CCR2 Orchestrates Preferential Homing and Therapeutic Efficacy of Gingival Mesenchymal Stem Cell-Derived Extracellular Vesicles in Rheumatoid Arthritis

Abstract

The clinical utility of mesenchymal stem cells (MSCs) is often limited by pulmonary entrapment and poor systemic distribution, particularly in diseases constrained by physiological barriers such as rheumatoid arthritis (RA), where joint accessibility restricts therapeutic efficacy. This study systematically compares the immunomodulatory capacity and inflammation-targeting potential of human gingiva-derived MSCs (GMSCs) and their extracellular vesicles (GMSC-EVs) in vivo. Using an experimental RA model, we demonstrate that GMSC-EVs exhibit superior tropism to inflamed joints compared to GMSCs, resulting in significantly greater amelioration of disease severity, including reduced joint swelling, bone destruction, and balanced pathogenic T-cell responses. Mechanistically, we identify C-C chemokine receptor type 2 (CCR2) as the critical molecular driver of this targeted homing. Genetic ablation of CCR2 via CRISPR-Cas9/sgRNA knockdown abolishes both the joint-specific accumulation of GMSC-EVs and their therapeutic efficacy. These findings elucidate the molecular basis for GMSC-EVs tropism to arthritic lesions and establish CCR2 as a pivotal target for developing precision-engineered EVs therapies with enhanced specificity for RA treatment.

Keywords

C-C chemokine receptor type 2 / chemotaxis / rheumatoid arthritis / extracellular vesicles / gingiva-derived mesenchymal stem cells / homing / inflammation

References

[1] Z. SzekaneczA and E. Koch, “Successes and Failures of Chemokine-Pathway Targeting in Rheumatoid Arthritis,” Nature Reviews Rheumatology 12, no. 1 (2016): 5–13.

[2] K. English, “Mechanisms of Mesenchymal Stromal Cell Immunomodulation,” Immunology and Cell Biology 91, no. 1 (2013): 19–26.

[3] R. L. Kadle, S. A. Abdou, A. P. Villarreal-Ponce, et al., “Microenvironmental Cues Enhance Mesenchymal Stem Cell-Mediated Immunomodulation and Regulatory T-Cell Expansion,” PLoS ONE 13, no. 3 (2018): e0193178.

[4] Q. Zhang, S. Shi, Y. Liu, et al., “Mesenchymal Stem Cells Derived From Human Gingiva Are Capable of Immunomodulatory Functions and Ameliorate Inflammation-Related Tissue Destruction in Experimental Colitis,” Journal of Immunology 183, no. 12 (2009): 7787–7798.

[5] G. B. Tomar, R. K. Srivastava, N. Gupta, et al., “Human Gingiva-Derived Mesenchymal Stem Cells Are Superior to Bone Marrow-Derived Mesenchymal Stem Cells for Cell Therapy in Regenerative Medicine,” Biochemical and Biophysical Research Communications 393, no. 3 (2010): 377–383.

[6] Q. Z. Zhang, W. R. Su, S. H. Shi, et al., “Human Gingiva-Derived Mesenchymal Stem Cells Elicit Polarization of m2 Macrophages and Enhance Cutaneous Wound Healing,” Stem Cells 28, no. 10 (2010): 1856–1868.

[7] W. R. Su, Q. Z. Zhang, S. H. Shi, et al., “Human Gingiva-Derived Mesenchymal Stromal Cells Attenuate Contact Hypersensitivity via Prostaglandin E2-Dependent Mechanisms,” Stem Cells 29, no. 11 (2011): 1849–1860.

[8] H. Li, Y. Deng, J. Liang, et al., “Mesenchymal Stromal Cells Attenuate Multiple Sclerosis via IDO-Dependent Increasing the Suppressive Proportion of CD5+ IL-10+ B Cells,” American Journal of Translational Research 11, no. 9 (2019): 5673–5688.

[9] J. Zhao, J. Chen, F. Huang, et al., “Human Gingiva Tissue-Derived MSC Ameliorates Immune-Mediated Bone Marrow Failure of Aplastic Anemia via Suppression of Th1 and Th17 Cells and Enhancement of CD4+ Foxp3+ Regulatory T Cells Differentiation,” American Journal of Translational Research 11, no. 12 (2019): 7627–7643.

[10] W. Wu, Z. Xiao, Y. Chen, et al., “CD39 Produced From Human GMSCs Regulates the Balance of Osteoclasts and Osteoblasts Through the Wnt/β-Catenin Pathway in Osteoporosis,” Molecular Therapy 28, no. 6 (2020): 1518–1532.

[11] L. Zangi, R. Margalit, S. Reich-Zeliger, et al., “Direct Imaging of Immune Rejection and Memory Induction by Allogeneic Mesenchymal Stromal Cells,” Stem Cells 27, no. 11 (2009): 2865–2874.

