The Role And Mechanism Of T Follicular Regulatory Cells In The Immune Disorder Of Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a chronic systemic autoimmune disease characterized by the production of autoantibodies including rheumatoid factor(RF)and anti-citrullinated protein antibody(ACPA).The typical clinical manifestation of RA is erosive arthritis,and it could also cause extra-articular organ damage.RA patients without proper treatment could suffer from joint deformities and dysfunction,which would greatly reduce their quality of life.In recent years,with the development of biologics,as well as the introduction and application of advanced concepts including "individualized treatment" and "treat-to-target",the prognosis of RA has been greatly improved.However,there are still many open issues in the clinical setting,such as poor response to the drugs and severe drug adverse reactions,making the current diagnosis and treatment of RA still a big challenge.So far,the etiology of RA has not been clarified,and it has been suggested that RA is closely related to many complex factors including genetics,environment and immune factors.The pathogenesis of RA mainly includes the following stages:Firstly,on the basis of complex interaction between genetic variation and environmental factors,immune disorder occurred and subsequently caused local inflammation infiltration and systemic immune reaction.Local inflammation infiltration would induce joint synovial inflammation and vascular crests formation which eventually lead to cartilage erosion and bone destruction.At the same time,the systemic immune reaction could induce rheumatoid nodule,vasculitis,fibrosis as well as other pathologic changes,which could cause damage to multiple organs such as heart,lungs,and kidneys.Immune disorder,which is closely associated with abnormal proliferation of autoreactive T cells,B cells,and overproduction of autoantibodies,is considered to be a central player in the pathogenesis of RA.T follicular regulatory cell(Tfr)subset is a new type of T regulatory cells(Treg)with immunosuppressive function.Tfr cells could simultaneously express Foxp3 and Bcl-6,as well as surface molecules including CD25,ICOS,PD-1 and CXCR5,presenting phenotypic characteristics of both Treg cells and T follicular helper cells(Tfh).Tfr cells were considered to play a key role in the regulation of Tfh cells,B cells,and antibody production.Research have demonstrated that the abnormal frequency and function of Tfr cells could disrupt the immune homeostasis,which is closely related to the occurrence and development of various diseases such as autoimmune disease,allergic disease,transplant rejection,and infectious disease.There are few studies conducted on Tfr cells in RA,and it is still unclear whether Tfr cells is defective and could mediate the occurrence and development of RA.In order to clarify this issue,we conducted a comprehensive investigation on three aspects of Tfr cells:the phenotypic characteristics of Tfr cells in RA and collagen-induced arthritis(CIA),the role of Tfr cells in the immune disorder of RA,and the factors restricting Tfr cells in RA as well as its underlying mechanisms.The results and conclusions of this study could provide new perspectives on understanding the pathogenesis of RA and theoretical basis on exploring treatment targets,which is beneficial for improving the diagnosis and treatment of RA.Part Ⅰ.The phenotypic characteristics of Tfr cells in RA patients and CIA modelObjective:To investigate the frequency,clinical significance,as well as the expression levels of specific molecules of Tfr cells on RA patients and CIA model.Method:Peripheral blood mononuclear cells(PBMC)from RA patients and healthy controls(HC)were isolated by density gradient centrifugation.The proportion of Tfr cells,their dynamic change with disease status,expression levels of specific molecules and proportion of functional Treg cells were detected by flow cytometry.Correlation analysis were conducted to explore the correlations between the proportion of Tfr cells and clinical parameters.Immunization of type II collagen on DBA/1 mouse was performed to establish CIA model.At the peak period of CIA disease,spleen lymphocytes were isolated and detected by flow cytometry to define the proportion of Tfr cells and expression level of specific molecules.Results:Compared with healthy controls(HC),the proportion of Tfr cells in the peripheral blood of RA patients was significantly decreased while the proportion of Tfh cells was significantly increased.The proportion of Tfr cells in RA was inversely correlated