Study On Glucose Metabolism Mechanism Of Peripheral B Cell Dysfunction In Patients With Rheumatoid Arthritis

BackgroundRheumatoid arthritis(RA)is a chronic,autoimmune inflammatory disease characterized by the unbalanced differentiation of T cell subsets,inflammatory cytokines over-production and autoantibodies production,which mechanism is not fully understood.The activation of immune cells requires a large amount of energy and metabolites to meet the needs of their biosynthesis and complete the proliferation,differentiation and effector functions.In addition,metabolic signals could also regulated the function of immune cells.Studies have shown that B cells could mediate their immune functions such as proliferation and antibody production by selecting appropriate metabolic pathways under different conditions.There are high levels of autoantibodies in the serum of RA patients,suggesting that autoreactive B cell dysfunction might be involved in the pathogenesis of RA.However,the peripheral B cell dysfunction mechanism in RA patients is still unclear.Follicular helper T(T follicular helper T,Tfh)cells are a group of T cell subgroups that are located in the germinal centers of secondary lymphoid organs,which provides help for B cell activation and antibody secretion.In recent years,Tfh-like cells have been found in non-lymphoid organs of RA and other autoimmune diseases,which can assist B cell responses and might play a more important role in the process of B cell activation,differentiation and antibody production.ObjectiveThis study is to reveal the types of energy metabolism of peripheral B cells in RA patients and their effects on B cell dysfunction,and to clarify the metabolic mechanism of RA inflammatory microenvironment regulating peripheral B cell dysfunction.Besides,this studdy further explore the key T cell subsets that help B cells produce antibodies in the periphery of RA patients.MethodsPeripheral blood mononuclear cells(PBMCs)were isolated from RA patients and healthy controls(Healthy control,HC),and plasmablasts(CD19~+CD27~+CD38hi)and plasma cells(CD19~+CD138~+)numbers,the expression of costimulatory molecules CD80 and CD86 on the surface of B cells,the phosphorylation level of the mammalian target of rapamycin(mTOR)signaling pathway,the hypoxia inducible factor-1?(Hypoxia inducible factor-1?,HIF-1?)expression,glucose uptake capacity and glycolysis-related rate-limiting enzyme expression were detected by flow cytometry assay;Isolated CD19~+B cells in peripheral blood of RA patients and HC with immunomagnetic bead assay and stimulate with anti-CD40/Cp G for 1-5 days,and real-time fluorescent quantitative PCR and flow cytometry assay were used to detect the expression of rate-limiting enzymes related to glycolysis and fatty acid oxidation on B cells,and the expression of costimulatory molecules CD80 and CD86on the surface of B cells,proliferation,apoptosis and the numners of B cells subsets were measured with flow cytometry assay.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of immunoglobulin and cytokines in the cell culture supernatant,and the colorimetric method was used to detect the content of lactic acid in the supernatant,in some experiments,glycolysis inhibitor 2-DG,mTOR inhibitor rapamycin or fatty acid oxidation inhibitor ecomoser were added into the culture system.CD19~+B cells were isolated from HC patients and stimulated with2%fetal bovine serum,2%RA patient serum and 2%healthy control serum for 1-5days,in some experiments,anti-IL-27 antibody was added to the culture system.Flow cytometry assay was used to detect B cell proliferation,activation,differentiation,glucose uptake capacity and phosphorylation level of mTOR signaling pathway.Real-time fluorescent quantitative PCR and flow cytometry assay were used to detect glycolysis related rate-limiting enzyme expression,and colorimetric method to detect the level of lactic acid in the cell culture supernatant.Collection of RA patients and HC serum,and protein chip method to detect the expression of 440 kinds of cytokines,ELISA assay to detect IL-27 and immunoglobulin Ig M and Ig G levels,and olorimetric method to detect the content of lactic acid.In addition,the correlation between the level of IL-27 in the serum of RA patients and clinical indicators were analysed.Isolate the CD19~+B cells from RA patients and stimulated with recombinant IL-27 in vitro for 1-5 days.In some experiments,anti-IL-27 antibody2-DG or rapamycin were added into the cell culture system.B cell proliferation,activation,differentiation,glucose uptake capacity,surface gp130,WSX-1 expression and mTOR signaling pathway phosphorylation level were measured with flow cytometry assay,real-time fluorescence quantification PCR and flow cytometry assay were used to detect the expression of glycolysis-related rate-limiting enzymes in B cells,the ELISA assay was used to detect the levels of immunoglobulin and cytokines in the supernatant,and the colorimetric method were used to detect the content of lactic acid in the supernatant.Isolate RA patients PBMCs,and the percentage of CD4~+PD-1~+T cells and CD4~+PD-1~+Foxp3~-T cells were detected with flow cytometry assay,and the correlation between the number of CD4~+PD-1~+Foxp3~-T cells and the clinical indicators of RA patients was analyzed.CD4~+PD-1~+T cells and CD4~+PD-1~+Foxp3~-T cell surface activation molecules(ICOS,CD40L,HLA-DR,CD38)expression and secrete