The Therapeutic Effect Of Exosomes With Membrane-associated TGF-β1from Genetically Modified Immature Dendritic Cells Inhibit EAE And The Underlying Mechanisms

DCs are unique professional APCs and important for the initiation and regulation of immune response. Immature DCs have lower levels of co-stimulatory molecules and usually induce antigen-specific T cell anergy. TGF-β1is a negative regulator of pro-inflammatory immune responses. Our previous study found that systemic administration of TGF-β1genetically modified immature DCs retarded the development of dextran sulfate sodium induced IBD in mice. Therefore, TGF-β1may modify DCs toward to regulatory DCs, inhibiting inflammation.Exosomes are small lipid bilayer vesicles with a size of50to100nm and released by almost all types of cells. They are formed by membrane budding into the lumen of an endocytic compartment, leading to the formation of multivesicular bodies. Fusion of multivesicular bodies to the plasma membrane leads to the extracellular release of exosomes. Exosomes have diverse functions, depending on the source. Exosomes from tumor cells can induce tumor-specific immunity to eradicate an established tumor in mice. In contrast, exosomes from highly metastatic melanomas promote tumor metastasis. Exosomes from gene modified parent cells may change their components and function. Indeed, exosomes from the DCs that have been transfected with the vIL-10, Fas L or IL-4gene inhibit delayed-type hypersensitivity and collagen-induced arthritis in mice. These results indicate that exosomes can be an effective vehicle to carry immunoregulatory molecules for the treatment of autoimmune diseases.Antigen-specific Thl and Th17cells are crucial for the development of tissue-specific autoimmune diseases, such as IBD, type1diabetes and EAE. In addition, a reduced frequency and activity of Tregs are associated with the development of autoimmune diseases. Hence, down-regulation of pro-inflammatory Thl and Thl7, and promotion of Treg responses can inhibit the development and progression of autoimmune diseases. Our previous study has shown that treatment with sTGF-β1-EXO attenuates Th17responses, but promotes Treg responses, leading to inhibition of IBD development in mice.However, the same treatment does not significantly modulate the progression of IBD in mice after onset of IBD. Given that the secreted cytokines are like to be packed in the exosomes, which may impair its function. We hypothesized that exosomes containing membrane-associated TGF-β1should have stronger activity in inhibiting pro-inflammatory T cell autoimmunity and inducing Treg responses. In this study, we generated recombinant adenovirus (Ad) that expressed membrane-associated TGF-β1(Ad mTGF-β1) and isolated mTGF-β1-EXO from Ad mTGF-β1-infected DCs derived from C57BL/6mice. Our results show that treatment of mice with mTGF-β1-EXO impaired antigen-specific Thl responses and IFN-y production, but promoted IL-10responses ex vivo. Treatment with mTGF-β1-EXO decreased the frequency of Th17cells in EAE mice, which might be associated with the down-regulation of p38, ERK, Stat3and NF-κB activation and IL-6expression in DCs. Treatment with mTGF-β1-EXO increased the frequency of splenic and inguinal lymph node Tregs, and adoptive transfer of CD4+CD25+T cells from the mTGF-β1-EXO-treated EAE mice dramatically prevented the development of EAE in the recipients. Moreover, treatment with mTGF-β1-EXO from C57BL/6mice effectively inhibited the proliferation of T cells from BALB/c mice and proteolipid protein (PLP) peptide-induced EAE in BALB/c mice. These indicate that mTGF-β1-EXO possess more powerful immunosuppressive ability and can effectively inhibit the development and progression of EAE in different strains of mice. Our findings may provide a basis for the design of new immunotherapies for autoimmune diseases.