Follicular Helper T Cells In Peyer's Patches Induce Iga-secreting B Lymphocyte Differentiation Into Plasma Cells And Participate In The Development Of Iga Nephropathy

PartⅠ. Establishment, identification and immunohistochemistry of an IgA nephropathy animal modelObjective To investigate the pathogenesis and role of Peyer's Patches (PPs) in IgA nephropathy in an animal model established by mucosal immunization. Methods The mice nephropathy model were induced by bovine serum, albumin (BSA) and staphylococcal enterotoxin B (SEB) injection. At 0,6,12 week, mice were placed in metabolic cages and 24-h urine samples were collected to determine the numbers of erythrocytes in the urine precipiate and 24-h urinary protein (24-h UP). Serum samples were obtained by gouged out eyeballs to detect the levels of total protein (TP), albumin (Alb), urea nitrogen (BUN), creatinine (Cr), Cystatin C (Cys C). Results (1) 24-h UP at 0,6,12 week were 0.61±0.26 mg,3.61±0.75 mg,4.15±0.82 mg in the IgAN group,0.67±0.22 mg,0.77±0.27 mg, 1.09±0.51 mg in the control group, respectively. At 0 week, There're not significant difference in 24-h UP between the IgAN group and the control group (P＞0.05), however, at 6 week and 12 week,24-h UP were significantly increased in the IgAN group compared with those in the control group (P＜0.01). There was not significant variation in the number of urine erythrocytes between the IgA group and the control group at 0,6,12 week (1.90±1.20 cells/HP vs 1.30±0.95 cells/HP,1.80±0.92 cells/HP vs 1.50±1.08 cells/HP, 2.50±1.18 cells/HP vs 2.00±0.82 cells/HP, P＞0.05). (2) There was mesangial accumulation of IgA in 33 of 40 in the IgA group, but in only 2 of 40 in the control group. Accumulation of IgA concentrated on mesangial area in glomerulus, presenting granular distribution of IgA. (3) Level of Cys C elevated (P＜0.05), and Alb decreased (P＜0.05), while TP, BUN and Cr not markedly difference (P＞0.05) in the IgAN group when compared with the control group. (4) In the PPs, IgA and Bcl-6 positive cells localized in bright and the dark areas junction of the germinal centers (GCs). Semi-quantitative analysis showed that the number of IgA and Bcl-6 positive cells were significant higher in the IgAN group when compared with the control group (P＜0.01). Conclusion These findings suggest that combining BSA with SEB can successfully induce experimental IgA nephropathy, which might be an ideal method to establish an animal model of IgA nephropathy, at the same time, mucosal immunization maybe involved in the IgA nephropathy.PartⅡ. IgA-secreting cells differentiation in PPs and the regulation of follicular helper T cellsObjective To investigate the development and differentiation of IgA-secreting B cells in Peyer's Patches, and to show the important role of PPs in the differentiation of IgA-secreting B cells, and the significance of follicular helper T cells to regulate B cell differentiation into IgA-secreting B cells. Methods In the IgA nephropathy model, PPs were isolated and cells were harvested. Cells were divided into 10 EP tubes as follows:①cells;②cells+CXCR5-APC;③cells+CD4-PE;④cells+CXCR5-APC+CD4-PE (Control);⑤ells+IgA-FITC;⑥cells+B220-perCP;⑦cells+IgM-PE-Cy7;⑧cells+ IgA-FITC+B220-PerCP+IgM-PE-Cy7 (Control);⑨cells+100ulPBS+ APC-CXCR5(4ul)+PE-CD4(4ul) (IgAN);⑩cells+100ulPBS+FITC-IgA(4ul)+ PerCP-B220(2.0ul)+PE-Cy7-IgM(1.25ul) (IgAN). The percentage of Tfh cells, B220+IgM+ B lymphocyte, B220+IgA+B lymphocyte, B220-IgA+plasmablast and IgA were analyzed with flow cytometry. Results The percentage of Tfh cells, B220+IgM+ B lymphocyte, B220+IgA+ B lymphocyte, B2201gA+ plasmablast and IgA were 10.6%±2.49%,34.7%±11.1%,7.92%±2.21%,5.57%±1.75%,13.5%±3.62% in the IgAN group, and 4.88%±1.41%,24.3%±7.84%,5.75%±2.10%,3.74%±1.46%,9.49%±2.14% in the control group, respectively. There were significant differences in the percentage of Tfh cells and IgA (P＜0.01), and B220+IgM+B lymphocyte, B220+IgA+B lymphocyte, B220"IgA+plasmablast (P＜0.05) between the IgAN group and the control group. Conclusion The percentage of Tfh cells increased in PPs in the IgA nephropathy model suggest that Tfh cells regulate B cell differentiation into IgA-secreting cells. PPs have an advantage over B cell development and differentiation into IgA