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

Part Ⅰ. Establishment, identification andimmunohistochemistry of an IgA nephropathy animal modelObjective To investigate the pathogenesis and role of Peyer’s Patches (PPs) in IgAnephropathy in an animal model established by mucosal immunization. Methods The micenephropathy model were induced by bovine serum albumin (BSA) and staphylococcalenterotoxin B (SEB) injection. At0,6,12week, mice were placed in metabolic cages and24-h urine samples were collected to determine the numbers of erythrocytes in the urineprecipiate and24-h urinary protein (24-h UP). Serum samples were obtained by gouged outeyeballs to detect the levels of total protein (TP), albumin (Alb), urea nitrogen (BUN),creatinine (Cr), Cystatin C (Cys C). Results (1)24-h UP at0,6,12week were0.61±0.26mg,3.61±0.75mg,4.15±0.82mg in the IgAN group,0.67±0.22mg,0.77±0.27mg,1.09±0.51mg in the control group, respectively. At0week, There’re not significantdifference in24-h UP between the IgAN group and the control group (P>0.05), however,at6week and12week,24-h UP were significantly increased in the IgAN group comparedwith those in the control group (P <0.01). There was not significant variation in thenumber of urine erythrocytes between the IgA group and the control group at0,6,12week(1.90±1.20cells/HP vs1.30±0.95cells/HP,1.80±0.92cells/HP vs1.50±1.08cells/HP, 2.50±1.18cells/HP vs2.00±0.82cells/HP, P>0.05).(2) There was mesangialaccumulation of IgA in33of40in the IgA group, but in only2of40in the control group.Accumulation of IgA concentrated on mesangial area in glomerulus, presenting granulardistribution 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 whencompared with the control group.（4）In the PPs, IgA and Bcl-6positive cells localized inbright and the dark areas junction of the germinal centers （GCs）. Semi-quantitativeanalysis showed that the number of IgA and Bcl-6positive cells were significant higher inthe IgAN group when compared with the control group (P <0.01). Conclusion Thesefindings suggest that combining BSA with SEB can successfully induce experimental IgAnephropathy, which might be an ideal method to establish an animal model of IgAnephropathy, at the same time, mucosal immunization maybe involved in the IgAnephropathy.Part Ⅱ. IgA-secreting cells differentiation in PPs and theregulation of follicular helper T cellsObjective To investigate the development and differentiation of IgA-secreting B cellsin Peyer’s Patches, and to show the important role of PPs in the differentiation ofIgA-secreting B cells, and the significance of follicular helper T cells to regulate B celldifferentiation into IgA-secreting B cells. Methods In the IgA nephropathy model, PPswere isolated and cells were harvested. Cells were divided into10EP tubes as follows:①c ells;②cells+CXCR5-APC;③c ells+CD4-PE;④c ells+CXCR5-APC+CD4-PE(Control);⑤cells+IgA-FITC;⑥c ells+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 wereanalyzed with flow cytometry. Results The percentage of Tfh cells, B220+IgM+B lymphocyte, B220+IgA+B lymphocyte, B220-IgA+plasmablast and IgA were10.6%±2.49%,34.7%±11.1%,7.92%±2.21%,5.57%±1.75%,13.5%±3.62%in the IgANgroup, and4.88%±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 thepercentage of Tfh cells and IgA (P <0.01), and B220+IgM+B lymphocyte, B220+IgA+Blymphocyte, B220-IgA+plasmablast (P <0.05) between the IgAN group and the controlgroup. Conclusion The percentage of Tfh cells increased in PPs in the IgA nephropathymodel suggest that Tfh cells regulate B cell differentiation into IgA-secreting cells. PPshave an advantage over B cell development and differentiation into IgA plasmablast. PPsare key site for IgA plasmablast producing, and provide the microenvironment for thedifferentiation of B cells into secreted