with DAS28,serum CRP level,ACPA titer and IgG level.The imbalance between Tfr cells and Tfh cells in RA could be partly reversed after treatment.Compared with HC,the expression levels of Foxp3,ICOS,and CCR7 as well as the proportion of effector Treg in Tfr cells were significantly reduced while the expression level of PD-1 were significantly increased.In addition,compared with control group,the proportion of Tfr cells in the spleen lymphocytes of CIA mice as well as the expression levels of Foxp3 and ICOS on Tfr cells were significantly reduced at the peak period of CIA disease.Conclusion:Compared with HC,the phenotypes of Tfr cells in RA and CIA model were significantly altered.These results indicated that the function of Tfr cells may be inhibited,which might contribute to the occurrence and development of immune disorder in RA.Part Ⅱ.The role of Tfr cells in the occurrence and development of immune disorder in RAObjective:To investigate whether Tfr cells could mediate the occurrence and development of immune disorder in RA.Method:Firstly,Tfr cells were labeled with CFSE and detected by flow cytometry to assess their proliferation capacity.Subsequently,a coculture system of sorted Tfr cells,Tfh cells,and B cells from HC and RA patients was successfully constructed.Concentration levels of IgG and IgM in the coculture supernatant were measured by enzyme-linked immunosorbent assay(ELISA).And the proliferation capacity of Tfh cells and proportion of B cell subsets were examined by flow cytometry.Results:Compared with HC,the proliferation capacity of blood Tfr cells in RA patients was significantly reduced.In the coculture system,compared to the group with addition of HC Tfr cells,the concentration levels of IgG and IgM in the supernatant,the proliferation capacity of Tfh cells,and the proportion of memory B cells in the group with addition of RA Tfr cells were significantly increased.Conclusion:In patients with RA,the proliferation level of Tfr cells and their capacity to inhibit Tfh cells and B cells were greatly weakened.The inhibition of Tfr cells could lead to the overreaction of Tfh cells,B cells,and overproduction of antibodies,which might subsequently mediate the occurrence and development of immune disorder in RA.Part Ⅲ.The effect and mechanism of inflammation on Tfr cellsObjective:To investigate the effect and mechanism of RA inflammation on the proliferation and function of Tfr cells.Method:Firstly,plasma cross-culture experiment,plasma cytokines detection,in vitro stimulation by recombinant cytokines and clinical correlation analysis were performed to explore the inflammatory cytokine(s)that could regulate Tfr cells.Tfr cells were then stimulated in vitro by the cytokine to determine its effect on cell proliferation and function.Finally,the regulatory mechanism of inflammatory cytokine on Tfr cells was clarified by detecting pathway protein and transcription factor involved in the pathological process.Results:In HC PBMC cultured with RA plasma,the proportion of Tfr cells was decreased while the proportion of Tfh cells was increased.While in RA PBMC cultured with HC plasma,the imbalance between Tfr cells and Tfh cells could be partly reversed.Compared with HC,the concerntration levels of IL-2,IL-6,IL-17,IL-21,and TNF-α in RA plasma were significantly higher.However,only PBMC stimulated with IL-21 could result in the imbalance between Tfr cells and Tfh cells.Correlation analysis suggested that the level of plasma IL-21 in RA patients was positively correlated with DAS28 and plasma IgM concentration,while inversely correlated with the proportion of Tfr cells and ratio of Tfr/Tfh.Function study showed that IL-21 could weaken the proliferation level of Tfr cells and their capacity to inhibit Tfh cells as well as antibody production.In addition,pathway protein detection revealed that IL-21 could reduce the expression level of Foxp3,the key transcription factor of Tfr cells,by downregulating pAkt,which would thus result in the phenotypic abnormality and functional inhibition of Tfr cells.Furthermore,the expression of Foxp3 on RA Tfr cells was more sensitive to pAkt downregulation mediated by IL-21.Conclusion:IL-21 concentration was significantly higher in RA plasma than that in HC plasma.IL-21 could reduce the expression of Foxp3 on Tfr cells by downregulating pAkt,which would thus inhibit the proliferation and immunosuppressive function of Tfr cells.The inhibition of Tfr cells could cause the overproliferation of Tfh cells and overproduction of antibodies,which would thereby mediate the occurrence and development of immune disorder in RA.