[12] S. Schrepfer, T. Deuse, H. Reichenspurner, et al., “Stem Cell Transplantation: The Lung Barrier,” Transplantation Proceedings 39, no. 2 (2007): 573–576.

[13] P. D. RobbinsA and E. Morelli, “Regulation of Immune Responses by Extracellular Vesicles,” Nature Reviews Immunology 14, no. 3 (2014): 195–208.

[14] T. Zhang, L. Zhang, X. Ma, et al., “The Tiny Giants of Regeneration: MSC-Derived Extracellular Vesicles as Next-Generation Therapeutics,” Frontiers in Cell and Developmental Biology 13 (2025): 1612589.

[15] T. O. Klyucherev, M. A. Peshkova, M. D. Yurkanova, et al., “Advances in Regenerative Therapies for Inflammatory Arthritis: Exploring the Potential of Mesenchymal Stem Cells and Extracellular Vesicles,” International Journal of Molecular Sciences 26, no. 12 (2025): 5766.

[16] G. S. Teo, Z. Yang, C. V. Carman, et al., “Intravital Imaging of Mesenchymal Stem Cell Trafficking and Association With Platelets and Neutrophils,” Stem Cells 33, no. 1 (2015):265–277.

[17] C. R. Harrell, C. Fellabaum, N. Jovicic, et al., “Molecular Mechanisms Responsible for Therapeutic Potential of Mesenchymal Stem Cell-Derived Secretome,” Cells 8, no. 5 (2019): 467.

[18] N. Milman and L. GininiZ Gil, “Exosomes and Their Role in Tumorigenesis and Anticancer Drug Resistance,” Drug Resistance Updates 45 (2019): 1–12.

[19] M. Gazdic, V. Volarevic, N. Arsenijevic, et al., “Mesenchymal Stem Cells: A Friend or Foe in Immune-mediated Diseases,” Stem Cell Reviews and Reports 11, no. 2 (2015): 280–287.

[20] E. Eggenhofer, F. Luk, M. H. Dahlke, et al., “The Life and Fate of Mesenchymal Stem Cells,” Frontiers in immunology 5 (2014): 148.

[21] A. SohniC M Verfaillie, “Mesenchymal Stem Cells Migration Homing and Tracking,” Stem Cells International 2013 (2013): 130763.

[22] M. Honczarenko, Y. Le, M. Swierkowski, et al., “Human Bone Marrow Stromal Cells Express a Distinct Set of Biologically Functional Chemokine Receptors,” Stem Cells 24, no. 4 (2006): 1030–1041.

[23] L. A. Marquez-Curtis and A. Janowska-Wieczorek, “Enhancing the Migration Ability of Mesenchymal Stromal Cells by Targeting the SDF-1/CXCR4 Axis,” BioMed Research International 2013 (2013): 561098.

[24] M. Chen, W. Su, X. Lin, et al., “Adoptive Transfer of Human Gingiva-Derived Mesenchymal Stem Cells Ameliorates Collagen-Induced Arthritis via Suppression of Th1 and Th17 Cells and Enhancement of Regulatory T Cell Differentiation,” Arthritis and Rheumatism 65, no. 5 (2013): 1181–1193.

[25] Y. Luo, W. Wu, J. Gu, et al., “Human Gingival Tissue-Derived MSC Suppress Osteoclastogenesis and Bone Erosion via CD39-Adenosine Signal Pathway in Autoimmune Arthritis,” EBioMedicine 43 (2019): 620–631.

[26] Y. J. LiZ and Y. Chen, “Cell-Based Therapies for Rheumatoid Arthritis: Opportunities and Challenges,” Therapeutic Advances in Musculoskeletal Disease 14, no. 2022: 1759720×221100294.

[27] Y. Shimizu, E. H. Ntege, C. Azuma, et al., “Management of Rheumatoid Arthritis: Possibilities and Challenges of Mesenchymal Stromal/Stem Cell-Based Therapies,” Cells 12, no. 14 (2023): 1905.

[28] C. R. Harrell, M. G. Jankovic, C. Fellabaum, et al., “Molecular Mechanisms Responsible for Anti-Inflammatory and Immunosuppressive Effects of Mesenchymal Stem Cell-Derived Factors,” Advances in Experimental Medicine and Biology 1084 (2019): 187–206.

[29] C. Grange, M. Tapparo, S. Bruno, et al., “Biodistribution of Mesenchymal Stem Cell-Derived Extracellular Vesicles in a Model of Acute Kidney Injury Monitored by Optical Imaging,” International Journal of Molecular Medicine 33, no. 5 (2014): 1055–1063.

