Construction And Application Of Tolerant DC Induced By NF-κB ODN Decoy In CIA Rat

Objective:To construct stable and tolerant dendritic cells derived from spleen in the process of collagen-induced arthritis(CIA)in rats using NF-κB decoy strategy;And using NF-κB ODN Decoy to induce BIIC-loaded DCs derived from the spleen of CIA rats and normal rats,which interferes with CIA rats and allogeneic CIA rats during the course of disease in vivo and observes the effect of its intervention,which seeks new targets and strategies for the treatment of rheumatoid arthritis(RA).Methods:SD rats were used as experimental animals.1.Splenic mononuclear cells from normal rats and CIA ratsr espectively were induced to transform into DC with GM-CSF and IL-4,and then which tolerance was induced by NF-κB ODN Decoy.It is divided into six groups according to the source of cell and the method of treatment,including DC group of normal rats(normal-Control-DC),decoy-induced DC in normal rats(normal-Decoy-DC),DC group of CIA rats(CIA-Control-DC),decoy-induced DC in CIA rats(CIA-Decoy-DC),LPS stimulated Decoy-DC group in CIA rats(CIA-LPS-Decoy-DC)and LPS stimulated DC group in CIA rats(CIA-LPS-DC).Observation of biological characters in each group:Morphological characteristics of cells observed by phase contrast microscope or Rayon staining;Trypan blue staining method for evaluating cell viability;DC specific markers of rat OX-62 were detected by flow cytometry to evaluate the purity of DC.At the same time,the expression of CD80 and CD86 was detected to evaluate the maturity of DC.Allogeneic mixed lymphocyte reaction was observed to promote T cell proliferation and detect IFN-γand IL-10 levels in cell culture supernatant,and to evaluate the inhibitory effect of NF-κB ODN Decoy on DC maturation.2.(1)Decoy-DC loaded with BIIC derived from CIA rats and normal rats was used to treat CIA rats during the course of disease in vitro.(2)Adult SD rats were injected intradermally with Bovine collagen type II(BIIC)and Freund’s complete adjuvant in a 1:1 emulsion and boosted by subcutaneous injection at two-week intervals to form collagen-induced arthritis.(3)On the 13th day after the primary immunization,half a spleen of SD rats was aseptically harvested and used to extract mononuclear cells to induce tolerogenic DCs.(4)On the 20th day after the initial immunization,DCs(5×10~6)were treated by tail vein injection in each group of experimental rats.The experimental rats were divided into six groups,including blank control group,CIA model group,BⅡC-Decoy DC group of CIA rats,BⅡC-Decoy DC control group of CIA rats,BⅡC-Decoy DC group of normal rats and BⅡC-Decoy DC control group of normal rats,there were eight rats in each group.5.Observed indicators and tests:From the beginning of modeling,the degree of joint swelling in rats was observed and the arthritis index(AI)was calculated weekly.At the 42nd day of the initial immunization,the rats were weighed and sacrificed.Ankle joints were examined by pathology and serum IFN-γand IL-10 levels were measured by ELISA.Results:1.(1)The activity of DC in each group was>90%;The expression rate of the rat specific marker OX-62was>55%.In group normal-Control-DC and CIA-Control-DC,the cell morphology was irregular,and the perimeter appeared more hairy protuberances.After the addition of LPS,there was a hair like increase around the cells.Normal-Decoy-DC group,CIA-Decoy-DC group and CIA-Decoy-DC group added with LPS were similar in shape,with less hairlike protrusions around the cells.(2)In normal rats and CIA rats,compared with Control-DC group and LPS-DC group,the positive expression rate and average fluorescence intensity of costimulatory molecules CD80 and CD86 in Decoy-DC group were significantly decreased(P<0.05);In the CIA rats,compared with the Decoy-DC group,the positive expression rate and and average fluorescence intensity of CD80 and CD86 in the Decoy-DC group were not significantly increased after LPS stimulation(P>0.05).(3)DCs from spleen of normal rats and CIA rats showed morphological similarities and no significant differences in the expression of co-stimulatory molecules CD80 and CD86.(4)In normal rats and CIA rats,compared with Control-DC group,the ability of decoy-DC to stimulate lymphocyte proliferation was significantly lower(P<0.05).At the same time,it stimulated lymphocytes to secrete lower IFN-γ,higher IL-10(P<0.05).Compared with decoy-DC in CIA rats,the ability of LPS-Decoy-DC group to stimulate lymphocytes did not increase significantly.2.(1)After intervention with DCs from CIA rats and normal rats treated with NF-κB ODN Decoy and loaded with BIIC on their own CIA rats and allogeneic CIA rats,The arthritic symptoms in the paws of CIA rats were significantly inhibited.(2)The serum levels of IFN-γin the rats of the CIA model group were significantly higher than those in the control group(P<0.01),and the levels of IL-10were significantly lower(P<0.01).After treatment with DCs loaded with NF-κB ODN Decoy and loaded with BIIC,the levels of IFN-γin the serum of rats with BIIC-Decoy DC were significantly lower than those in the model group(P<0.01),while the levels of IL-10 were significantly higher Model group(P<0.01).Conclusion:(1)There was no significant difference in the morphology and characteristics of spleen-derived DC between normal rats and CIA rats.(2)The comparison of CD80 and CD86 positive rates and mean fluorescence intensities on DC surface demonstrated that mononuclear cells derived from CIA rats were successfully treated with NF-κB ODN Decoy strategy to obtain tolerogenic DCs with stable morphology and phenotype.(3)The mixed lymphocyte reaction indicated that Decoy-DC derived from CIA rats had lower ability to stimulate allogeneic lymphocyte reaction,further indicating that it had a relatively immature immunophenotype,namely tolerant DC.(4)Arthritis index was scored by observing the swelling of four joints in each group of rats,and the histopathological appearance of the joint was observed.It displays DCs treated with NF-κB ODN Decoy and loaded with BIIC from the course of CIA rats and normal rats has a good therapeutic effect on CIA rats in the course of the disease,resulting in a certain degree of reversal and improvement in symptoms,signs,and histopathological manifestations.