. Lv, Z.et al.TRPV1 alleviates osteoarthritis by inhibiting M1 macrophage polarization via Ca2+/CaMKII/Nrf2 signaling pathway. Cell Death Dis. 12, 504(2021).
33. Zhang, Y.et al.Reprogramming of mitochondrial respiratory chain complex by targeting SIRT3-COX4I2 axis attenuates osteoarthritis progression. Adv. Sci. 10, e2206144(2023).
34. Vergadi, E.et al.Akt signaling pathway in macrophage activation and M1/M2 polarization. J. Immunol. 198, 1006-1014 (2017).
35. Zhao, S. J.et al. Macrophage MSR1 promotes BMSC osteogenic differentiation and M2-like polarization by activating PI3K/AKT/GSK3β/β-catenin pathway. Theranostics 10, 17-35 (2020).
36. Bode, J. G., Ehlting, C.& Häussinger, D. The macrophage response towards LPS and its control through the p38(MAPK)-STAT3 axis. Cell Signal. 24, 1185-1194 (2012).
37. Wang, L.et al. Gut microbial bile acid metabolite skews macrophage polarization and contributes to high-fat diet-induced colonic inflammation. Gut Microbes 12, 1-20 (2020).
38. Chen, J. Y.et al.CTRP9 induces macrophages polarization into M1 phenotype through activating JNK pathway and enhances VSMCs apoptosis in macrophages and VSMCs co-culture system. Exp. Cell Res. 395, 112194(2020).
39. Zhang, H., Cai, D.& Bai, X. Macrophages regulate the progression of osteoarthritis. Osteoarthr. Cartil. 28, 555-561 (2020).
40. Gao C.& Chen, Y. G. Dishevelled: the hub of Wnt signaling. Cell Signal. 22, 717-727 (2010).
41. Wu D.& Pan, W. GSK3: a multifaceted kinase in Wnt signaling. Trends Biochem. Sci. 35, 161-168 (2010).
42. Denk, D.et al. Expansion of T memory stem cells with superior anti-tumor immunity by Urolithin A-induced mitophagy. Immunity 55, 2059-2073.e8 (2022).
43. Bernkopf, D. B.et al.Pgam5 released from damaged mitochondria induces mitochondrial biogenesis via Wnt signaling. J. Cell Biol. 217, 1383-1394 (2018).
44. Lv, J.et al.Fluorination promotes the cytosolic delivery of genes, proteins, and peptides. Acc. Chem. Res. 55, 722-733 (2022).
45. Lv J.& Cheng, Y. Fluoropolymers in biomedical applications: state-of-the-art and future perspectives. Chem. Soc. Rev. 50, 5435-5467 (2021).
46. Cummings R. D.The mannose receptor ligands and the macrophage glycome. Curr. Opin. Struct. Biol. 75, 102394(2022).
47. Zhang, W.et al.Current research on pharmacologic and regenerative therapies for osteoarthritis. Bone Res. 4, 15040(2016).
48. Cao, W.et al.Tumor suppressor adenomatous polyposis coli sustains dendritic cell tolerance through IL-10 in a β-catenin-dependent manner. J. Immunol. 210, 1589-1597 (2023).
49. Wang, M.et al.A fluorinated dendrimer achieves excellent gene transfection efficacy at extremely low nitrogen to phosphorus ratios. Nat. Commun. 5, 3053(2014).

Funding

Yiyun Cheng ([e-mail]) or Wei Lu ([e-mail]) or Xiaoling Zhang ([e-mail])