[30] L. Hejretová, M. Čedíková, M. Dolejšová, et al., “Comparison of the Immunomodulatory Effect of Single MSC Batches Versus Pooled MSC Products,” Cell and Tissue Banking 21, no. 1 (2020): 119–129.

[31] D. Kaplan, E. Christian, S. P. Pope, et al., “Analyte Heterogeneity Analysis as a Possible Potency Parameter for MSC,” Best Practice & Research, Clinical Haematology 37, no. 4 (2024): 101596.

[32] J. Talbot, F. J. Bianchini, D. C. Nascimento, et al., “CCR2 Expression in Neutrophils Plays a Critical Role in Their Migration into the Joints in Rheumatoid Arthritis,” Arthritis & Rheumatology 67, no. 7 (2015): 1751–1759.

[33] B. Wang, B. H. Zinselmeyer, H. A. Runnels, et al., “In Vivo Imaging Implicates CCR2(+) Monocytes as Regulators of Neutrophil Recruitment During Arthritis,” Cellular Immunology 278, no. 1–2 (2012): 103–112.

[34] J. Gan, X. Zhang, G. Chen, et al., “CXCR4-Expressing Mesenchymal Stem Cells Derived Nanovesicles for Rheumatoid Arthritis Treatment,” Advanced Healthcare Materials 13, no. 9 (2024): e2303300.

[35] W. Wang, Z. Deng, G. Liu, et al., “Platelet-Derived Extracellular Vesicles Promote the Migration and Invasion of Rheumatoid Arthritis Fibroblast-Like Synoviocytes via CXCR2 Signaling,” Experimental and Therapeutic Medicine 22, no. 4 (2021): 1120.

[36] S. Li, L. Ding, Z. Kong, et al., “Elucidating the Molecular Mechanisms of CCR5-Containing Extracellular Vesicles in Vitro and in a Rat Model of Experimental Rheumatoid Arthritis,” International Immunopharmacology 146 (2025): 113869.

[37] C. Lechanteur, A. Briquet, V. Bettonville, et al., “MSC Manufacturing for Academic Clinical Trials: From a Clinical-Grade to a Full GMP-Compliant Process,” Cells 10, no. 6 (2021): 1320.

[38] J. Chen, F. Huang, Y. Hou, et al., “TGF-Beta-Induced CD4+ FoxP3+ Regulatory T Cell-Derived Extracellular Vesicles Modulate Notch1 Signaling Through miR-449a and Prevent Collagen-Induced Arthritis in a Murine Model,” Cellular & Molecular Immunology 18, no. 11 (2021): 2516–2529.

[39] F. Huang, M. Chen, W. Chen, et al., “Human Gingiva-Derived Mesenchymal Stem Cells Inhibit Xeno-Graft-Versus-Host Disease via CD39-CD73-Adenosine and IDO Signals,” Frontiers in Immunology 8 (2017): 68.

[40] W. Fu, C. Lei, S. Liu, et al., “CAR Exosomes Derived From Effector CAR-T Cells Have Potent Antitumour Effects and Low Toxicity,” Nature Communications 10, no. 1 (2019): 4355.

[41] J. Chen, X. Shi, Y. Deng, et al., “miRNA-148a-Containing GMSC-Derived EVs Modulate Treg/Th17 Balance via IKKB/NF-κB Pathway and Treat a Rheumatoid Arthritis Model,” JCI Insight 9, no. 10 (2024): e177841.

[42] P. Lai, X. Chen, L. Guo, et al., “A Potent Immunomodulatory Role of Exosomes Derived From Mesenchymal Stromal Cells in Preventing cGVHD,” Journal of Hematology & Oncology 11, no. 1 (2018): 135.

[43] D. D. Brand, K. A. Latham, and E. F. Rosloniec, “Collagen-Induced Arthritis,” Nature Protocols 2, no. 5 (2007): 1269–1275.

[44] N. Kong, Q. Lan, W. Su, et al., “Induced T Regulatory Cells Suppress Osteoclastogenesis and Bone Erosion in Collagen-Induced Arthritis Better Than Natural T Regulatory Cells,” Annals of the Rheumatic Diseases 71, no. 9 (2012): 1567–1572.

[45] N. Kong, Q. Lan, M. Chen, et al., “Antigen-Specific Transforming Growth Factor β-Induced Treg Cells, but Not Natural Treg Cells, Ameliorate Autoimmune Arthritis in Mice by Shifting the Th17/Treg Cell Balance From Th17 Predominance to Treg Cell Predominance,” Arthritis and Rheumatism 64, no. 8 (2012): 2548–2558.

[46] L. Lu, Q. Lan, Z. Li, et al., “Critical Role of All-trans Retinoic Acid in Stabilizing Human Natural Regulatory T Cells Under Inflammatory Conditions,” PNAS 111, no. 33 (2014): E3432–E3440.

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