| Objective:Rheumatoid arthritis(RA)is a chronic systemic autoimmune disease and inflammatory response induced by activated CD4+T cells is the main pathogenesis of this disease.Tolllike receptor 2(TLR2)is highly expressed on the surface of innate immune cells,which can regulate the adaptive immune response by regulating the activation of innate immune cells and the antigen presentation process,and can also regulate cell function by regulating cell metabolism.It has been suggested that TLR2 also expressed on CD4+T cells,but how it regulates RA CD4+T cells function remains unclear.In addition,some studies have shown that CD4+T cells lose the expression of costimulatory molecule CD28 and become into CD4+CD28-T cells when they are continuously stimulated by antigens or exist in the inflammatory environment for a long time.CD4+CD28-T cells show unique killing and proinflammatory functions in some autoimmune diseases and cardiovascular diseases.However,the specific activation pathway and its function in RA remain to be studied.Here,we aimed to investigate the role of TLR2 signaling in the metabolism and function of peripheral CD4+T cells of RA,as well as the activation pathway of CD4+CD28-T cells and its specific function in the pathogenesis of RA,so as to provide new ideas for the study of the pathogenesis and new strategies for the prevention and treatment of RA.Methods:1.Serum samples were collected from RA patients and healthy controls,and the levels of soluble TLR2 were detected by ELISA.2.Peripheral blood mononuclear cells(PBMC)were isolated from RA patients and healthy controls,and TLR2 expression on CD4+T cells and activation of CD4+T cells were detected by flow cytometry.3.CD4+T cells from RA patients were purified by immunomagnetic beads and stimulated with TLR2 ligand Pam3CSK4.The activation,production of cytokines and reactive oxygen species(ROS),and glucose uptake function of CD4+T cells in RA patients were detected by flow cytometry.The expression of glucose and lipid metabolism-related enzymes were detected by qPCR.The lactic acid content of culture supernatant was detected by detection kit.4.CD4+T cells of RA patients were cultured in the presence of Pam3CSK4 and inhibited mitochondrial ROS production,and the changes of cell functional were detected by flow cytometry.After Pam3CSK4 stimulation,CD4+T cells of RA patients were treated with 2-DG or rotenone or G6PDi-1 to inhibit glucose metabolism,aerobic oxidation of glucose and pentose phosphate pathways,respectively,and the changes of cell functional were detected by flow cytometry.5.TLR2 and CD28 expression levels on CD4+T cells and CD8+T cells of RA patients and healthy controls were detected by flow cytometry.The activation and cytokine production of CD4+CD28+T cells and CD4+CD28-T cells in RA patients were detected by flow cytometry.Purified CD4+T cells of RA patients were stimulated with anti-CD3 antibody and Pam3CSK4 alone or in combination.The activation of CD4+CD28-T cells and the production of cytokines were detected by flow cytometry.Results:1.The level of soluble TLR2 in serum of RA patients was higher than that in serum of healthy controls,and was positively correlated with the serum levels of C-reactive protein and rheumatoid factor.The expression of TLR2 on CD4+T cells in RA patients was higher than that in healthy controls.2.CD4+TLR2+T cells expressed higher levels of CD25,CD40L,CD69,PD-1 and ICOS than CD4+TLR2-T cells in RA patients.3.Pam3CSK4 could up-regulate the expression of CD25,CD40L,CD69 and PD-1 on CD4+T cells in RA patients,and promote the production of tumor necrosis factor-α(TNF-α),total intracellular ROS and mitochondrial ROS.The expression of CD25 and CD40L and the production of TNF-α of CD4+T cells in RA patients were downregulated after ROS depletion,which could not be restored when cultured with Pam3CSK4.4.After TLR2 was activated by Pam3CSK4,the glucose uptake ability of CD4+T cells in RA patients was enhanced,the expression of hexokinase 2 was up-regulated,the expression of phosphofructokinase-2/fructose-2,6-biphosphatase 3 and lactate production were decreased,but the lipid metabolism of CD4+T cells in RA patients did not be affected significantly.5.The total ROS and mitochondrial ROS production,CD25 and CD40L expression and TNF-α secretion of CD4+T cells in RA patients were inhibited when inhibit hexokinase 2,which could not be restored by Pam3CSK4 stimulation.The activation of CD4+T cells in RA patients were partially inhibited when inhibit the glucose aerobic oxidation and pentose phosphate pathway,but there was no significant effect on the production of TNF-α.6.The expression of TLR2 on CD4+CD28-T cells was higher than that in CD4+CD28+T cells in RA patients and healthy controls.The proportion of CD4+CD28-TLR2+T cells in RA patients was higher than that in healthy controls.The expression of TLR2 on CD8+CD28-T cells was higher than that in CD8+CD28+T cells,but there was no significant difference in the proportion of CD8+CD28-TLR2+T cells between RA patients and healthy controls.7.The proportion of CD4+CD28-T cells in peripheral blood of RA patients was higher than that of healthy controls,and the proportion of CD8+CD28-T cells in peripheral blood of RA patients was not significantly different to healthy controls.The proportion of CD4+CD28-T cells in peripheral blood of RA patients was positively correlated with rheumatoid factor concentration in serum of RA patients and erythrocyte sedimentation rate.The proportion of CD8+CD28-T cells was positively correlated with age of RA patients.8.Compared with CD4+CD28+T cells,CD4+CD28-T cells in RA patients showed a decreased expression of CD25,increased expression of CD40L,and production large amounts of perforin and TNF-α.There was no significant difference in the expression of CD25,CD40L and the production of perforin and TNF-α in CD4+CD28-T cells from RA patients when controlled with Pam3CSK4.Conclusion:1.The expression of TLR2 on peripheral CD4+T cells in RA patients is increased when compared with healthy controls.TLR2 signaling promotes the activation of CD4+T cells and the production of TNF-α in RA patients.2.TLR2 signaling promotes mitochondrial ROS production by up-regulating glucose metabolism of CD4+T cells in RA patients,which induces the activation and the production of TNF-α in CD4+T cells from RA patients.3.The proportion of CD4+CD28-T cells in RA patients is higher than that in healthy controls,and is positively correlated with RF and ESR.4.The CD4+CD28-T cells of RA patients have cytotoxic and pro-inflammatory functions.However,TLR2 cannot replace CD28 to activate CD4+CD28-T cells. |
