The Mechanism Of Low IgG Level Caused By B Cell Dysfunction In Children With Primary Nephrotic Syndrome

PART ? ABNORMALITIES OF B CELLS AND THEIR RELATIONSHIP WITH SERUM IGG LEVEL IN CHILDREN WITH PRIMARY NEPHROTIC SYNDROMEObjective:The serum level of IgG in PNS patients significantly decreased which is closely related to secondary infection and relapse,frequent recurrence.The urinary loss could not explain the reason of low IgG in PNS fully indicating the damaged immunoglobulin production of B cells.This part of experiment is to determine whether B cells are dysfunction and involved in the occurrence of low IgG level.Methods:117 PNS and 97 healthy control children were enrolled in nephrology department of Children's Hospital of Chongqing Medical University?due to the need of large blood collection to text of all index,we didn't use one blood sample from the same patient and healthy control to complete the measurement of whole experimental contents?.The percentage of CD19⁺B cells,CD27⁺memory B cells,CD27^hiCD38^hii plasmablasts,CD138⁺plasma cells,CD27^-CD24^hiCD38^hii transitional B cells,CD24⁺CD38⁺mature-naive B cells and CD38⁺IgD^-GC-like cells as well as the Median fluorescence intensity?MFI?expression levels of CD80,CD86 and CD95 on CD19⁺B cells were analysed by flow cytometry.We detected the percentage of CD19⁺sIgG⁺B cells,CD19⁺cIgG⁺B cells,CD19⁺sIgM⁺B cells,CD19⁺cIgM⁺B cells,CD27⁺sIgG⁺memory B cells and CD38⁺cIgG⁺plasma cells.The CD19⁺B Cells apoptosis was determined using Annexin V-FITC/PI Apoptosis Detection Kit.Besides,we analysed the relationship between CD27^hiCD38^hii plasmablasts,CD138⁺plasma cells,CD19⁺sIgG⁺B cells as well as CD38⁺cIgG⁺B cells and serum IgG level and the relative percentage of CD19⁺sIgG⁺B cells and CD19⁺cIgG⁺B cells among the CD19⁺IgG⁺and CD19⁺IgM⁺B cells.Results:1.Compared with healthy controls,the percentage of CD27^hii CD38^hii plasmablasts,CD138⁺plasma cells and CD24⁺CD38⁺mature-naive B cells were increased?P<0.05?,while the ratio of CD38⁺IgD^-GC-like cells was decreased?P<0.05?in PNS group.2.The proportions of surface CD19⁺sIgG⁺B cells,CD19⁺cIgG⁺B cells,CD27⁺sIgG⁺B cells and CD38⁺cIgG⁺B cells were lower in the patient group than in the HC Group?P<0.05?.3.The patients with PNS showed a trend towards a decreased relative percentage of CD19⁺sIgG⁺B cells and CD19⁺cIgG⁺B cells among the CD19⁺IgG⁺and CD19⁺IgM⁺B cells.4.There was a strong correlation between the proportion of CD19⁺sIgG⁺B cells as well as CD19⁺cIgG⁺B cells and serum IgG levels?r=0.449,P=0.04;r=0.535,P=0.009?.5.The expression of CD86 on CD19⁺B cells increased in patients with PNS compared to HC subjects?P<0.05?and the expression of CD95 and apoptosis rate had no difference between the two groups?P>0.05?.Conclusion:There are disturbance of B cells subsets and decreased percentage of switched IgG⁺cells which may participate in the occurrence of low IgG level in PNS.PART ? THE EFFECT OF CIRCULATING FOLLICULAR T HELPER CELLS ON IMPAIRED IMMUNOGLOBULIN CLASS-SWITCH RECOMBINATION OF B CELLS IN CHILDREN WITH PRIMARY NEPHROTIC SYNDROMEObjective: The abnormity of B cells and the relationship with IgG were found in part I.Current studies have found the TFH cells provide key signal for the proliferation,differentiation and Ig production of B cells.Therefore,we determine whether TFH cells are involved in the damaged Ig generation or CSR of B cells.Methods: The percentage of CD4+CXCR5+ TFH cells,TFH1 cells,TFH2 cells and TFH17 cells as well as the Median fluorescence intensity?MFI?expression levels of ICOS on TFH cells and their three subsets were analysed by flow cytometry.The percentage of IL-4+ TFH cells and IL-21+TFH cells was measured after stimulation with PMA+I+BFA in vitro.Besides,the vital functional molecule CD40 L on TFH Cells and