Detection Of Plasma Exosome Protein Profiles In IgG4-related Diseases And Research On B Cell And T Cell Differentiation And Tissue Damage

Part 1 Investigation of plasma exosome differentially expressed proteins in IgG4-RDBackground and aimIgG4-related disease(IgG4-RD)is a newly recognized chronic fibroinflammatory disease,characteriszed by tumefactive swelling of affected organs,infiltration of dense lymphoplasmatic cells especially IgG4 positive plasma cells in involved organs and serum IgG4 is always elevated(>1350mg/L).IgG4-RD is multi-organ affected disease with etiology needed to be eludicated.The abnormality of B cells and plasmablast plays pivotal role in the disturbance of humoral immunity in IgG4-RD.However,the regulatory mechanism of B cell activation and differentiation to plasmablast needs to be elucidated.Exosome Exosomes are nanoscale extracellular vesicles with sizes of 30 to 100 nm(in diameter)that are secreted by most mammalian cells.Exosome exists in body fluids which induces inflammatory cytokines by secreting or direct interation.In that way,multiple immune cells are activated indicating that the immune response is strenthgend.So we aim to elucidate the mechanism of exosome on the activation of B cells of IgG4-RD,and also the role of exosome on B cell class switch and differentiation to plasmablast.Materials and methodssixty-two untreated IgG4-RD patients and sex,age matched healthy controls(HCs)were enrolled in this study.All patients fulfilled the fulfilled the 2011 comprehensive diagnostic criteria of IgG4-RD.10 milliliter peripheral blood of patients and HCs were collected.Plasma was obtained and stored in minus 80 degrees refrigerator.The plasma samples from patients and normal volunteers were pooled together,respectively,to perform isolation of exosomes by ultracentrifugation and Total Exosome Isolation Reagent for further liquid chromatography(LC)-MS/MS analysis.The identified proteins were classified by FunRich software version 3 and gene ontology(GO)based on the protein's molecular function,cellular components,and biological process.Enrichment analysis was performed using the web-based Gene Set AnaLysis Toolkit.The statistical significance of a pathway was based on P<0.05 and the presence of at least four target genes in the Wiki Pathway.Meanwhile,STRING was performed to find the potential biological function for protein-protein interaction.Next,exosome extracted from plasma of 32 IgG4-RD patients and matched HCs for Enzyme linked immunosorbent assay(ELISA)and western blot analysis(WB)to validate the differentially expressed proteins.Results1.By bioinformational analysis,we identified a total of 329 proteins.Compared with HCs,the expression ratio of each protein was acquired by comparing IgG4-RD patients group with normal volunteers group,and ratios greater than 1.2-fold or less than 0.83-fold were defined as up-or downregulated,respectively.In total,82 proteins were upregulated and 96 proteins were downregulated in IgG4-RD patients group as compared with normal volunteers group.2.Differentially expressed genes were enriched in exosome,complement function and immune response by GO analysis.It was found that target protein enrichment in immunodeficiency diseases ranked third of reliability through the correlation analysis of the disease.Wikipathway analysis revealed that complement cascade pathway in exosome of IgG4-RD was activated,together with reduced C3 complement and C5 complement,while SERPING1 and CFI of complement activation pathway were significantly up-regulated.The above results indicated complement activation in exosome may be related to IgG4-RD.3.C3 in exosome of IgG4-RD was lower compared withHCs(70.97±28.93 ?g/ml vs 113.49±37.51 ?g/ml,P<0.0001).In addition,C5a in exosome of IgG4-RD was lower compared with HCs as well(6.14±6.46 ng/ml vs 9.41±8.17 ng/ml,P=0.02).Whereas,C1q in exosome was comparable between HCs and IgG4-RD(119.37±55.11 ?g/ml vs 105.28±36.95?g/ml,P=0.242).4.C3 in exosome of IgG4-RD correlated negatively with ESR,serum IgG,IgG1 and IgG4,r=-0.465,r=-0.450,r=-0.416 and r=-0.391 respectively;P value was P=0.01,P=0.02,P=0.03,P=0.04 respectively.C5a in exosome of IgG4-RD correlated negatively with serum IgG1 and IgG4-RD RI,r=-0.524 and r=-0.405 respectively,P=0.006 and P=0.04 respectively.5.Detection of exosomal complement C3,C5 and C1 inhibitors by WB.Results revealed that exosomal C3 expression was lower in IgG4-RD patients than in HC(57.53±19.89 vs 84.41±35.95),P=0.02.The expression of exosomes C5 in HC exosomes(142.96±43.13)was higher than that of IgG4-RD(114.15±36.06),P=0.03.While SERPING1 was higher in IgG4-RD exosomes than HC(199.47±37.05 vs 159.64±51.65),P=0.04.ConclusionThere were 82 differentially expressed upregulated proteins and 96 downregulated proteins in lgG4-RD exosome compared with HCs.Differentially expressed genes were enriched in exosome,complement function and immune response indicating complement activation in exosome may be related to IgG4-RD.Reduced C3 and C5 expression in exosome of IgG4-RD may indicate the disease severity.Part 2 IgG4-RD exosomes participated in the B cells,T cells differentiation and