Regulatory T cell (Treg)‐derived extracellular vesicles (EVs) represent a contact‐independent mechanism by which Tregs suppress dysregulated immune responses. These EVs carry diverse immunomodulatory molecules, including CD73, an ectoenzyme that hydrolyses AMP into adenosine. Adenosine subsequently acts as a potent immunosuppressive mediator that inhibits effector CD4⁺ T cell activation and controls pathological inflammation. Periodontitis is a highly prevalent inflammatory disease characterised by the accumulation of IL‐17A‐expressing CD4⁺ T cells in response to dysbiotic oral bacterial biofilms, ultimately leading to RANKL‐mediated alveolar bone resorption and tooth loss. We tested the hypothesis that CD73⁺ Treg‐derived EVs, isolated from Tregs induced with polarising cytokines in the presence of retinoic acid, could limit inflammation and prevent alveolar bone loss in periodontitis. Our findings demonstrate that Tregs induced with polarising cytokines in the presence of retinoic acid express high levels of CD73 and secrete adenosine‐producing suppressive CD73 ⁺ EVs. Furthermore, local administration of these CD73⁺ Treg‐derived EVs in a murine periodontitis model reduced activated CD4⁺ T cell infiltration, decreased IL‐17A and RANKL expression, and attenuated osteoclast‐mediated alveolar bone loss. In conclusion, retinoic acid‐induced Treg‐derived EVs suppress CD4⁺ T cell‐driven inflammation and ameliorate periodontitis, at least in part through CD73/adenosine‐dependent immunomodulatory mechanisms.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1β, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1β, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

Periodontitis is a chronic inflammatory disease characterized by alveolar bone loss driven by dysregulated immune responses. We previously showed that extracellular vesicles derived from retinoic acid-induced regulatory T lymphocytes (RA-Treg EVs) suppress pathogenic CD4⁺ T-lymphocyte responses and reduce alveolar bone loss during periodontitis. Herein, we investigated whether RA-Treg EVs also modulate macrophage and CD8⁺ T-lymphocyte responses during experimental periodontitis. Ligature-induced periodontitis was generated in mice, followed by local administration of RA-Treg EVs. Alveolar bone loss was analyzed by micro-computed tomography, and periodontal tissues and cervical lymph nodes were analyzed by flow cytometry to quantify antigen-presenting cells, macrophages, macrophage subsets, and CD8⁺ T lymphocytes. The direct effects of RA-Treg EVs on macrophage phenotype and CD8⁺ T-cell proliferation and activation were assessed in vitro. RA-Treg EV treatment attenuated alveolar bone loss and preserved trabecular microarchitecture. This effect was associated with reduced macrophage infiltration into periodontal tissues, modulation of macrophage polarization, and restoration of CD8⁺ T-cell abundance in periodontal tissues and draining cervical lymph nodes, without major changes in CD8⁺IFN-γ⁺ or CD8⁺RANKL⁺ cells. In vitro, RA-Treg EVs induced heterogeneous macrophage phenotypes distinct from the classical M1/M2 polarization states while markedly enhancing CD8⁺ T-cell proliferation and activation. These findings indicate that RA-Treg EVs preserve alveolar bone during experimental periodontitis while selectively modulating macrophage and CD8⁺ T-lymphocyte responses.