Porphyromonas gingivalis-derived extracellular vesicles aggravate bone destruction in rheumatoid arthritis by promoting Syk-dependent osteoclastogenesis

Abstract

Rheumatoid arthritis (RA) is an autoimmune disorder that triggers progressive joint destruction by inducing excessive osteoclastogenesis. Porphyromonas gingivalis (Pg), the main pathogenic bacterium involved in periodontitis (PD), is closely related to RA. Pg can secrete extracellular vesicles (EVs), which carry numerous virulence factors. The aim of this study was to investigate whether Pg-derived EVs can be transported and exacerbate bone destruction in RA by promoting osteoclastogenesis and to elucidate the underlying mechanisms involved. EVs derived from Porphyromonas endodontalis (Pe), which is weakly associated with PD or RA, were used as controls. Pg and Pe EVs interact with osteoclasts after translocating into the marrow and metacarpal joints of mice. In vitro, Pg EVs induce osteoclastogenesis via various components, such as lipopolysaccharide, proteins, lipoproteins, and proteases. TNF-α, IL-1β, and IL-6 promote but cannot independently control Pg EV-induced osteoclastogenesis. RNA sequencing and verification experiments further demonstrated that Pg EVs induced osteoclastogenesis by promoting the phosphorylation of spleen tyrosine kinase (Syk). In vivo, Pg EVs exacerbated RA-induced bone destruction by activating Syk-dependent osteoclastogenesis. R406, a Syk inhibitor, significantly attenuated Pg EV-induced RA osteoclastogenesis and bone destruction. However, Pe-derived EVs presented an extremely weak ability to promote osteoclastogenesis and RA. Our findings reveal a new mechanism by which Pg EVs can exacerbate RA via transport through the circulation and the promote Syk-dependent osteoclastogenesis. This study deepens our understanding of the significant pathogenic role of EVs derived from oral bacterial in RA and explores targeted therapeutic strategies by inhibiting the activation of Syk.

References

[1] Firestein GS. Evolving concepts of rheumatoid arthritis. Nature, 2003, 423: 356-361

[2] Bartold PM, Lopez-Oliva I. Periodontitis and rheumatoid arthritis: an update 2012-2017. Periodontology, 2020, 83: 189-212

[3] Kaur S, White S, Bartold PM. Periodontal disease and rheumatoid arthritis: a systematic review. J. Dent. Res., 2013, 92: 399-408

[4] Okano Tet al.. Caspase-11 mediated inflammasome activation in macrophages by systemic infection of A. actinomycetemcomitans exacerbates arthritis. Int. J. Oral. Sci., 2024, 16: 54

[5] Holt SC, Ebersole JL. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: the “red complex”, a prototype polybacterial pathogenic consortium in periodontitis. Periodontology, 2005, 38: 72-122

[6] Courbon Get al.. Porphyromonas gingivalis experimentally induces periodontis and an anti-CCP2-associated arthritis in the rat. Ann. Rheum. Dis., 2019, 78: 594-599

[7] Munenaga Set al.. The involvement of C5a in the progression of experimental arthritis with Porphyromonas gingivalis infection in SKG mice. Arthritis Res. Ther., 2018, 20: 247

[8] Jeong SHet al.. Interrupting oral infection of Porphyromonas gingivalis with anti-FimA antibody attenuates bacterial dissemination to the arthritic joint and improves experimental arthritis. Exp. Mol. Med., 2018, 50 e460

[9] Martinez-Martinez REet al.. Detection of periodontal bacterial DNA in serum and synovial fluid in refractory rheumatoid arthritis patients. J. Clin. Periodontol., 2009, 36: 1004-1010

[10] Reichert Set al.. Detection of oral bacterial DNA in synovial fluid. J. Clin. Periodontol., 2013, 40: 591-598

[11] Farrugia C, Stafford GP, Murdoch C. Porphyromonas gingivalis outer membrane vesicles increase vascular permeability. J. Dent. Res., 2020, 99: 1494-1501

