| Background:The function of follicular helper T (Tfh) cells specialized in helping B cells differentiation, maturation and high affinity antibody production. The abnormal expansion of Tfh cells may be involved in the pathogenesis of systemic lupus erythematosus (SLE). Mesenchymal stem cells (MSCs) are a kind of stem cells with immunoregulatory function. MSC transplantation could alleviate disease progression in both lupus mice and patients. The mechanism is largely unknown.Objective:To investigate modulatory effects of human umbilical cord MSCs (hUC-MSCs) on Tfh cells in B6.MRL-Faslpr lupus mice (B6.lpr mice) and underlying mechanism.Methods:The percentages of splenic CD4+CXCR5+PD-1high Tfh cells in B6.lpr lupus mice and C57BL/6 (B6) mice were examined by flow cytometry. Serum IL-21 levels were measured by ELISA. The correlations between the percentages of splenic Tfh cells, serum IL-21 concentrations and lupus manifestations of B6.lpr mice were analyzed respectively. hUC-MSCs were isolated from human umbilical cord. CD4+ T cells were isolated from the spleen of B6.lpr mice by magnetic activated cell sorting (MACS) and co-cultured with hUC-MSCs for three days, and then the frequencies of Tfh cells were determined by flow cytometry. Purified splenic CD4+CXCR5+ T cells were sorted by MACS and co-cultured with hUC-MSCs. The apoptosis (percentage of Annexin V+ cells) and proliferation (percentage of Brdu+ cells) of CD4+CXCR5+ T were examined by flow cytometry after co-cultured with hUC-MSCs for three days and five days respectively. CD4+CD62L+ naive T cells were isolated from the spleen of lupus mice and then co-cultured with hUC-MSCs in a Tfh differentiation-inducing condition. Four days later, the frequencies of Tfh cells were determined by flow cytometry. Fifteen B6.1pr mice were randomly divided into three groups. 1×106 hUC-MSCs were injected into lupus mice via tail vein in one group and the two control groups were administered with 1×106 human synovial fibroblasts (FLSs) and PBS respectively. Four weeks later, all the mice were sacrificed. Serum IgG, IL-21 and anti-dsDNA antibody were detected by ELISA. Histopathology of kidneys was observed by HE staining and glomerular IgG deposition were determined by immunofluorescence. The frequencies of splenic Tfh cells and plasma cells were examined by flow cytometry. CD4+CXCR5+T cells were isolated from spleens of all the administered mice and co-cultured with B cells isolated from spleens of B6 mice. Five days later, IgG levels of the co-culture supernatants were measured by ELISA. The RNA was extracted from hUC-MSCs after they were co-cultured with CD4+T cells isolated from the spleen of lupus mice. The mRNA expression of inducible nitric oxide synthase (iNOS), hepatocyte growth factor (HGF), CC-chemokine ligand 2(CCL2), transforming growth factor-β (TGF-β), human leukocyte antigen-G (HLA-G), indoleamine 2,3-dioxygenase (IDO), cyclooxygenase2 (COX2) and IL-10 were detected by real time polymerase chain reaction (RT-PCR). Anti-HGF antibody or iNOS inhibitor L-NMMA were added to the hUC-MSC-CD4+T cell co-culture medium, and the frequencies of Tfh cells were determined by flow cytometry after three days incubation.Results:The percentages of splenic Tfh cells and serum IL-21 concentrations in B6.lpr lupus mice were significantly higher than those in B6 mice. The frequencies of splenic Tfh cells were positively correlated with spleen weights, serum IgG levels and anti-dsDNA antibody concentrations in lupus mice respectively. hUC-MSCs could down-regulate the frequency of Tfh cells in vitro. Both the differentiation and proliferation of Tfh cells could be inhibited by hUC-MSCs in vitro. Serum IgG concentrations of mice in MSC treated group decreased significantly, as well as the inflammatory cell infiltration in kidney and glomerular IgG fluorescence intensity. The percentages of splenic Tfh cells and plasma cells of mice in MSC treated group also decreased significantly compared with control groups. Supernatants of B cells co-cultured with CD4+CXCR5+ T cells isolated from spleens of hUC-MSC treated lupus mice had lower levels of IgG than control groups. After being co-cultured with splenic CD4+ T cells of lupus mice, hUC-MSCs expressed significantly higher levels of iNOS and HGF mRNA. The inhibitory effect of hUC-MSCs on Tfh cells generation can be reversed after L-NMMA being added to the hUC-MSC-CD4+T cell co-culture medium.Conclusions:hUC-MSCs could down-regulate Tfh cells in lupus mice through secreting nitric oxide (NO), which may be one of the mechanisms of hUC-MSC transplantation alleviating lupus. |
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