| BackgroundSystemic lupus erythematosus (SLE) is a chronic autoimmune disease that is always accompanied by muti-organs and muti-systems involved. SLE is more common detected in women of childbearing age, the incidence rate of our country is far higher than that of foreign. The accurate mechanism of SLE is still unknown. But it is clear that the immune disorder is involved in the pathogenesis of SLE. Therefore, it is meaningful to reveal the immunological mechanisams of SLE and find new therapeutic targets and methods, which can help us to improve clinical treatment level for SLE.Follicular helper T cell (Tfh), a newly discovered T subsets with the ability to secrete IL-21, can help the differentiation, maturation, and antibodies secretion of effector B cells, Tfh cell plays an important role in development of autoimmune diseases. It was reported that the percentage of Tfh cells was increased in SLE patients, but the role of expanded Tfh cells in the disease progress of SLE is still unknown. Regulatory B cell (Breg) has the immune regulatory function via secreting inhibitor cytokines, which plays an important regulatory role in murine models of collagen-induced arthritis (CIA), inflammatory bowel disease (IBD) and experimental autoimmune encephalomyelitis (EAE). But the definite percentage and role of Breg cells in the pathogenesis of SLE is not known. Furthermore, the role of Tfh cells in the differentiaton of Breg cells in SLE is also unclear.In addition, mesenchymal stem cells (MSC) are recently used for the treatment of lupus prone murine models, which exert immunesuppression function via secreted cytokines or cell contact. But the effect of MSC treatment on the differentiation of Tfh and Breg cells needs further research.ObjectivePart one:To make clear the percentage of Tfh and Breg cells in SLE, the correlation between Tfh/Breg cells and SLE disease activity were investigated.Part two:To investigate the role of Tfh and Breg cells in the pathogensis of MRL/Ipr lupus mice.Part three:To study the role and mechanism of Tfh cell-derived IL-21on the differentiation of Breg cells.Part four:To investigate the therapeutic effect of MSC on lupus prone MRL/lpr mice, and regulatory effect of MSC on Tfh and Breg cells.MethodsPart one:Using fluorescence microscope to detect the presence of Tfh and Breg cells in peripheral blood monouclear cells (PBMC) of SLE patients. IL-10positive B cells infiltration in patient skin were detected by immunohistochemistry. Thirty adult patients with SLE and fifteen normal people were included for blood sample collection. Samples were processed to detect Tfh and Breg cells by flow cytometry, related protein and transcription factor genes expression by real-time reverse transcription-polymerase (real-time RT-PCR), and IL-21, IL-10, ANA and ds-DNA production were detected by enzyme linked immunosorbent assay (ELISA). The correlation between Tfh and Breg cells was analyzed; the role of Tfh and Breg cells in the course of SLE disease progress was evaluated.Part two:The proportion of Tfh and Breg cells in splenocytes of MRL/Ipr lupus mice was analyzed by flow cytometry, the gene expression of related cytokine and transcription factor of Tfh and Breg cells were detected by real-time RT-PCR, and the concentration of IL-21, IL-10, ANA and ds-DNA were detected by ELISA, the protein expression of PNA, IL-21and IL-10in spleen was detected by immunohistochemistry. Finally, the correlation between Tfh/Breg cells and the lupus disease activity in lupus prone MRL/Ipr mice was evaluated.Part three:Naive B cells were stimulated in the presence of supernatants from cultured Tfh cells for2-3days, the differentiation of Breg cells was analyzed by flow cytometry, the concentration of IL-10in supernatants was detected by ELISA. Naive B cells were stimulated in the presence of IL-21, the differentiation of Breg cells and secretion of IL-10were examined. The signal pathway of JAK-STAT3in IL-21-mediated differentiation of Breg cells was analyzed when blockade of JAK and p-STAT3by specific inhibitors. In additioin, the immunosuppressive function of IL-21-induced Breg cells was further clarified in vitro.Part four:The total bone marrow adherent method was used for separation MSC, MSC was purified and cultured to the forth generation (P4).8surface markers were identified by flow cytometry. After co-culture with MSC, the percentage of Tfh and Breg cells in splenocytes was detected by flow cytometry. Sorted CD4+T cells and CD19+B cells were co-cutured with MSC in vitro, the related gene expression of cytokines and transcription factors in T and B cells was detected by real-time RT-PCR. Purified MSC was injected into MRL/Ipr mice by tail vein,8weeks later, the disease activity of MRL/lpr mice was evaluated, and the percentage of Tfh and Breg cells in splenocytes of MRL/Ipr mice was analyzed.ResultsPart one:First, we demonstrated the presence of Tfh and Breg cells in PBMC of SLE patient, the ration of theses cells were increased and positively correlated. Furthermore, increased IL-10positive B cells infiltration in skin tissue of SLE patients were detected. Secondly, the gene expression and the protein secretion of IL-21and IL-10were significantly increased in SLE patients. IL-21was not only related to autoantibody production, but also related to the secretion of IL-10.Part two:The spleen of MRL/Ipr mice proliferated obviously, the ratio of Tfh and Breg cells were increased; the relative cytokines and transcription factors gene expression were significantly increased. The expansion of Tfh cells in spleen was not only related to the formation of germinal center (GC), autoantibodies production, but also closely related to the amplification of Breg cells.Part three:The supernatants from Tfh cells could directly induce the differentiation of Breg cells and secretion of IL-10in vitro, IL-21alone could promote the differentiation of Breg cells and secretion of IL-10time and dose-dependent. IL-21promoted the differentiation of Breg cells and secretion of IL-10via activation of phosphorylation of STAT3. Blocking JAK and p-STAT3signal pathway could prevent IL-21-induced IL-10secretion in Breg cells. Finally, we proved that IL-21-induced Breg cells still hold the immunosuppressive function.Part four:First, high purity of bone marrow-derived MSC could be harvested via forth generation. MSC could inhibit the differentiation of Tfh cells and Breg cellsin vitro. MSC treatment in vivo could effectively relieve the progress of disease; inhibit the differentiation of Tfh cells. Unfortunately, MSC could not effectively promote the differentiation of Breg cells in vitro and in vivo.ConclusionPart one:Tfh and Breg cells existed in SLE patients, the ration of these two groups were significantly increased, and related to the activity of SLE. The amplified Breg cells were close related to the expansion of Tfh cells in SLE.Part two:The ratio of Tfh and Breg cells were increased in MRL/Ipr lupus mice, and the amplified Breg was related to Tfh cells.Part three:Tfh cells can induce the Breg cell differentiation and IL-10secretion via IL-21. IL-21promotes the differentiation of Breg cells through activation of STAT3.Part four:MSC treatment can effectively relieve the lupus progress in MRL/lpr mice, which exert antiinflammtory function mainly by inhibiting Tfh cells rather than promoting the Breg cells. |
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