[12] Okamura Het al.. Outer membrane vesicles of Porphyromonas gingivalis: novel communication tool and strategy. Jpn Dent. Sci. Rev., 2021, 57: 138-146

[13] Amano Aet al.. Genetic characteristics and pathogenic mechanisms of periodontal pathogens. Adv. Dent. Res., 2014, 26: 15-22

[14] Ha JY, Choi SY, Lee JH, Hong SH, Lee HJ. Delivery of periodontopathogenic extracellular vesicles to brain monocytes and microglial IL-6 promotion by RNA cargo. Front. Mol. Biosci., 2020, 7: 596366

[15] Hascoet Eet al.. New insights into inflammatory osteoclast precursors as therapeutic targets for rheumatoid arthritis and periodontitis. Bone Res., 2023, 11: 26

[16] Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature, 2003, 423: 337-342

[17] Akkaoui Jet al.. Contribution of Porphyromonas gingivalis lipopolysaccharide to experimental periodontitis in relation to aging. Geroscience, 2021, 43: 367-376

[18] Olsen I, Nichols FC. Are sphingolipids and serine dipeptide lipids underestimated virulence factors of Porphyromonas gingivalis?. Infect Immun., 2018, 86: e00035-18

[19] Suzuki Y. Porphyromonas gingivalis Fimbriae Induce Osteoclastogenesis via Toll-like Receptors in RAW264 Cells. Int. J. Mol. Sci., 2022, 23: 15293

[20] Lombardo Bedran TBet al.. Porphyromonas endodontalis in chronic periodontitis: a clinical and microbiological cross-sectional study. J. Oral Microbiol., 2012, 4: 10123

[21] Esberg A, Johansson L, Johansson I, Dahlqvist SR. Oral microbiota identifies patients in early onset rheumatoid arthritis. Microorganisms, 2021, 9: 1657

[22] Fleetwood AJet al.. Metabolic remodeling, inflammasome activation, and pyroptosis in macrophages stimulated by porphyromonas gingivalis and its outer membrane vesicles. Front. Cell Infect. Microbiol., 2017, 7: 351

[23] Chen G, Sun Q, Cai Q, Zhou H. Outer membrane vesicles from fusobacterium nucleatum switch M0-like macrophages toward the M1 phenotype to destroy periodontal tissues in mice. Front. Microbiol., 2022, 13: 815638

[24] Toyofuku M, Nomura N, Eberl L. Types and origins of bacterial membrane vesicles. Nat. Rev. Microbiol., 2019, 17: 13-24

[25] Welsh JAet al.. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J. Extracell. Vesicles, 2024, 13 e12404

[26] Zhang Z, Liu D, Liu S, Zhang S, Pan Y. The role of porphyromonas gingivalis outer membrane vesicles in periodontal disease and related systemic diseases. Front. Cell Infect. Microbiol., 2020, 10: 585917

[27] Han ECet al.. Extracellular RNAs in periodontopathogenic outer membrane vesicles promote TNF-alpha production in human macrophages and cross the blood-brain barrier in mice. FASEB J., 2019, 33: 13412-13422

[28] Li Fet al.. Gingipains may be one of the key virulence factors of Porphyromonas gingivalis to impair cognition and enhance blood-brain barrier permeability: an animal study. J. Clin. Periodontol., 2024, 51: 818-839

[29] Seyama Met al.. Outer membrane vesicles of Porphyromonas gingivalis attenuate insulin sensitivity by delivering gingipains to the liver. Biochim. Biophys. Acta Mol. Basis Dis., 2020, 1866: 165731

[30] Kim HY, Song MK, Gho YS, Kim HH, Choi BK. Extracellular vesicles derived from the periodontal pathogen Filifactor alocis induce systemic bone loss through Toll-like receptor 2. J. Extracell. Vesicles, 2021, 10: e12157

[31] Liu Yet al.. Polymicrobial infection induces adipose tissue dysfunction via gingival extracellular vesicles. J. Dent. Res., 2024, 103: 187-196

