The Mechanism Of BLyS On The Activation Of Lymphocytes In MRL/lpr Mice And The Effect Of TACI-Ig

Systemic lupus erythematosus (SLE) is an autoimmune disease involved in multiple genes, systems and organs. The pathogenesis of SLE is complicated, which is generally thought to be associated with genes, hormone and the environment. Although the pathogenesis is unclear, recent studies suggest that the T and B lymphocytes immune disorder in SLE patients, leading to a large number of pathogenic autoantibodies, formation of immune complex deposition in the skin, joints, brain and kidney and other organs or tissues, is the main cause of the disease and even death. Because B lymphocyte is the main cell which produces a large number of pathological autoantibody, and participate in the process of SLE, so targeting on B cells becomes a research hotspot of SLE treatment.B lymphocyte stimulator (BLyS), which is also known as B-cell activation factor (BAFF), is a family of tumor necrosis factor (TNF) found in 1999. It is mainly secreted by monocytes, macrophages, dendritic cells, and neutrophils. BLyS plays an important role in regulating B lymphocytes maturation, promoting the immune globulin type conversion, helper T cell activation and participating in immune function through binding to its receptor. Studies reported that there was an increased level of BLys in the serum of SLE patients and lupus-prone animal models, and decreased BLys level could improve the symptoms of SLE. In 2011, a monoclonal antibody (Belimumab) for SLE treatment which targeted on BLyS became the only FDA approved new drug for more than 50 years. Although there are many studies have been carried out on BLyS, but the mechanism of BLyS accelerates the transformation of plasma cells need to be further studied.Transmembrane activator-1 and calcium modulator and cyclophilin ligand-interactor (TACI) is one of the three receptors of BLys. TACI receptor fusion protein (TACI-Ig) is a decoy receptor of BLyS, which can combine with BLyS and decrease the BLyS level in SLE patients or animal model. Our researches found that TACI-Ig could obviously reduce albuminuria, augmented spleen and lymph nodes, proliferative B lymphocytes, and germinal center barriers in lupus-prone mice, which suggested a good perspective for the treatment of SLE mice. Based on the researches, we explored the molecular mechanisms of BLyS on the promotion of plasma cell differentiation from B cells and antibodies production, and observed the effects of BLyS on phenotypes and functions of B lymphocyte and T follicular helper cells (Tfh), meanwhile the effects of TACI-Ig were investigated. We tried to provide experimental basis to elucidate the mechanism of BLyS participating in SLE pathological process through B cell differentiation and discover new drugs targeted of BLyS.ObjectiveTo investigate the therapeutic effects of TACI-Ig on the treatment of spontaneous lupus model MRL/lpr mice, the influence of BLyS on the plasma cell differentiation were observed by pathological morphology, molecular immunology and cell signaling. Moreover, the effects of BLyS on the Bcl-6 protein of follicular helper T lymphocytes were also observed to elucidate the mechanism of BLyS participating in SLE pathological process through B cell differentiation and Tfh cell activation. At the same time, TACI-Ig, a new drugs targeted of BLyS, its therapeutic role was evaluated.Methods1. Experiments in vivoMRL/lpr mice were divided into 5 groups including model group, TACI-Ig group (3.75, 7.5,15mg/kg) and positive control group (prednisone,5mg/kg). The animal activities, state of hair, feces signs of the animals during the 8-week’s treatment were observed and recorded. The urine protein changes were detected by albustix paper, and HE staining was used to examine the kidney and spleen pathology. ELISA kit was used to investigate the changes of serum IgGl, IgG2a, ANA, anti-dsDNA, BLyS, IL-10 and IL-21. The subsets of T and B cells were detected by flow cytometry. Blimp-1 and Bcl-6 protein in splenic tissue were detected by Western blot.2. Experiments in vitroTo define the appropriate concentration and co-culture time of BLyS, the proliferation of MRL/lpr splenic lymphocytes was detected by MTT, the receptors of BLyS in Tfh cell and the altered subsets of spleen T/B lyphocytes which stimulated with BLyS were measured by flow cytometry. The levels of IL-21 and IgG were detected by ELISA kit while the level of Bcl-6 protein expression and gene expression in Tfh cell were detected by Western blot RT-qPCR, respectively.Results1. Effects of TACI-Ig on MRL/lpr miceTACI-Ig (7.5,15 mg/kg) subcutaneously injection could obviously reduce the urine protein level compared with model group, effectively improve hair condition, and reduce the damage index in mice. TACI-Ig could also significantly improve the renal glomerular mesangial cell proliferation, inflammatory cells infiltration and other lesions, inhibit the proliferation of spleen enlargement and germinal center formation, and decrease the increased spleen index compared with the model group. The results suggested that TACI-Ig (7.5,15 