Experimental Research On The Effect Of Thymic INKT Cells On Th1/Th2/Th17 Subsets And Cytokines In Rheumatoid Arthritis Mice

ObjectiveIn order to investigate the effect of iNKT cells on the immune balance of rheumatoid arthritis(RA),we established a mouse model of rheumatoid arthritis(RA)and detected the frequency of iNKT cells,Th1/Th2/Th17 subsets and cytokines.In vitro induction of thymocyte differentiated into i NKT cells,adoptive infusion of cells into RA mice and explore the intervention effect of iNKT cells in RA mice,and the influence of Th1/Th2/Th17 immune imbalance.Discussing the influence mechanism of iNKT cells on RA,and provide a new theoretical basis for exploring the pathogenesis and treatment methods of RA.Method1.To establish a mouse model of RA and identify it.HGPI325-339,hGPI469-483 peptide fragments of 1:1 mixed,emulsified with complete Freund’s adjuvant.DBA/1 mice were given subcutaneous injection of the immunogens and pertussis toxin was injected into the abdominal cavity to strengthen the immunity at 0h and48 h.In healthy mice as the control group,observe the changes of body weight,ankle joint swelling of ankle joint.The joint tissues stained with hematoxylin and eosin(HE)was used to evaluate the inflammatory cells.Fluorescence-activated cell sorting(FACS)was used to detect the frequency of peripheral blood and spleen iNKT cells and Th1/Th2/Th17 subsets.Cytometric bead array(CBA)was used to analyze the changes of serum cytokines IL-4、IL-10、TNF-α、IFN-γ、IL-17A、IL-2、IL-6.2.Induction of thymus derived iNKT cells and detection of immune function.The thymus of DBA/1 mice was isolated and the lymphocyte suspension was obtained.IL-2,IL-4 and-GalCer were added to stimulate the iNKT cells in vitro.One week later,i NKT cells were separated by Magnetic Cell Sorting(MACS),FACS was used to detect the frequency of iNKT cells and the cell subsets,and the level of cytokines in the supernatant was detected by CBA.With deep red cell membrane fluorescent probe(DiR)staining cells,intravenous infusion of adoptive to normal mice,small animal imaging system to observe thecell distribution,and to evaluate the mice weight,fur,skin,activity and posture,determine the transfusion safety.3.To observe the effect of thymus derived iNKT cells on Th1/Th2/Th17 in RA mice.RA mice were treated with thymus derived iNKT cells.With-GalCer as positive control.To observe the changes of body weight and ankle swelling changes.The joint tissues stained with HE was used to evaluate the changes of inflammatory cells.FACS was used to detect the frequency of iNKT cells and the changes of cell subsets.The levels of cytokines in the serum of each group were detected by CBA.Result1.Comparison between healthy group and model group,a slow increase of body weight of RA mice.The hind paw of the RA mice treated with mixed peptide fragments first began to appear redness and swelling on 7th day,then accentuated gradually to the limbs,the swelling reached peak on 14 th d,and was then gradually relieved.Inflammatory cells infiltration were found inankle joint by HE.At the peak of inflammation,the frequency of i NKT cells decreased significantly(P<0.05).In the early stage of inflammation,Th1 and Th17 increased significantly(P<0.05).In the middle and late stages of inflammation,Th2 group increased significantly(P<0.05).At the peak of inflammation,the levels of serum TNF-α、IFN-γ,IL-17 A,IL-2 and IL-6 were significantly higher(P<0.05).The frequency of i NKT in RA mice was negatively correlated with the levels of TNF-α,IL-6 and Th1.2.In thymus lymphocytes,CD4+CD8+ cells were the main ones.After one week of stimulation,the frequency of i NKT cells was 7.07% ± 1.12%.After purification,the frequency was 63.37% ± 5.84%.The isolated cells were mainly Th2 cells,which were stimulated to secrete IL-4.The cells were distributed in the liver and spleen after infusion into normal mice.The scores of mice were normal.3.RA mouse model of slow increase of body weight and swelling joints can be significantly improved by alpha-GalCer and cell infusion.In addition,they reduced the ankle joint infiltration of inflammatory cells,increased the frequency of iNKT cells and regulated Th1/Th2/Th17 subsets and cytokines disorders.Conclusion1.GPI mixed peptide induced RA model mice in iNKT cells and Th1/Th2/Th17 cells are more closely related to RA and can be used as a good tool for the research and treatment of RA immune mechanism..2.The double positive T cells in thymus can be induced into iNKT cells in vitro,which provides a reference for the development of iNKT cells and the establishment of iNKT based cell therapy.3.Cell transfusion can correct the immune balance disorder of Th1/Th2/Th17 subsets and cytokines in RA mice,maintain immune tolerance,and exert immune regulation function.It has potential clinical application value.