[32] Reyes L, Getachew H, Dunn WA, Progulske-Fox A. Porphyromonas gingivalis W83 traffics via ICAM1 in microvascular endothelial cells and alters capillary organization in vivo. J. Oral. Microbiol., 2020, 12: 1742528

[33] Niu C, Huang J, Wei L, Wang J, Ran S. Proinflammatory effect of membrane vesicles derived from Enterococcus faecalis at neutral and alkaline pH. J. Endod., 2024, 50: 1602-1611.e1610

[34] Furuta Net al.. Porphyromonas gingivalis outer membrane vesicles enter human epithelial cells via an endocytic pathway and are sorted to lysosomal compartments. Infect. Immun., 2009, 77: 4187-4196

[35] O’Donoghue EJ, Krachler AM. Mechanisms of outer membrane vesicle entry into host cells. Cell Microbiol., 2016, 18: 1508-1517

[36] Kassem Aet al.. Porphyromonas gingivalis Stimulates Bone Resorption by Enhancing RANKL (Receptor Activator of NF-kappaB Ligand) through Activation of Toll-like Receptor 2 in Osteoblasts. J. Biol. Chem., 2015, 290: 20147-20158

[37] Wielento Aet al.. TLR2 activation by Porphyromonas gingivalis requires both PPAD activity and fimbriae. Front. Immunol., 2022, 13: 823685

[38] Kim HYet al.. Effects of extracellular vesicles derived from oral bacteria on osteoclast differentiation and activation. Sci. Rep., 2022, 12 14239

[39] Kim Jet al.. Lipoproteins are an important bacterial component responsible for bone destruction through the induction of osteoclast differentiation and activation. J. Bone Min. Res., 2013, 28: 2381-2391

[40] Yoshitaka Tet al.. Enhanced TLR-MYD88 signaling stimulates autoinflammation in SH3BP2 cherubism mice and defines the etiology of cherubism. Cell Rep., 2014, 8: 1752-1766

[41] Aoki Y, Tabeta K, Murakami Y, Yoshimura F, Yamazaki K. Analysis of immunostimulatory activity of Porphyromonas gingivalis fimbriae conferred by Toll-like receptor 2. Biochem Biophys. Res. Commun., 2010, 398: 86-91

[42] Jain S, Coats SR, Chang AM, Darveau RP. A novel class of lipoprotein lipase-sensitive molecules mediates Toll-like receptor 2 activation by Porphyromonas gingivalis. Infect. Immun., 2013, 81: 1277-1286

[43] Chow YCet al.. Implications of Porphyromonas gingivalis peptidyl arginine deiminase and gingipain R in human health and diseases. Front. Cell Infect. Microbiol., 2022, 12: 987683

[44] Qiu Qet al.. Porphyromonas gingivalis extracellular vesicles exacerbated osteoporosis by disrupting osteoblast mitochondrial dynamics and inhibiting Cpt2-regulated fatty acid oxidation. J. Nanobiotechnol., 2025, 23 635

[45] Akiyama Tet al.. Porphyromonas gingivalis-derived lysine gingipain enhances osteoclast differentiation induced by tumor necrosis factor-alpha and interleukin-1beta but suppresses that by interleukin-17A: importance of proteolytic degradation of osteoprotegerin by lysine gingipain. J. Biol. Chem., 2014, 289: 15621-15630

[46] Sabokbar A, Mahoney DJ, Hemingway F, Athanasou NA. Non-canonical (RANKL-Independent) pathways of osteoclast differentiation and their role in musculoskeletal diseases. Clin. Rev. Allergy Immunol., 2016, 51: 16-26

[47] Pan W, Wang Q, Chen Q. The cytokine network involved in the host immune response to periodontitis. Int. J. Oral Sci., 2019, 11: 30

[48] Chen LL, Yan J. Porphyromonas gingivalis lipopolysaccharide activated bone resorption of osteoclasts by inducing IL-1, TNF, and PGE. Acta Pharm. Sin., 2001, 22: 614-618

[49] Iwata Set al.. Activation of Syk in peripheral blood B cells in patients with rheumatoid arthritis: a potential target for abatacept therapy. Arthritis Rheumatol., 2015, 67: 63-73

