Effects Of Low-dose Interleukin-2 On Gut Microbiota And Tfh/tfr In Collagen-induced Arthritis(CIA)mice

Background:Rheumatoid arthritis(RA)is a systemic autoimmune condition with protean manifestations.Although the cause of rheumatoid arthritis is not very clear,the lack of autoimmune tolerance is an important factor of RA.Maintaining autoimmune tolerance and inhibiting autoimmune response are very promising therapeutic strategies for rh eumatoid arthritis.Follicular helper T cell(Tfh)is a unique CD4~+helper T cell subgroup,which promotes the differentiation and maturation of B cells and plasma cells,so as to regulate of antibodies.Tfr is a newly discovered subgroup of regulatory T cells.Tfr cell plays a negative regulatory role in inhibiting the excessive proliferation,class conver sion and high affinity antibody production of B cells in Tfh and GC.The latest rese arch shows that the balance between Tfh and Tfr is directly related to the production of autoantibodies in RA and is closely related to disease activity.Interleukin-2(IL-2)is a cytokine with multiple immune regulatory functions.It has been agreed that IL-2 can specifically and directly expand Treg cells,and then increase the directional transformed Tfr to inhibit Tfh,reverse Tfh/Tfr imbalance and c ontrol the progression of RA.Some studies have shown that the gut microbiota of RA is disturbed.Gut micr obiota imbalance is considered to be an indispensable environmental factor in the process of immune tolerance defects.Therefore,the role gut microbiota of regulating in controlling the progression of rheumatoid arthritis has attracted more and more atte ntion.In order to better understand the role of IL-2 in controlling RA and further observe the effects of low-dose IL-2 on gut microbiota,Tfh/Tfr and metabolite,it is ex pected to provide a new thinking for the treatment of RA with low-dose IL-2.Part Ⅰ:Low dose IL-2 alleviates arthritis symptoms in CIA miceObjective:To observe the remission of arthritis symptoms in CIA mice by low-dose IL-2Methods:DBA/1 male mice were randomly divided into 5 groups:Control group,Model group,Model-MTX group,Model-IL-2-a group and Model-IL-2-b group.MTX was given by gavage once a week,and IL-2 was injected intraperitoneally every other day.Four weeks after the initial immunization,the arthritis index of mice AI>4 began to be administered,and the in vivo experiment was carried out for four weeks.During the course of administration,the joint swelling degree of mouse hind limbs was continuously measured,the degree of joint inflammation was scored,and the mouse joints were photographed and X-rayed.After 4 weeks of treatment,the mice were euthanized and the peripheral blood and joint specimens were taken.The mouse joints were fifixed,embedded and HE.Then HE staining was performed to observe t he changes of joint synovium.The content of cytokines in serum was detected by ELISA.Results:1.Arthritis index(AI):Compared with the Model group,the arthritis index of other groups decreased significantly.There was no difference between Model-IL-2-a group,Model-IL-2-b group and Model-MTX group.2.Pictures of mouse joints:The swelling of foot joints and ulnar deviation wer e obvious in Model group,no swelling of joints and soft tissues in Model-MTX group,and mild swelling of joints and soft tissues in Model-IL-2-a group and Model-IL-2-b group.3.Radiography:The X-ray changes of Model-MTX group,Model-IL-2-a group and Model-IL-2-b group were less than those of Model group.4.Histopathology:In Model group,severe lymphocyte infifiltration,cartilage erosion,bone destruction,and narrowing of the joint space in the hematoxylin and eosin-stained pathological sections of the Model group,which were signifificantly allev iated in both the IL-2-a and IL-2-b groups.5.ELISA:The concentration of IL-2 in the model group was lower(P<0.05).After treatment with IL-2 and MTX,the concentration of IL-2 increased in mice and IL-2-b group increased significantly.Conclusion:LD-IL-2 can alleviate joint inflammation in CIA mice. Part Ⅱ:Effects of low-dose IL-2 on Tfh/Tfr cells in CIA miceObjective:To observe the distribution changes of Tfh and Tfr cells in spleen,intestine and joint synovium of CIA mice before and after low-dose IL-2 treatment.Methods:The distribution of Tfh and Tfr cells in spleen,intestinal mucosa and synovium of mice were observed by immunofluorescence