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Dendritic Cell Derived Exosomes Loaded with Immunoregulatory Cargo Reprogram local Immune Responses and Inhibit Degenerative Bone Disease In vivoElashiry, Mahmoud; Biomedical Sciences (Augusta University, 2020-12)Background: Histopathological study of periodontitis (PD) lesions at sites of bone loss reveals infiltration with dendritic cells (DC) CD4+ T cell clusters and other inflammatory cells. DCs can direct bone protective T-regulatory cell (Tregs) responses, or bone destructive T-helper 17 (Th17). The use of exosomes (EXO), natural nanoparticles released by DCs and other cells, are under intense scrutiny in clinical trials for autoimmune diseases and cancer, but no studies to date have harnessed DC-derived EXO to regulate alveolar bone loss in PD. Aim: To determine the ability of custom DC-derived EXO to reprogram immune cell functions of recipient DCs and T cells and mitigate inflammatory bone loss in mice. Methods: Murine bone marrow derived donor DC subtypes, including immune regulatory DCs (regDC), immature DCs (iDC) and immune stimulatory (stimDC) DCs were the source of purified DC EXO. Reg DC EXO were actively loaded with TGFB1/IL10 using ultrasonication. Preliminary in vitro studies of EXO cargo, stability and resistance of cytokine cargo to proteolysis, as well as immune functions and osteoclastogenesis was investigated. The following DC EXO subtypes were then tested in vivo in six groups of mice, in the ligature induced PD model: Group 1, no ligature, Groups 2, 3, 4, 5 and 6, 8 ligature plus gingival injection of, respectively, PBS, regDC EXO, iDC EXO, stimDC EXO and free TGFB1/IL10. Biodistribution and in vivo uptake of EXO by gingival recipient DCs and T cells were tracked. The ability of DC EXO to modulate gingival recipient DC and CD4 T cells and cytokine expression was confirmed. TRAP staining of histological sections measured osteoclast number, while bone loss volume was measured in 3-D by micro-CT. Results: Injected EXO showed a high affinity for gingival site of inflammatory bone loss. RegDC EXO containing TGFb/IL-10 cargo, protected cargo against proteolytic degradation and were taken up by recipient DCs and T cells in vivo, promoting Tregs, while inhibiting Th17 recruitment and inhibiting bone loss. In contrast, EXO subtypes lacking TGFb/IL-10 or free TGFB/IL-10 did not shift the Treg-Th17 balance and did not inhibit bone loss. Mechanistically, a key role for TGFb1 in induction of Tregs by regDC EXO was found using blocking antibodies to TGFb and/or IL-10. T.E.M. analysis revealed TGFb1 localized in the EXO lumen and in the transmembrane domain, which sustained signaling in recipient DCs. Blocking experiments revealed that sustainable prolonged TGFb1 signaling required initial interaction between regDCs EXO and TGFBR1 complex on acceptor cells, followed by internalization of regDC EXO with TGFB1-TGFBR1 complex for sustained SMAD2/3 phosphorylation. Conclusion: This is the first study to demonstrate the efficacy of DCs exosomes for inhibition of experimental bone loss and the cellular immune mechanisms involved. This provides the basis for a future novel immunotherapeutic strategy for PD in humans.