[50] Gyori DS, Mocsai A. Osteoclast signal transduction during bone metastasis formation. Front. Cell Dev. Biol., 2020, 8: 507

[51] Mocsai A, Ruland J, Tybulewicz VL. The SYK tyrosine kinase: a crucial player in diverse biological functions. Nat. Rev. Immunol., 2010, 10: 387-402

[52] Mueller Het al.. Tyrosine kinase Btk regulates E-selectin-mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) gamma2 and PI3Kgamma pathways. Blood, 2010, 115: 3118-3127

[53] Braselmann Set al.. R406, an orally available spleen tyrosine kinase inhibitor blocks FC receptor signaling and reduces immune complex-mediated inflammation. J. Pharm. Exp. Ther., 2006, 319: 998-1008

[54] Fan Z, Su D, Li ZC, Sun S, Ge Z. Metformin attenuates central sensitization by regulating neuroinflammation through the TREM2-SYK signaling pathway in a mouse model of chronic migraine. J. Neuroinflammation, 2024, 21 318

[55] Connell NT, Berliner N. Fostamatinib for the treatment of chronic immune thrombocytopenia. Blood, 2019, 133: 2027-2030

[56] Tanaka Y, Millson D, Iwata S, Nakayamada S. Safety and efficacy of fostamatinib in rheumatoid arthritis patients with an inadequate response to methotrexate in phase II OSKIRA-ASIA-1 and OSKIRA-ASIA-1X study. Rheumatology, 2021, 60: 2884-2895

[57] Yamasaki Tet al.. The dectin 1 agonist curdlan regulates osteoclastogenesis by inhibiting nuclear factor of activated T cells cytoplasmic 1 (NFATc1) through Syk kinase. J. Biol. Chem., 2014, 289: 19191-19203

[58] Wang Let al.. Gain-of-function variants in SYK cause immune dysregulation and systemic inflammation in humans and mice. Nat. Genet., 2021, 53: 500-510

[59] Li SLet al.. Peroxiredoxin 1 aggravates acute kidney injury by promoting inflammation through Mincle/Syk/NF-KB signaling. Kidney Int., 2023, 104: 305-323

[60] Gravallese EMet al.. Synovial tissue in rheumatoid arthritis is a source of osteoclast differentiation factor. Arthritis Rheum., 2000, 43: 250-258

[61] Komatsu N, Takayanagi H. Mechanisms of joint destruction in rheumatoid arthritis - immune cell-fibroblast-bone interactions. Nat. Rev. Rheumatol., 2022, 18: 415-429

[62] Takayanagi Het al.. T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. Nature, 2000, 408: 600-605

[63] Meednu Net al.. Production of RANKL by memory B cells: a link between B Cells and bone erosion in rheumatoid arthritis. Arthritis Rheumatol., 2016, 68: 805-816

[64] Mekata Met al.. Porphyromonas gingivalis outer membrane vesicles increase vascular permeability by inducing stress fiber formation and degrading vascular endothelial-cadherin in endothelial cells. FEBS J., 2025, 292: 1696-1709

[65] Huang Set al.. Porphyromonas gingivalis outer membrane vesicles exacerbate retinal microvascular endothelial cell dysfunction in diabetic retinopathy. Front. Microbiol., 2023, 14: 1167160

[66] Luan Jet al.. Applicability and implementation of the collagen-induced arthritis mouse model, including protocols (Review). Exp. Ther. Med., 2021, 22: 939

[67] Hayer Set al.. ‘SMASH’ recommendations for standardised microscopic arthritis scoring of histological sections from inflammatory arthritis animal models. Ann. Rheum. Dis., 2021, 80: 714-726

Funding

National Natural Science Foundation of China (National Science Foundation of China)(81870752)

China Postdoctoral Science Foundation(2021MD703905)

Natural Science Foundation of Liaoning Province (Liaoning Provincial Natural Science Foundation)(2023-BS-097)

RIGHTS & PERMISSIONS

The Author(s)