mg/kg) could improve lupus nephritis and splenomegaly, which showed a better therapeutic effect.2. Effects of TACI-Ig on the indicators related to immune in MRL/Ipr miceTACI-Ig could significantly reduce the elevated serum BLyS, IgG1, ANA, anti dsDNA, IL-10 and IL-21, with ANA and BLyS level in a dose dependent way. The results suggested that TACI-Ig as a decoy receptor could combine with the high level BLyS, reduce the free BLyS level in the body, significantly reduce the production of autoantibodies and pathogenic factor of inflammation. Further research results showed that TACI-Ig (7.5,15 mg/kg) could significantly decrease the number of total B (CD 19+) cells, mature B (CD19+IgD+) cells and plasma cells (CD19-CD138+), and decrease the percentage of Tfh (CD4+PD1+CXCR54) cells and express activation induced molecular Th lymphocytes (CD4+CD69+). Additionaly, TACI-Ig (7.5,15 mg/kg) also decreased the splenic protein expression of Blimp-1 and Bcl-6. These results indicated that TACI-Ig could inhibit T and B lymphocyte subsets activation, maybe correlated with the regulation of Blimp-land Bcl-6 protein expression.3. BLyS promoting plasma differentiation and the effects of TACI-IgAfter treatment with BLyS (1μg/ml) for 48h, the percentage of total B (CD 19^ cells, mature B (CD19+IgD+) cells and plasma cells (CD19-CD138+) and serum IgGl level were increased. TACI-Ig (1.0, 10μg/ml) could inhibit the activation of B cells, and decrease the percentage of total B cells, mature B cells, early B cells and plasma cells stimulated with BLyS, and decrease the section of IgG But it had no obvious effect on memory B cells and IgM. The results suggested that BLyS in vitro could promote B lymphocyte activation, promote the transformation of B lymphocyte into plasma cells and antibodies production in vitro. TACI-Ig (1.0, 10μg/ml) could inhibit B lymphocyte activation and reduce antibodies production.4. Influence of BLyS on Tfh cell proliferation and cytokine production and the effects of TACI-IgAfter treatment with BLyS (1μg/ml) for 24h, the proportion of splenic Tfh (CD4+PD1+CXCR5+) cells and express activation induced molecular Th lymphocytes (CD4+CD69+) and serum IL-21 level were increased. TACI-Ig (1.0,10μg/ml) could inhibit the activation of related T cells and decrease the IL-21 level. The above results suggested that BLyS could promote T cells activation and inflammation factor secretion, further exacerbating the T/B cell disorder of immune response.5. Influence of BLyS on the proliferation of Tfh cells, cytokine secretion and the effect of TACI-IgTfh subset is the main T cell subtype which helps B cell differentiation. The CD40L and ICOS on Tfh cells can respectively bind with CD40 and ICOSL on B cells, and Tfh cells secrete IL-21. It plays an important role in B cell proliferation, differentiation and immune globulin type conversion. BLyS (1μg/ml) treatment for 48h in vitro could significantly promote the Tfh spleen lymphocyte proliferation and the production of IL-21. TACI-Ig (1.0,10.0μg/ml) could inhibit the Tfh cells proliferation induced by BLyS, reduce elevated levels of IL-21. Further study found that on the surface of Tfh cells, there was high expression of BAFF-R and BCMA, but TACI expression was less. TACI-Ig could inhibit the Tfh cells proliferation induced by BLyS, and reduce the supernatant IL-21 level. The results suggested that BLyS could promote the Tfh cells proliferation and the secretion of IL-21 through combined with BAFF-R and BCMA on Tfh cells, and further promote the activation of B lymphocytes and plasma cells differentiation.6. Influence of BLyS on the Bcl-6 expression of Tfh cells and the effect of TACI-IgB cell lymphoma-6 (Bcl-6) is the key factor of Tfh cells growth and germinal center formation, it plays an important role in the B lymphocytes differentiating into plasma cells. BLyS (1μg/ml) treatment 48h could obviously improve the Bcl-6 gene expression and protein level of Tfh cells in vitro, and TACI-Ig could decrease the expression of Bcl-6. The above results suggested that the upregulation of Bcl-6 expression induced by BLyS associated with promoting B cell differentiation into plasma cells.Conclusions1. TACI-Ig (7.5,15 mg/kg) treatment has obvious therapeutic effect on MRL/Lpr mice, its role may be associated with the improvement in kidney and spleen pathology, inhibition inflammatory cytokines and regulating immune cell subsets and its functions and so on.2. BR3 and BCMA are the major receptors of BLyS in Tfh cell which first time were observed in the study. BLyS and its signals through the two receptors could promote the proliferation and IL-21 production of Tfh cells, which maybe play an important role during the differentiation from B cells into plasma cells.3. BLyS could promote Bcl-6 expression in Tfh cell, which could further accelerate the B lymphocytes differentiate into plasma cells and germinal center formation, maybe one of the important mechanisms of SLE.4. TACI-Ig, as a decoy receptor, would be a prospective therapeutic agent for SLE, which could reduce the high level of BLyS, inhibit the B cell mature and activation, regulate Tfh functions.