tissue damageBackground and aimIn our previous study,complement system was activated in IgG4-RD exosome compared with healthy controls.The abnormality of complement in IgG4-RD exosome correlated with immunoglobulin and IgG4-RD responder index.Dense infiltration of T cells and B cells,especially Th2 cells and plasmablasts in IgG4-RD affected tissues were found to participate in tissue damage.The decrease of serum IgG4-RD complement level may indicate disease activity.In this study,we further investigate the potential role of IgG4-RD exosome in B cell and T cells in order to elucidate whether exosome participate in the pathogenesis of IgG4-RD.Materials and methodsFifty-eighty untreated IgG4-RD patients and sex-age matched 73 healthy controls were enrolled in this study.The effects of IgG4-RD exosome on B and T cell activation,proliferation,apoptosis and differentiation was measured by flowcytometry.The changes of B cell antibody class-switch genes was measured by qPCR.To further clarify the effect of IgG4-RD exosome on B cell,proteome analysis was performed.The expression of exosomes in the submandibular gland tissues of IgG4-RD patients was measured by LC-MS and compared non-specific chronic salivitis patients.Results1.IgG4-RD exosome in B cell activation,proliferation and apoptosis was comparable with HC exosome.2.CD19+IgD+CD38+/-naive B deceased by stimulation of IgG4-RD exosome compared with HCs(57.75%±7.09%vs 63.23%±6.89%),P=0.01.While CD19+IgD-CD27+memory B cells increased by IgG4-RD exosome stimulation compared with HCs(10.48%±3.15%vs 7.52%±2.98%),P=0.007.CD19+IgD-CD38hi plasmablast was also increased by IgG4-RD exosome stimulation than HC exosome(4.07%±1.92%vs2.47%±1.34%),P=0.006.3.XBP1 relative expression was elevated by IgG4-RD exosome stimulation than HC exosome in primary B cells(2.388±1.332 vs 1.023±0.249),P=0.049.AICDA was elevated by IgG4-RD exosome stimulation than HC exosome in Ramos B cells(1.415±0.234 vs 1.014±0.185),P=0.01.4.Compared with HC exosome stimulation,98 differentially expressed proteins was indentified in B cells stimulated by IgG4-RD exosome,including 60 upregulated proteins and 38 downregulated proteins.5.In order to clarify the interaction of B cells differentially expressed proteins,we performed Cytoscape analysis.Totally 56 differentially expressed proteins were successfully matched in STRING database.In the protein interaction network,the protein CYCS was located at the central node,which is significantly upregulated in B cells stimulated by IgG4-RD patients' exosomes and participates in multiple histone interactions.Pathway analysis was performed,and it was found that the oxidative damage pathway protein CYCS was significantly up-regulated and complement system was down-regulated in B cells stimulated by IgG4-RD exosome.These results revealed that the stimulation of B cells by IgG4-RD exosomes may activate the auto-oxidative damage pathway of B cells.6.ROS in CD19+B cells was increased in IgG4-RD compared with HCs(23.85%±13.94%vs 10.94%±7.57%),P=0.001.ROS MFI in CD19+B cells was also elevated in IgG4-RD than HCs(2720±1223 vs 1873±764),P=0.04.By further investigation in B cell subsets,we found ROS in CD19+CD24-CD38hi plasmablast/plasma cells was higher in IgG4-RD compared with HCs(59.17%±23.73%vs 30.68%±20.15%),P=0.003.ROS iMFI was also higher in CD19+CD24-CD38hi plasmablast/plasma cells of IgG4-RD than HCs[336(74-1416)vs 124(22-174)],P=0.004.In addition,ROS expression in CD19+CD24intCD38int naive B cells of IgG4-RD patients was higher than that of HC(17.52%±13.35%vs 8.44%±4.50%),P=0.02.ROS staining of memory B cells in IgG4-RD patients,suggesting that ROS expression of CD19+CD24hiCD38-memory B cells in IgG4-RD patients also increased compared with HC(22.21%±19.85%vs 10.44±5.72%),P=0.01.7.CD4+IL-4+Th2 cells were higher when stimulated with IgG4-RD exosomes than HC exosome(9.29%±3.68%vs 7.34%±2.70%),P=0.0497.Compared with HC exosomes,IgG4-RD exosomes had no significant effect on T cell activation,proliferation,and apoptosis.8.Compared with results from patients with chronic non-specific submaxillitis,555 differential proteins,including 346 up-regulated proteins and 209 down-regulated proteins were found in submandibular glands from IgG4-RD.GO analysis showed that the differential proteins are mainly involved in biological processes such as lysosomes,exosomes,and cytoplasm et al.Pathway enrichment revealed that multiple proteins in the B cell receptor signaling pathway,including LCK,SYK,HCLS1,LYN,GRB2,BTK,VAV1,PTPN6,and PLCG2 were significantly up-regulated,revealing that changes in IgG4-RD submandibular gland tissue may be related to B cell receptor activation.Whether exosomes are involved in B cell abnormalities of tissues or participated in tissue damage requires further study.ConclusionOur research suggests that IgG4-RD exosomes can participate in the transformation of IgG4-RD B cell antibody classes,activate the B cell auto-oxidative damage pathway and potentially participate in the pathogenesis of IgG4-RD.Besides,IgG4-RD exosome could induce the differentiation of naive T into Th2 cells.Exosome in tissues may participated in IgG4-RD tissue damage.