BAFF Receptor TACI Regulates The IL-35 Secretion Of MLR/lpr Mouse Splenic B Cells And Its Mechanism

Background and ObjectiveSystemic lupus erythematosus (SLE) is a systemic autoimmune disease involving multiple systems and organs. The disease is characterized by excessive activation of B cells and production of a large number of autoantibodies. Treatment of SLE is mainly with glucocorticoid and immunosuppressive agents, which cause side effects commonly. In 2011, the United States FDA approved the B cell activating factor (BAFF) antagonist belimumab for the treatment of SLE, so that the BAFF system has become a hot research topic. But the BAFF system has three receptors, BR3, TACI, BCMA, and their effects are not the same. According to the different functions of BAFF receptors, blocking receptors that are capable of promoting autoimmunity may be more efficient than blocking BAFF. The study on the functions of BAFF receptors will be helpful to the development of B cells-targeted treatment for SLE. MRL//pr mouse is an animal model of SLE with an Ipr gene mutation, which makes the expression defect of Fas antigen, and thereby B cells cannot be natural apoptosis, resulting in SLE. The purpose of our study was to investigate whether BAFF has regulatory effects on the IL-35 secretion of MRL//pr mouse splenic B cells and its mechanism.Methods1. MRL/lpr mouse splenic B cells were sorted by magnetic isolation Kit. B cells of experimental group were cultured with BAFF and control group with PBS. After B cells were cultured for 72 hours, the expressions of P35 and EBI3, which are two chains consisting IL-35 were detected by Western blot. The frequencies of EBI3+P35+B cell were detected by Flow cytometry. Concentrations of IL-35 in supernatant were detected by ELISA. Cultured cell smears were stained with anti-EBI3 and anti-P35 fluorescence antibodies, and then observed under fluorescence microscope and photographed. The MRL/Ipr mice were divided into experimental group and control group. Blocking BAFF monoclonal antibody and PBS were injected into the tail veins of mice in experimental group and control group, respectively. After 72 hours, mouse spleens were extracted. Parts of the spleens were made into paraffin section with anti-CD19, anti-EBI3 and anti-P35 immunofluorescence staining, observed under the fluorescence microscope and photographed. The proportions of CD19+EBI3+P35+cells were detected by flow cytometry after removal of red blood cells from other parts of the spleens.2. MRL/Ipr mouse splenic marginal zone B (MZ) B cells and follicular area (FO) B cells were sorted by magnetic beads. We performed in vitro experiments on these two B cell subsets, respectively. The experimental group B cells cultured with BAFF and control group B cells with equal volume PBS. After 72 hours, the frequencies of EBI3+P35+B cells were detected with flow cytometry, and the concentrations of IL-35 in the supernatant with ELISA.3. MRL/Ipr mouse splenic B cells were sorted by magnetic beads. B cells were divided into 3 group with BAFF, BAFF+9B9 (9b9 is a monoclonal antibody for blocking BR3) and PBS added respectively. The expressions of P35 and EBI3 were detected by Western blot and the frequencies of EBI3+P35+B cells by flow cytometry. Then, the mouse spleen B cells were divided into 3 groups, adding BAFF, ML120B (small molecule inhibitor of the classical type NF-κB pathway)+BAFF, PBS respectively. The expressions of EBI3 and P35 were detected by Western blot. Data were expressed as mean ± standard error, and the difference between two groups were compared by student t test. One-way ANOVA was used to multiple group comparisons. p<0.05 was considered to have statistical significance.Results1. The expression levels of EBI3 and P35 were the highest in BAFF 20ng/ml, and the differences between experimental group and control group were statistically significant (p<0.01). Flow cytometry of In vitro study showed the frequency of EBI3+P35+ B cells in experimental group was higher than that of the control group (p<0.05), and of in vivo study the frequency of EBI3+P35+ B cells in experimental group was lower than that of control group (p<0.05). ELISA showed that the IL-35 level in supernatant of experimental group was higher than that of control group (p<0.05).2. flow cytometry showed that the freqency of EBI3+P35+B cells in the experimental group was higher than that in the control group (p<0.05). ELISA showed that the IL-35 level in supernatant of experimental group was higher than that of control group (p<0.05).3. Results of Western blot showed that the expressions of EBI3 and P35 in BAFF group and (BAFF+9B9) group were higher than those in PBS group (p<0.05). The expressions of EBI3 and P35 in BAFF group were slightly higher than those in (BAFF+9B9) group, but the differences were of no statistical significance (p>0.05). With the increase of ML120B concentration, the expressions of EBI3 and P35 decreased, and the expressions of EBI3 and P35 at MLB120 10μM were significantly lower than those of BAFF group (p<0.01). Flow cytometry showed that the frequencies of EBI3+P35+B cells in BAFF group and (BAFF+9B9) group were higher than that in PBS group (p<0.05), while the frequency of EBI3+P35+B cells in (BAFF+9B9) group was slightly higher than that in BAFF group, but the difference was not statistically significant (p>0.05).ConclusionsBAFF promotes MRL//pr mouse splenic B cells, mainly MZ B cells to secrete IL-35. BAFF receptor TACI mediates this effect by activating the classical NF-κB pathway. Because TACl has inhibitory effect on the immune system, blocking BR3 and simultaneously activating TACl in the treatment of SLE may be more beneficial than inhibiting BAFF alone.