Identification of FOXO1 as a geroprotector in human synovium through single-nucleus transcriptomic profiling

Abstract

The synovium, a thin layer of tissue that is adjacent to the joints and secretes synovial fluid, undergoes changes in aging that contribute to intense shoulder pain and other joint diseases. However, the mechanism underlying human synovial aging remains poorly characterized. Here, we generated a comprehensive transcriptomic profile of synovial cells present in the subacromial synovium from young and aged individuals. By delineating aging-related transcriptomic changes across different cell types and their associated regulatory networks, we identified two subsets of mesenchymal stromal cells (MSCs) in human synovium, which are lining and sublining MSCs, and found that angiogenesis and fibrosis-associated genes were upregulated whereas genes associated with cell adhesion and cartilage development were downregulated in aged MSCs. Moreover, the specific cell-cell communications in aged synovium mirrors that of aging-related inflammation and tissue remodeling, including vascular hyperplasia and tissue fibrosis. In particular, we identified forkhead box O1 (FOXO1) as one of the major regulons for aging differentially expressed genes (DEGs) in synovial MSCs, and validated its downregulation in both lining and sublining MSC populations of the aged synovium. In human FOXO1-depleted MSCs derived from human embryonic stem cells, we recapitulated the senescent phenotype observed in the subacromial synovium of aged donors. These data indicate an important role of FOXO1 in the regulation of human synovial aging. Overall, our study improves our understanding of synovial aging during joint degeneration, thereby informing the development of novel intervention strategies aimed at rejuvenating the aged joint.

Key words： aging; single-nucleus RNA sequencing; synovium; FOXO1; senescence

References

1 Agarwal SK, Brenner MB. Role of adhesion molecules in synovial inflammation. Curr Opin Rheumatol 2006;18:268–76.

2 Aging Atlas C. Aging atlas: a multi-omics database for aging biology. Nucleic Acids Res 2021;49:D825–30.

3 Aging Biomarker C, Suo J, Gan Y et al. A framework of biomarkers for skeletal aging: a consensus statement by the Aging Biomarker Consortium. Life Med 2023.

4 Aibar S, González-Blas CB, Moerman T et al. SCENIC: single-cell regulatory network inference and clustering. Nat Methods 2017;14:1083–6.

5 Akasaki Y, Hasegawa A, Saito M et al. Dysregulated FOXO transcription factors in articular cartilage in aging and osteoarthritis. Osteoarthritis Cartilage 2014;22:162–70.

6 Alivernini S, MacDonald L, Elmesmari A et al. Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis. Nat Med 2020;26:1295–306.

7 Alvarez-Garcia O, Matsuzaki T, Olmer M et al. Age-related reduction in the expression of FOXO transcription factors and correlations with intervertebral disc degeneration. J Orthop Res 2017;35:2682–91.

8 Alvarez-Garcia O, Matsuzaki T, Olmer M et al. FOXO are required for intervertebral disk homeostasis during aging and their deficiency promotes disk degeneration. Aging Cell 2018;17:e12800.

9 Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics (Oxford, England) 2015;31:166–9.

10 Aging Biomarker Consortium, Bao H, Cao J, Chen M et al. Biomarkers of aging. Sci China Life Sci 2023;66:893–1066.

11 Bartok B, Firestein GS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev 2010;233:233–55.

12 Bertin FR, Lemarié CA, Robins RS et al. Growth arrest-specific 6 regulates thrombin-induced expression of vascular cell adhesion molecule-1 through forkhead box O1 in endothelial cells. J Thromb Haemost 2015;13:2260–72.

13 Bhattaram P, Chandrasekharan U. The joint synovium: a critical determinant of articular cartilage fate in inflammatory joint diseases. Semin Cell Dev Biol 2017;62:86–93.

14 Bi S, Liu Z, Wu Z et al. SIRT7 antagonizes human stem cell aging as a heterochromatin stabilizer. Protein Cell 2020;11:483–504.

15 Cai Y, Song W, Li J et al. The landscape of aging. Sci China Life Sciences 2022;65:2354–454.

16 Chou C-H, Jain V, Gibson J et al. Synovial cell cross-talk with cartilage plays a major role in the pathogenesis of osteoarthritis. Sci Rep 2020;10:10868.

17 Chu Q, Liu F, He Y et al. mTORC2/RICTOR exerts differential levels of metabolic control in human embryonic, mesenchymal and neural stem cells. Protein Cell 2022;13:676–82.

18 Collins FL, Roelofs AJ, Symons RA et al. Taxonomy of fibroblasts and progenitors in the synovial joint at single-cell resolution. Ann Rheum Dis 2023;82:428–37.

19 Culemann S, Grüneboom A, Nicolás-Ávila J et al. Locally renewing resident synovial macrophages provide a protective barrier for the joint. Nature 2019;572:670–5.

20 Dahl IM, Husby G. Hyaluronic acid production in vitro by synovial lining cells from normal and rheumatoid joints. Ann Rheum Dis 1985;44:647–57.