plasmablast. PPs are key site for IgA plasmablast producing, and provide the microenvironment for the differentiation of B cells into secreted IgA plasmablast and the pathogenesis of IgA nephropathy.PartⅢ. Assay the function of Tfh cells in PPsObjective To determinate the anti-BSA antibody production in PPs, indirectly reflect Tfh cells function, and investigate the function change of Tfh cells in PPs with IgA nephropathy. Methods Magnetic bead was performed to sort Tfh cells. Tfh cells coculture with spleen cells itself, and the supernatants were collected at day 8 to measure the level of anti-BSA antibody, which reflect the function of Tfh cells. Results The content of anti-BSA antibody in supernate of cell culture was 0.58±0.10 in IgAN group,0.31±0.08 in control group. When compared with the control group, the level of anti-BSA antibody in the IgAN group were markedly increased (t=2.97, P=0.01). Conclusion The result indicated that not only the percentage but only the function of Tfh cells was increase in PPs with IgA nephropathy, suggest that Tfh cells play an important role in the differentiation of B cells into secreted IgA plasmablast.PartⅣ. Expression of Tfh cells associated transcription factors and cell factors in PPsObjective To determinate the mRNA expressions of interleukin-21 (IL-21), transforming growth factor-β1 (TGF-β1) and the protein expression of IL-21, Bcl-6, Blimp-1. To probe the function of Tfh cells related cells factors and the role in IgA nephropathy. Methods PPs were isolated, and total RNA was extracted, and synthesis cDNA. Real-time fluorescence quantitative PCR to detect the the mRNA expressions of IL-21 and TGF-β1. Supernatants of PPs containing proteins were collected. The protein expression of IL-21, Bcl-6 and Blimp-1 were assayed by Western blot. Results The mRNA expressions of IL-21 and TGF-β1 were 1.67±0.13 and 1.21±0.09, respectively, in the IgA nephropathy mice.1.49±0.13 and 1.10±0.10, respectively, in the control group. The mRNA expressions of IL-21 and TGF-β1 were significantly increased in the IgA group compared with the control group (t=2.730, P=0.016;t=2.416, P=0.03). The relative protein expressions of IL-21, Bcl-6 and Blimp-1 were 0.67±0.21,0.60±0.19 and 1.03±0.07, respectively, in IgAN mice.0.45±0.10,0.34±0.21 and 0.67±0.07, respectively, in the control group, Compared with the control group, the relative protein expressions of IL-21 and Bcl-6 were increased (t=2.628, P=0.025; t=2.665, P=0.019); significantly increased in the Blimp-1 protein expression (t=10.128, P=0.000). Conclusion The expression of Tfh cells associated transcription factors and cell factors in PPs are increased in mice with IgA nephropathy, which facilitate B cells differentiation into secreted IgA plasmablast. Tfh cells and associated cells factors may be involved in the pathogenesis of IgA nephropathy.PartⅤ. Tfh cells related cell factors promote naive B cells differentiation mature and secretion of galactose-deficient IgAlObjective To investigate the mature process of naive B cells induced by Tfh cells associated cells factors in children with IgA nephropathy, and discuss the role of Tfh cells in the pathogenesis of IgA nephropathy. Methods Magnetic bead was performed to sort peripheral blood naive B cells (CD27IgD+) in children with IgA nephropathy, and were cultured with IL-21 and TGF-β1. Supernatant was collected to detect the expression of J chain, the excretion of IgA and galactose-deficient IgA1 (Gd-IgA1). Results Expression of J chain, excretion of IgA and level of Gd-IgAl were 0.85±0.16,6.64±0.85 pg/ml,85.93±7.91 U/ml, respectively, in children with IgA nephropathy, and 0.63±0.28,6.43±0.51 pg/ml,73.1±8.24 U/ml, respectively, in the control group. The levels of Gd-IgA1 in supernatant of activate human B cell in children with IgA nephropathy were much higher than those of the control group (t=3.182,P=0.007), while no significant difference in the expression of J chain and IgA level (t=1.914, P=0.076;t=0.592,P=0.563). Conclusion Naive B cells can matured in peripheral blood, and produce Gd-IgA1. Tfh cells and related cell factors may play an important role in B cells differentiation into secreted IgA plasmablast and producing Gd-IgAl, and participates in the pathogenesis of IgA nephropathy.