IgA plasmablast and the pathogenesis of IgAnephropathy.Part Ⅲ. Assay the function of Tfh cells in PPsObjective To determinate the anti-BSA antibody production in PPs, indirectly reflectTfh cells function, and investigate the function change of Tfh cells in PPs with IgAnephropathy. Methods Magnetic bead was performed to sort Tfh cells. Tfh cells coculturewith spleen cells itself, and the supernatants were collected at day8to measure the level ofanti-BSA antibody, which reflect the function of Tfh cells. Results The content of anti-BSAantibody in supernate of cell culture was0.58±0.10in IgAN group,0.31±0.08incontrol group. When compared with the control group, the level of anti-BSA antibody inthe IgAN group were markedly increased (t=2.97, P=0.01). Conclusion The resultindicated that not only the percentage but only the function of Tfh cells was increase in PPswith IgA nephropathy, suggest that Tfh cells play an important role in the differentiation ofB cells into secreted IgA plasmablast.Part Ⅳ. 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 IgAnephropathy. Methods PPs were isolated, and total RNA was extracted, and synthesiscDNA. Real-time fluorescence quantitative PCR to detect the the mRNA expressions ofIL-21and TGF-β1. Supernatants of PPs containing proteins were collected. The proteinexpression of IL-21, Bcl-6and Blimp-1were assayed by Western blot. Results The mRNAexpressions of IL-21and TGF-β1were1.67±0.13and1.21±0.09, respectively, in the IgAnephropathy mice.1.49±0.13and1.10±0.10, respectively, in the control group. ThemRNA expressions of IL-21and TGF-β1were significantly increased in the IgA groupcompared with the control group (t=2.730, P=0.016; t=2.416, P=0.03). The relative proteinexpressions of IL-21, Bcl-6and Blimp-1were0.67±0.21,0.60±0.19and1.03±0.07,respectively, in IgAN mice.0.45±0.10,0.34±0.21and0.67±0.07, respectively, in thecontrol group, Compared with the control group, the relative protein expressions of IL-21and Bcl-6were increased (t=2.628, P=0.025; t=2.665, P=0.019); significantly increased inthe Blimp-1protein expression (t=10.128, P=0.000). Conclusion The expression of Tfhcells associated transcription factors and cell factors in PPs are increased in mice with IgAnephropathy, which facilitate B cells differentiation into secreted IgA plasmablast. Tfh cellsand associated cells factors may be involved in the pathogenesis of IgA nephropathy.Part Ⅴ. Tfh cells related cell factors promote naive B cellsdifferentiation mature and secretion of galactose-deficient IgA1Objective To investigate the mature process of naive B cells induced by Tfh cellsassociated cells factors in children with IgA nephropathy, and discuss the role of Tfh cellsin the pathogenesis of IgA nephropathy. Methods Magnetic bead was performed to sortperipheral blood naive B cells（CD27-IgD+）in children with IgA nephropathy, and were cultured with IL-21and TGF-β1. Supernatant was collected to detect the expression of Jchain, the excretion of IgA and galactose-deficient IgA1(Gd-IgA1). Results Expression ofJ chain, excretion of IgA and level of Gd-IgA1were0.85±0.16,6.64±0.85pg/ml,85.93±7.91U/ml, respectively, in children with IgA nephropathy, and0.63±0.28,6.43±0.51pg/ml,73.1±8.24U/ml, respectively, in the control group. The levels of Gd-IgA1insupernatant of activate human B cell in children with IgA nephropathy were much higherthan those of the control group（t=3.182，P=0.007）, while no significant difference in theexpression of J chain and IgA level (t=1.914，P=0.076；t=0.592，P=0.563). ConclusionNaive B cells can matured in peripheral blood, and produce Gd-IgA1. Tfh cells and relatedcell factors may play an important role in B cells differentiation into secreted IgAplasmablast and producing Gd-IgA1, and participates in the pathogenesis of IgAnephropathy.