cytokines(IL-4,IL-6,IL-10,IL-21)were detected with flow cytometry.CD4~+PD-1~-T cells,CD4~+PD-1~+CD25~-T cells,and CD4~+PD-1~+CD25~+T cells were measured with flow cytometry and co-cultured with allogeneic B cells for 8 days,and the percentage of plasmablasts and plasma cells were measured with flow cytometry assay,and the levels of immunoglobulin Ig M and Ig G were in the cell culture supernatant was measured by ELISA.CD4~+T cells from the RA patients were separated by magnetic beads,and anti-TNFR2 antibody(0.25?g/ml),anti-IL-1?R antibody(0.25?g/ml)and anti-IL-6R antibody(0.5?g/ml)combined with anti-CD3antibody/anti-CD28 antibody(2?g/ml)was added to the culture system separately.After 3 days of culture,flow cytometry was used to detect the percentage,activation and cytokine secretion of CD4~+PD-1~+Foxp3~-T cells were measured.Results1.Study on the enhancement of peripheral B cell mTOR-glycolysis signaling pathway to promote B cell dysfunction in patients with rheumatoid arthritis:Compared with HC,the proliferation and activation of peripheral B cells in RA patients were increased significantly,and the percentages of plasmablasts and plasma cells were upregulated.However,there was no significant difference in the percentage of apoptotic cells of B cells;Serum levels of immunoglobulin Ig M and Ig G were increased in RA patients;The expression of glycolysis-related rate-limiting enzymes,the glucose uptake capacity and lactate levels were increased on RA B cells.The mTOR signaling pathway phosphorylation were enhanced on RA B cells,and blocking glycolysis or inhibiting the mTOR signaling pathway could improve B cell dysfunction in RA patients;The expression of rate-limiting enzymes related to fatty acid oxidation in the periphery B cells of RA patients were up-regulated,while inhibited fatty acid oxidation has no effect on B cell dysfunction of RA patients.2.IL-27 is a key factor for the RA inflammatory microenvironment to promote B cell glycolysis and dysfunction:Compared with the healthy serum treatment group,the serum treatment of RA patients can significantly promote B cell proliferation,activation,plasmablast and plasma cell differentiation as well as antibody production;Furthermore,RA serum treatment could promote glycolysis-related rate-limiting enzyme expression,glucose uptake capacity,lactate secretion,and phosphorylation of mTOR signaling pathway;Protein chip and ELISA assay showed that serum IL-27levels in RA patients were significantly increased and the level of IL-27 was correlated with disease activity,autoantibody level,plasma cell percentage and immunoglobulin content,and adding anti-IL-27 antibody to the culture system of RA patient serum treatment could inhibit B cells dysfunction,glycolysis-related rate-limiting enzyme expression,glucose uptake capacity,lactate secretion and mTOR signaling pathway phosphorylation caused by RA serum.3.IL-27 promotes the dysfunction of peripheral B cells in RA patients by activating mTOR-glycolysis signaling pathway:Compared with healthy controls,the expression of IL-27 receptor gp130 and WSX-1 in peripheral B cells of RA patients were increased.In vitro,recombinant IL-27 stimulation can up-regulate the IL-27 receptor expression in peripheral B cells of RA patients;Compared with the control group,recombinant IL-27 stimulation can promote the proliferation,activation,and plasmablastogenesis of peripheral B cells in RA patients;Moreover,recombinant IL-27 stimulation could also promote glycolysis-related rate-limiting enzyme expression,glucose uptake capacity,lactate secretion and mTOR signaling pathway phosphorylation.Add anti-IL-27 antibody,glycolysis inhibitor or mTOR pathway inhibitors could significantly inhibit B cell dysfunction,glycolysis and mTOR signaling pathway phosphorylation caused by recombinant IL-27.4.Study of CD4~+PD-1~+Foxp3~-T cells helps the production of antibodies by peripheral B cells in RA patients:Compared with healthy controls,the percentage of CD4~+PD-1~+Foxp3~-T cells in the periphery of RA patients was significantly increased,and their number was positively correlated with autoantibody level,plasma cell percentage and immunoglobulin level.Compared with CD4~+PD-1~+Foxp3~+T cells,the expression of ICOS,HLA-DR,CD40L and CD38 on the surface of CD4~+PD-1~+Foxp3~-T cells in RA patients was significantly increased,and they secreted cytokines IFN-?,IL-4,IL-6,IL-10 and IL-21 were significantly enhanced;The expression of Ki67 in CD4~+PD-1~+Foxp3~-T cells of RA patients was significantly increased,and their surface did not expressed CD25,but partially expressed CXCR5;Compared with CD4~+PD-1~+CD25~+T cells,CD4~+PD-1~+CD25~-T cells could significantly promote the differentiation of B cells into plasmablasts and plasma cells,and promote the production of immunoglobulin Ig M;Compared with untreated CD4~+T cells,the addition of anti-IL-6R antibody could inhibit the percentage,activation and cytokine secretion of CD4~+PD-1~+Foxp3~-T cells of RA patients,while the addition of anti-TNFR2 antibody and anti-IL-1b R antibody has no effect on CD4~+PD-1~+Foxp3~-T cells generation.ConclusionIncreased mTOR-glycolysis signaling pathway in peripheral B cells of RA patients promotes B cell dysfunction,and IL-27 is a key factor in this process;In addition,the number of CD4~+PD-1~+Foxp3~-T cells in the periphery of RA patients was increased,which can participate in the pathogenesis of RA by assisting B cells to produce antibodies.