21 Diao Z, Ji Q, Wu Z et al. SIRT3 consolidates heterochromatin and counteracts senescence. Nucleic Acids Res 2021;49:4203–19.

22 Efremova M, Vento-Tormo M, Teichmann SA et al. CellPhoneDB: inferring cell–cell communication from combined expression of multi-subunit ligand-receptor complexes. Nat Protocols 2020;15:1484–506.

23 Fleming SJ, Chaffin MD, Arduini A et al. Unsupervised removal of systematic background noise from droplet- based single-cell experiments using CellBender. Nat Methods 2023;20:1323–35.

24 Frank-Bertoncelj M, Gay S. The epigenome of synovial fibroblasts: an underestimated therapeutic target in rheumatoid arthritis. Arthritis Res Ther 2014;16:117.

25 Geng L, Zhang B, Liu H et al. A comparative study of metformin and nicotinamide riboside in alleviating tissue aging in rats. Life Med 2023;2:lnac045.

26 Giorgino R, Albano D, Fusco S et al. Knee osteoarthritis: epidemiology, pathogenesis, and mesenchymal stem cells: What else is new? an update. Int J Mol Sci 2023;24:6405.

27 Hao Y, Hao S, Andersen-Nissen E et al. Integrated analysis of multimodal single-cell data. Cell 2021;184:3573–3587.e29.

28 Huang D, Zhao Q, Yang K et al. CRL2^APPBP2-mediated TSPYL2 degradation counteracts human mesenchymal stem cell senescence. Sci China Life Sciences 2022.

29 Hubbard EL, Catalina MD, Heuer S et al. Analysis of gene expression from systemic lupus erythematosus synovium reveals myeloid cell-driven pathogenesis of lupus arthritis. Sci Rep 2020;10:17361.

30 Huynh-Thu VA, Irrthum A, Wehenkel L et al. Inferring regulatory networks from expression data using tree-based methods. PLoS One 2010;5:e12776.

31 Kahn AJ. FOXO3 and related transcription factors in development, aging, and exceptional longevity. J Gerontol A Biol Sci Med Sci 2015;70:421–5.

32 Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods 2015;12:357–60.

33 Kishi JY, Lapan SW, Beliveau BJ et al. SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nat Methods 2019;16:533–44.

34 Knights AJ, Farrell EC, Ellis OM et al. Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 in osteoarthritis. Ann Rheum Dis 2023;82:272–82.

35 Kousteni S. FoxO1: a molecule for all seasons. J Bone Miner Res 2011;26:912–7.

36 Li F, Tang Y, Song B et al. Nomenclature clarification: synovial fibroblasts and synovial mesenchymal stem cells. Stem Cell Res Ther 2019;10:260.

37 Li F, Chen J, Gong M et al. Isolation and characterization of human synovial fluid-derived mesenchymal stromal cells from popliteal cyst. Stem Cells Int 2020a;2020:7416493.

38 Li N, Gao J, Mi L et al. Synovial membrane mesenchymal stem cells: past life, current situation, and application in bone and joint diseases. Stem Cell Res Ther 2020b;11:381.

39 Li Q, Wen Y, Wang L et al. Hyperglycemia-induced accumulation of advanced glycosylation end products in fibroblast-like synoviocytes promotes knee osteoarthritis. Exp Mol Med 2021;53:1735–47.

40 Liang C, Liu Z, Song M et al. Stabilization of heterochromatin by CLOCK promotes stem cell rejuvenation and cartilage regeneration. Cell Res 2021;31:187–205.

41 Lin X, Bell RD, Catheline SE et al. Targeting synovial lymphatic function as a novel therapeutic intervention for age-related osteoarthritis in mice. Arthritis Rheumatol (Hoboken, NJ) 2023;75:923–36.

42 Liu X, Jiao H, Zhang B et al. Migrasomes trigger innate immune activation and mediate transmission of senescence signals across human cells. Life Med 2023a.

43 Liu X, Liu Z, Wu Z et al. Resurrection of endogenous retroviruses during aging reinforces senescence. Cell 2023b;186:287–304.e26.

44 López-Otín C, Blasco MA, Partridge L et al. The hallmarks of aging. Cell 2013;153:1194–217.

45 Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014;15:550.

46 Lowin T, Straub RH. Integrins and their ligands in rheumatoid arthritis. Arthritis Res Ther 2011;13:244.

47 Ludikhuize J, de Launay D, Groot D et al. Inhibition of fork-head box class O family member transcription factors in rheumatoid synovial tissue. Arthritis Rheum 2007;56:2180–91.

48 McGinnis CS, Murrow LM, Gartner ZJ. DoubletFinder: doublet detection in single-cell RNA sequencing data using artificial nearest neighbors. Cell Syst 2019;8:329–337.e4.

49 McInnes IB, Schett G. Pathogenetic insights from the treatment of rheumatoid arthritis. Lancet (London, England) 2017;389:2328–37.

50 Migita K, Iwanaga N, Izumi Y et al. TNF-α-induced miR-155 regulates IL-6 signaling in rheumatoid synovial fibroblasts. BMC Res Notes 2017;10:403.