technique.Results:1.Spleen:After low-dose IL-2 treatment,Tfr cells increased,Tfh level did not change significantly,and there was little difference between IL-2-a group and IL-2-b group.After MTX treatment,the two kinds of cells decreased significantly,but the scattered distribution of Tfh and Tfr could still be observed.2.Intestinal mucosa:After low-dose IL-2 treatment,there was a large amount o f orange fluorescence aggregation in the intestinal mucosa,the number of Tfr increa sed and the number of Tfh decreased significantly.There was little difference betwe en IL-2-a group and IL-2-b group.Tfr was also increased in the intestinal mucosa of mice treated with MTX.3、Joint synovium:Only a small amount of Tfh cells were observed in the joint cavity.Conclusion:Low dose IL-2 can affect the distribution and quantity of Tfh and Tfr in peripheral lymphoid tissue,spleen and intestinal mucosal lymphoid tissue of CIA mice.Part Ⅲ:Effect of LD-IL-2 on Gut Microbiota of CIA miceObjective:To observe the effects of LD-IL-2 on the abundance,species and function of gut microbiota in CIA mice.Methods:The feces of mice were collected at the 8th week of the experiment,16SrRNA sequencing,species annotation,diversity analysis and function prediction were carried out to explore the effect of low-dose IL-2 on the gut microbiota of CIA mice.Results:1、Diversity analysisThe results of alpha diversity analysis show that the microbial community richness of Model group decreased significantly,there was no significant difference amon g Model-MTX group,IL-2-a group and IL-2-b group,and there was significant diff erence in microbial diversity between Model group and Control group、Model-MTX group、IL-2-a group and IL-2-b group.Beta diversity analysis was evaluated by Bray Curtis,weighted Uni Frac distances and unweighted Uni Frac distances.The compositi on of microbiota in Control group,Model-MTX group,IL-2-a and IL-2-b group was significantly different from that in Model group.The consist of microbiota in MTX model group,IL-2-a group and IL-2-b group was not significantly different.2.Changes of gut microbiota compositionThe number of species in the feces of the Control group was the most abundant in the seven levels of boundary,while the number of species in the feces of the Model group was the least.After low-dose IL-2 treatment,the gut microbiota of CI A mice recovered.3.Functional predictionAfter ld-il-2 treatment,the difference of metabolic signal pathway in model mice was reduced compared with that in Control group,including glutathione metabolism,porphyrin and chlorophyll metabolism,purine metabolism,drug metabolism,cytochr ome P450 and cytochrome P450 metabolism of exogenous substances.The functional differences involved most aspects of physical function,especially metabolism,cell process,environmental processing,genetic processing,human diseases,body system etc.Conclusion:Low dose IL-2 can partially restore the species,composition and biological dysfunction of gut microbiota in CIA mice.Part Ⅳ:Effects of low-dose IL-2 on metabolites in CIA miceObjective:To observe the effect of ld-IL-2 on short chain fatty acids,a metabolite of gut microbiota in CIA mice.Methods:The feces of mice in each group were collected at the 8th week of the experiment,and the concentration of SCFA in the feces of mice in each group was d etermined by GC-MS.Results:The total content of SCFA in the Model group was the lowest.The total content of SCFA in Model-MTX group and IL-2 group increased after treatment,among which IL-2-a group was the highest.Except hexanoic acid,other SCFA increased after IL-2 and MTX treatment,increasing trend of butyric acid and isovaleric acid in IL-2 group was more obvious than that in Model-MTX group.Conclusion:It is suggested that ld-IL-2 treatment can improve the metabolic disorde r of SCFAs caused by CIA.Part Ⅴ:Correlation between gut microbiota and immune metabolism in CIA miceObjective:The correlation between gut microbiota and short chain fatty acids and cytokines was analyzed to find the internal relationship.Methods:RDA analysis,Spearman correlation analysis and other statistical methods.Results:The results showed that ld-IL-2 had the highest correlation with microbiota,followed by TNF-ɑ,IL-6,IL-10,IL-17and IL-21.SCFA also had correlation with micro biota.Conclusion:It is suggested that ld-IL-2 has a significant effect on the microbiota.