The Variation Of T Cell Subsets In Peripheral Blood And Galectin-9 In Moderate And Severe Rheumatoid Arthritis Treated With Tacrolimus

ObjectiveRheumatoid arthritis(RA)is a chronic systemic autoimmune disease whose mainly manifestation is inflammatory synovitis.It is characterized by symmetric,invasive polyarticular inflammation that can lead to joint deformities and loss of function.The therapeutic strategy of RA is mainly to reduce joint inflammation,prevent irreversible bone destruction,and achieve complete remission or reduce disease activity.Tacrolimus(TAC),also known as FK506,is a new generation of calcineurin inhibitors that inhibits T lymphocyte proliferation and inhibiting by specifically inhibiting calcineurin(CN)activity and completely inhibits T lymphocyte activation and secretion of cytokines by inhibiting the release of Interleukin IL-2.There have been many reports confirming the effectiveness and safety of TAC in the treatment of RA at present and it can effectively control the condition in a short time,especially in patients with severe RA,thereby reducing joint damage and improving the quality of patients’ life.Galectin-9(Gal-9)is a mammalian lectin secreted by endothelial cells.It can chemoattract eosinophils,cancer cells,and immune cells,and plays various roles in regulating immunity,proliferation,and the like.Gal-9 is a ligand of T cell immunoglobulin domain and mucin-3(Tim-3).When combined,they can downregulate the expression of IFN-y and induce apoptosis of CD4+ T cells.Studies have found that Gal is highly expressed in the synovial tissue and synovial fluid of patients with RA[1].Gal-9 can inhibit the development of arthritis[2]by blocking the production of tumor necrosis factor(TNF-a)and IL-1 or through immune complexes activating phagocytic cells to produce IL-10.Therefore,Gal-9 is thought to be a negative regulator of RA.However,this view has been constantly questioned by the new results of Gal-9 function.In recent years,it has been found that the C-terminal and N-terminal CRDs of Gal-9 have different binding modes and affinity to Tim-3,and the Gal-9-N terminal can activate plasmacytoid dendritic cell(DCs)[3]and enhance NK cytotoxicity and induce TNF-a production to initiate innate immunity.Vascular endothelial cells(EC)express Gal-9,but literature reports that different splicing variants of Gal-9 have different effects on EC cell biology,and may be affected by intracellular localization,concentration and cell environment of Gal-9[4].This shows that Gal-9 can promote innate immunity,regulate adaptive immunity and cell proliferation through different concentrations and different pathways.At present,clinical reports on the discipline of expression and changes of Gal-9 in the peripheral blood of RA patients are few.The purpose of this study is to observethe variation of the disease activity,peripheral blood T-cell subsets,and expression of Gal-9 before and after using TAC specifically changing T-cell function in patients with moderate-severe RA,and to further clarify the correlation between Gal-9 and activity of RA and verify the role of Gal-9 as a biomarker for the change of RA condition and the mechanism of Gal-9 involvement in immune inflammation and angiogenesis of RA.MethodsModerate and severe patients of RA were collected at rheumatoid department and outpatients in Shandong University Qilu Hospital,including 77 patients before treatment by TAC,58 after treatment and 33 self-control cases before and after treatment.According to the reasons for enrollment,patients were divided into no-response group(NR group)and and not-tolerance group(NT group),with extra articular performance group(WG group)and no extra articular performance group(NG group),According to the strategy for treatment,patients were divided into with MTX group(WM group)and no MTX group(NM group),with hormone group(WP group)and no hormone group(NP group),all patients were followed up to 12 weeks(w),and we collect their gender、age、peripheral blood WBC count,lymphocyte ratio,ESR and CRP values,number of joint pain(T28)and number of swelling(SW28)at baseline(Ow)and 12w,record health assessment questionnaire(HAQ),patient global assessment(PGA)and physician global assessment(PHGA),and calculate the patient’s disease activity index,namely DAS28-ESR,DAS28-CRP,SDAI,and CDAI.At the same time,58 healthy examinees of the same age and sex of the physical examination center in our hospital were collected as a healthy donors group(HD group).Flow Cytometry was used to detect peripheral T cell subsets including CD4+、CD8+、CD4+CD8+(Double positive T cell,DPT)、CD4-CD8-(Double negative T cell,DNT)、Treg was marked by CD3+CD4+CD25+CD127low、CD14+(monocytes)and expression level of Gal-9 in the T cell subsets above-mentioned.The expression levels of Gal-9、VEGF、IL-2、IL-6 and TNG-α in plasma were detected by enzyme-linked immunosorbent assay(ELISA),related statistical analysis was performed by SPSS 22.0 software.Results1.Inflammation index and disease activity index of all enrolled patients after 12 weeks treatment by TAC were significantly decreased compared with baseline(P<0.01);2.The percentage of Treg cells in peripheral blood of 33 RA patients was higher than that in HD group,and decreased after 12 weeks treatment(P<0.01);3.The ELISA results showed that the baseline expression levels of Gal-9,IL-6,TNF-a,VEGF in plasma of 33 RA patients were significantly higher than those in the HD group(P<0.01).After treatment,plasma Gal-9 levels in RA patients were decreased(P<0.05).4.Through analysis of the correlation between indexes of 77 pretherapy patients、58 posttreatment patients and 33 self-control patients before and after treatment,we both founded a positive correlation between Gal-9 and CDAI、DAS28-CRP in 77 pretherapy patients、58 posttreatment patients and 33 patients after treatment.The proportion of Treg in CD4+ T cell subsets was positively correlated with the expression of Gal-9 on multiple T subsets.The relationship between Treg and DPT+Gal was positively correlated in all different groups of RA above mentioned.The correlation analysis of 58 posttreatment patients with RA showed that Gal-9 expression level in plasma is a positive related with that in varoious T subsets.Conclusions1.The treatment with tacrolimus in RA is effective,the clinical symptoms,inflammatory indexes and disease activity indexes of all patients with RA have been significantly reduced.2.The proportion of Treg in CD4+ T cell subsets in 33 pretherapy RA patients was higher than that in HD group,indicating that Treg immunoregulation was also increased in high disease activity levels with RA;decreased Treg ratio after treatment indicated the negative adjustment function of Treg was also weakened after decresed disease activity caused by TAC immunosuppression.3.Gal-9 in plasma of pretherapy RA patients was significantly elevated than HD group and decreased significantly after treatment.The decrease was positively correlated with disease activity,suggesting that Gal-9,a sensitive indicator of disease activity,could be used as a new biological Marker of RA.4.At the time point before and after RA treatment,correlation analysis showed that Treg was positively correlated with Gal-9 expression on multiple T subsets,and both of them decreased in the same direction before and after treatment.it suggested that Gal-9 is closely related the number and function of T subsets,especially the ratio and function of Treg,it is an important way to participate in the immune regulation of RA.