51 Nygaard G, Firestein GS. Restoring synovial homeostasis in rheumatoid arthritis by targeting fibroblast-like synoviocytes. Nat Rev Rheumatol 2020;16:316–33.

52 Onuora S. Draining OA from the synovium. Nat Rev Rheumatol 2023;19:130–130.

53 Orr C, Vieira-Sousa E, Boyle DL et al. Synovial tissue research: a state-of-the-art review. Nat Rev Rheumatol 2017;13:463–75.

54 Oyabu M, Takigawa K, Mizutani S et al. FOXO1 cooperates with C/EBPδ and ATF4 to regulate skeletal muscle atrophy transcriptional program during fasting. FASEB J 2022;36:e22152.

55 Pal S, Tyler JK. Epigenetics and aging. Sci Adv 2016;2:e1600584.

56 Pasquali-Ronchetti I, Frizziero L, Guerra D et al. Aging of the human synovium: an in vivo and ex vivo morphological study. Semin Arthritis Rheum 1992;21:400–14.

57 Peng H, Yu Y, Gu H et al. MicroRNA-483-5p inhibits osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting the RPL31-mediated RAS/MEK/ ERK signaling pathway. Cell Signal 2022;93:110298.

58 Rached MT, Kode A, Xu L et al. FoxO1 is a positive regulator of bone formation by favoring protein synthesis and resistance to oxidative stress in osteoblasts. Cell Metab 2010;11:147–60.

59 Ramezankhani R, Minaei N, Haddadi M et al. The impact of sex on susceptibility to systemic lupus erythematosus and rheumatoid arthritis; a bioinformatics point of view. Cell Signal 2021;88:110171.

60 Robinson WH, Lepus CM, Wang Q et al. Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis. Nat Rev Rheumatol 2016;12:580–92.

61 Sanchez-Lopez E, Coras R, Torres A et al. Synovial inflammation in osteoarthritis progression. Nat Rev Rheumatol 2022;18:258–75.

62 Shannon P, Markiel A, Ozier O et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 2003;13:2498–504.

63 Smolen JS, Aletaha D, McInnes IB. Rheumatoid arthritis. Lancet (London, England) 2016;388:2023–38.

64 Sun Y, Li Q, Kirkland JL. Targeting senescent cells for a healthier longevity: the roadmap for an era of global aging. Life Med 2022a;1:103–19.

65 Sun Y, Zhao J, Wu Q et al. Chondrogenic primed extracellular vesicles activate miR-455/SOX11/FOXO axis for cartilage regeneration and osteoarthritis treatment. NPJ Regen Med 2022b;7:53.

66 Tsurumi A, Li WX. Global heterochromatin loss: a unifying theory of aging? Epigenetics 2012;7:680–8.

67 Wang S, Song R, Wang Z et al. S100A8/A9 in Inflammation. Front Immunol 2018;9:1298.

68 Wang C, Shen J, Ying J et al. FoxO1 is a crucial mediator of TGF-beta/TAK1 signaling and protects against osteoarthritis by maintaining articular cartilage homeostasis. Proc Natl Acad Sci U S A 2020;117:30488–97.

69 Wei K, Korsunsky I, Marshall JL et al. Notch signalling drives synovial fibroblast identity and arthritis pathology. Nature 2020;582:259–64.

70 Wu T, Hu E, Xu S et al. clusterProfiler 40: a universal enrichment tool for interpreting omics data. Innovation (Cambridge (Mass)) 2021;2:100141.

71 Yang S, Liu C, Jiang M et al. A single-nucleus transcriptomic atlas of primate liver aging uncovers the pro-senescence role of SREBP2 in hepatocytes. Protein Cell 2023.

72 Yue J, Aobulikasimu A, Sun W et al. Targeted regulation of FoxO1 in chondrocytes prevents age-related osteoarthritis via autophagy mechanism. J Cell Mol Med 2022;26:3075–82.

73 Zhang H, Li J, Ren J et al. Single-nucleus transcriptomic landscape of primate hippocampal aging. Protein Cell 2021;12:695–716.

74 Zhang B, Yan H, Liu X et al. SenoIndex: S100A8/S100A9 as a novel aging biomarker. Life Med 2023a;2.

75 Zhang Y, Zheng Y, Wang S et al. Single-nucleus transcriptomics reveals a gatekeeper role for FOXP1 in primate cardiac aging. Protein Cell 2023b;14:279–93.

76 Zheng J, Wang J, Liu H et al. Alarmins S100A8/A9 promote intervertebral disc degeneration and inflammation-related pain in a rat model through toll-like receptor-4 and activation of the NF-κB signaling pathway. Osteoarthr Cartil 2022;30:998–1011.

77 Zhou Y, Zhou B, Pache L et al. Metascape provides a biologist-oriented resource for the analysis of systemslevel datasets. Nat Commun 2019;10:1523.

78 Zhou S, Liu L, Lu X. Endogenous retroviruses make aging go viral. Life Med 2023;2:lnad001.