CD40 on CD19+ B Cells was determined.Besides.The AID m RNA level was measured directly and after 5 days culture in vitro including blank group,CD40 L stimulation group and CD40L+anti-CD40 inhibition group by RT-PCR,respectively.Results: Compared with healthy controls,the percentage of TFH cells and their three subsets were similar?P>0.05?,while expression of ICOS on TFH cells and their three subsets increased in patients?P<0.05?.There was no difference of IL-4+ TFH and IL-21+ TFH between the two groups?P>0.05?.Moreover,we did not find a difference in the CD40 expression level on the CD19+ cells of the two groups?P>0.05?,while a significant decrease in the frequency of CD40L+ TFH was present in the patients with PNS?P<0.05?.No difference of AID m RNA was observed without culture in vitro in two groups.The isolated PBMCs had significantly increased AID m RNA expression following CD40 L stimulation in PNS group?P<0.05?,however,the increased AID m RNA level was still significantly lower in patients with PNS than HC subjects?P<0.05?.Conclusion: TFH cells are possibly associated with low levels of serum immunoglobulin G due to impaired immunoglobulin class-switch recombination of B cells through CD40-CD40L/AID in children with primary nephrotic syndrome.PART ? SEARCH FOR THE DIFFERENT EXPRESSION GENES?DEGs?AND POTENTIAL CANDIDATE PATHWAYS ON CD19+ B CELLS BY TRANSCRIPTOME AND BIOINFORMATICS ANALYSIS IN CHILDREN WITH PRIMARY NEPHROTIC SYNDROMEObjective: With the aid of bioinformatics and related analysis tools,the CD19+ B cells from PNS children and healthy control were analyzed by RNA-seq to figure out the differential expression genes especially for the key molecules and signal pathway closely related to the production of immunoglobulins and immunoglobulin class-switch recombination of B cells.Methods: 11 blood samples was collected from patients diagnosed as PNS at onset and 11 healthy control children were recruited during the same period.The CD19+ B cells were purified using a FACS cell sorter and DEGs were screened out by high-throughput transcriptome sequencing using Illumina platform and analyzed by EBSeq2 in R software between two groups.The expression level of gene was normalized with TPM and the distribution of gene expression level was analyzed.The up regulated and down regulated genes were clustering analysis and enriched analysis by GO?Gene Ontology?and KEGG pathway?Kyoto Encyclopedia of genes and genes?.The protein-protein interaction?PPI?for differential genes were constructed using string database and visualized to identify hub genes.Results: The distribution of gene expression level was equal and suitable to the next analysis.There were 664 of differential expression genes?DEGs?,including 547 up-regulated DEGs and 117 down-regulated DEGs?p-adjust < 0.05 & |log2FC|>= 1?.The difference of gene expression pattern between the two groups was found by the clustering analysis.The enrichment analysis of DEGs by GO and KEGG pathway revealed that B cell receptor signal,activation and antigen bind as well as PI3K-Akt pathway and NF-?b pathway were the significant enrichment?p-adjust <0.05?.The close relationship between PNS phenotype and IgG memory and Ig M memory as well as plasma cell and naive B cell was obtained by GSEA analysis?|NES|>1 & FDR q-val<0.25?.According to the degree centrality of DEGs,top six hub genes were selected from the PPI network analysis and visualization.They were CDK1,PLK1,BUB1,BUB1 B,MADIL1 and CCNB1,which are all involved in mitosis process.Conclusion: The RNA-seq data demonstrate there are vital effect of B cell receptor signal in PNS.At the same time,we should pay attention to the PI3K-Akt pathway,NF-?b pathway and cell division related CSR events for the mechanism research of low IgG in the future.