Galectin-9Ameliorates Anti-GBM Nephritis By Shifting The Balance Of Activated CD4~+T Cell Immune Response In Mice

Objective Anti-glomerular basement membrane (GBM) glomerulonephritis (GN) is a kind of autoimmune disease with extensive glomerular capillary damage via anti-GBM antibody, which performed as crescentic GN of type1, leading to severe and rapidly progressive renal injury and poor prognosis. A number of studies indicate that Th1-and Th17-predominant immunity play an important role in the pathogenesis of anti-GBM GN. T-cell immunoglobulin and mucin domain-containing molecule3(Tim-3) is expressed mostly on terminally differentiated Thl and Th17cells. It can transmit inhibitory signals causing cell apoptosis when activated and lead to a series of immune responses finally. Galectin-9(Gal-9), a β-galactoside binding lectin, identified as the ligand for Tim-3, which can induce apoptosis of activated CD4+T cells through the Ca2+-calpain-caspase1pathway via Gal-9/Tim-3interaction. Therefore, the purpose of this study was to investigate the effect of CD4+T cell-mediated immune response in C57BL/6mice anti-GBM glomerulonephritis and whether Gal-9administration ameliorated anti-GBM GN through inhibiting Thl and Th17cell-mediated immune responses and shifting the immune balance. Furthermore, we aimed to investigate the potential mechanisms.Methods1. C57BL/6mice were allocated to induce anti-GBM GN. All the mice were observed for Scr, BUN and proteinuria/creatinine at the7th day, the14th day, the21st day and the28th day, respectively. At the same time, the kidneys were collected for the observation of the pathological changes. The infiltration of immune cells in lymphoid organs and renal tissues was detected by FACS. The level of serum IFN-y, IL-4, IL-17A, IL-12, IL-10and TGF-β1were determined by ELISA and the mRNA of IFN-γ, IL-4, IL-17A, TGF-β1, CCL-2, CCL-4and CCL-5in kidney were detected by real time PCR.2. Renal injury and infiltration of immune cells in lymphoid organs and renal tissues were observed after Gal-9administration, as well as the expression of cytokines.3. Lymphocytes were harvested from C57BL/6mice, from which CD4+naive T cells were isolated with MACS and activated, then stimulated to Thl/Th2/Th17differentiation for5days. All cells were divided into3groups:control group, Gal-9group and Gal-9+a-lactose group. Apoptosis of CD4+T cells and the proportion of the subset cells were detected by FACS. The expression of cytokines and transcription factors was detected. The gene expression of Tim-3, T-bet, GATA-3, RORyt and Foxp3was evaluated by real time PCR.Results1. Scr and BUN were obviously increased on day21in nephritic group. Compared to normal control group, mice in nephritic group with Scr (2.68±0.98), BUN (34.15±2.61), proteinuria/creatinine (15.85±3.20) on day28appeared obviously increased. Meanwhile, crescent formation, the thickening and breakage of GBM, glomerular mesangial cell and matrix proliferation, protein casts and inflammatory cells were observed in nephritic group. Immunofluorescence examination showed that rabbit IgG and mouse IgG linearly deposited along the GBM. Then the distribution of immunologic cells in spleen showed that CD4+CD69+T cells, Thl and Th17cells increased in nephritic group; the infiltration of Thl and Th17in kidneys were also increased in nephritic group. The expression of IFN-y, IL-4, IL-17A and TGF-β1in kidney and serum were also up-regulated in nephritic group.2. Gal-9administration attenuated and delayed the renal damages in anti-GBM GN, which included less crescent formation, protein casts, glomerular mesangial cell and matrix proliferation and inflammatory cells, as well as the benefits to mouse IgG deposition and renal function. Gal-9led to the decrease of CD4+Tim-3+T cells, Th1and Th17cells in spleen, so as in kidney. Meanwhile the enhanced Th2cell-mediated immune response was disclosed in spleen and kidney. The expression of IFN-y and IL-17A was decreased in serum and kidney, as well as chemokines CCL2, CCL5, CXCL-9, CXCR-3, CXCL-10and CCL20in kidney.3. Compared with control group and Gal-9+a-lactose group, in Gal-9group, apoptosis of activated CD4+T cells was increased, the proportions of Thl and Th17cells were decreased, as well as the levels of IFN-y and IL-17A and the expression of T-bet, RORyt and Tim-3. After stimulating to Thl/Th2/Th17differentiation, the proportions of Thl and Th17cells were decreased, no significant difference in Th2cells and the expression of Treg cells was increased; the gene levels of T-bet and RORyt were deceased, no significant difference of GATA-3and the expression of Foxp3was increased.Conclusions CD4+T cell-mediated immune response can contribute to anti-GBM GN in mice and play an important promotion in the progress of pathology. It also demonstrated that Gal-9could ameliorate and delay the renal injury of anti-GBM GN in mice through inhibiting Thl and Th17cell-mediated immune responses, which depends on the reaction with Tim-3partly.