Effects Of NF-κB ODN Decoy-induced Bone Marrow-derived Tolerogenic DCs On T Cell Subsets In CIA Rats

Objective:To establish tolerogenic dendritic cells(DCs)induced by NF-κB ODN Decoy and expore the stability in vitro and effects on T cell subsets in CIA rats.Methods:Rat bone marrow-derived rat dendritic cells were generated in the presence of granulocyte-macrophage colony stimulating factor(GM-CSF)and interleukin 4(IL-4)and induced into tolerogenic DCs by NF-κB ODN Decoy.The morphology of DCs were observed by giemsa staining and cell viability was observed by staining with placenta blue.Flow cytometry was used to detect the expression of rat dendritic cells specific maker OX-62 and costimulatory molecules CD80 and CD86 on the surface of DCs to determine the purity and maturity of DCs.We conducted mixed lymphocyte response(MLR)to assess the ability of tolerogenic DCs for T cells proliferation.NF-κB ODN Decoy induced tolerogenic DCs were applied to CIA rats by tail vein on the 19th day after the primary immunization.Joint swelling was observed and arthritis index score was obtained;On the 42nd day of primary immunization,HE staining was performed on the pathological sections of the joints to compare the pathological changes of the joints and flow cytometry was used to analyze the changes of Th1(CD3~+CD8~-IFN-γ~+),Th2(CD3~+CD8~-IL-4~+),Th17(CD3~+CD8~-IL-17~+)and Treg(CD3~+CD8~-Foxp3~+)subsets in spleen.Results:The viability of both NF-κB ODN Decoy induced DC and control group were more than95%with no difference.Compared with control group,the tolerogenic DCs were with shorter and thicker hair-like projections on the cell surface.The percentage of OX-62positive cells were both above 95%,indicating that all the DCs we generated were with high purity.The expression of costimulatory molecules CD80 and CD86 were both much higher than control group with or without LPS stimulation.Tolerogenic DCs have lower ability to stimulate allogeneic lymphocytes.In CIA rat model,the tolerogenic DCs treated group exhibited less severity regarding to arthritis phenotype.Furthermore,Th1 and Th17 cells populations were decreased while Th2 and Treg cells were increased in rats with tolerogenic DCs treatment.Conclusions:Dendritic cells(DCs)induced by NF-κB ODN Decoy were featured with commendable stability and tolerance.Tolerogenic dendritic cells carrying bovine type II collagen could suppress the autoimmune inflammatory response by regulating the T cell subsets,at least to some degree,attenuating CIA in rat.