Study On The Abnormal Mechanism Of Exosome Complement In IgG4-related Disease

Background and objective:IgG4 related disease(IgG4-RD)is a chronic fibroinflammatory disease characterized by tufecive swelling of involved organs,elevated serum IgG4 and lymphocytes,plasma cells,especially IgG4+plasma cells infiltration in involved organs.The most commonly affected organs are submandibular glands,lacrimal glands,parotid gland,pancreas,bile ducts,lung,retroperitoneum/aorta.Allergic symptoms are frequently present in about 60%of IgG4-RD,patients with allergy have higher tendency of head-neck involvement and eosinophila.Irreversible organ function damage occurs due to persistent tissue fibrosis.So,to comprehensively investigate the pathogenesis of IgG4-RD is of urgent need.Protein profiling of plasma-derived exosomes is not elucidated in IgG4-RD.Therefore,the aim of this study is to investigate the protein profiling of plasma-derived exosomes by Liquid chromatography-tandem mass spectrometry(LC-MS/MS).Next,proteomics of IgG4-RD patients with/without allergy were also investigated in order to demonstrate the immune background,relationship and mechanism of allergy to organ specific damage.Submandibular glands(SMGs)are commonly used as the research specimens in patients with IgG4-RD for tissue protein mass spectrometry verification.Finally,the protein expression profiling of submandibular glands in IgG4-RD were evaluated to define whether exosome participated a role in organ damage.Materials and methods134 untreated patients with IgG4-RD and 150 gender-age matched healthy controls were enrolled in our study.This study was approved by the ethnic committee,written informed consent were obtained for all patients and controls enrolled.Exosomes were extracted by ultra-centrifuge and exosome extract kit.Exosome proteins were labled by TMT and detected by LC-MS/MS.Differentially expressed proteins(DEPs)were validated by ELISA and WB.B cell differentiation and ROS expression level were detected by flowcytometry analysis.In addition,B cells cultured by IgG4-RD and HC exosomes were detected by LC-MS/MS.Besides,plasma protein profiling of 15 IgG4-RD patients with allergy,15 IgG4-RD without allergy and 15 gender-age matched were detected.Finally,proteomics expression was detected in 4 biopsy-proven IgG4-RD and 4 biopsy-proven chronic sialadentis by isobaric tags for relative and absolute quantitation(iTRAQ).ResultsA total of 178 differentially expressed proteins were identified in plasma derived exosomes in IgG4-RD patients compared with HCs,and these proteins were enriched predominantly in the complement cascade pathway.Furthermore,reduced expression levels of complement components C3 and C5 in IgG4-RD were correlated with clinical parameters.Following stimulation with IgG4-RD plasma derived exosomes,the percentages of na(?)ve B cells decreased,while those of memory B cells and plasmablasts increased;the levels of cytochrome c,somatic(CYCS)and downstream complement system activation also increased.The proteome revealed integrin pathway and complement cascade pathway were downregulated(inhibited)of IgG4-RD patients with allergy compared with those without allergy,indicating complement system and integrin pathway participated in pathogenesis of IgG4-RD with allergy.A total of 269 DEPs were identified in IgG4-RD SMGs compared with controls,including 141 upregulated proteins and 128 downregulated proteins.In terms of cell subsets infiltration,T cells,B cells,macrophages and platelets were activated significantly.Immune related proteins were altered tremendously in IgG4-RD compared with controls.GO analysis indicated altered proteins enriched in signal transduction,cell communication and immune response in biological process.KEGG pathway analysis showed DEPs mainly enriched in immune pathway activation,cytotoxicity,Fc gamma R-mediated phagocytosis,and metabolic abnormalities.According to disease correlation analysis,altered proteins associated with immune system disease,lymphoproliferative disorders,immunologic deficiency syndromes,homological malignancies.LYN,CR2,SYK,CD72 and PTPN6 were identified as hub proteins,indicating BCR signaling pathway activation.In addition,exosomes were also enriched in SMGs of IgG4-RD.ConclusionProteomic profiling revealed that complement cascade pathway activated plasma derived exosome proteins,which may participate in the pathogenesis of IgG4-RD through complement activation and may be involved in B cell differentiation and activation of the B cell auto-oxidative damage pathway.Inhibition of complement and integrin pathways in plasma exosome of IgG4-RD patients with allergy,indicating complement and integrin involved in disease progression.Exosome enriched in SMGs which may indicate exosome parcipated in tissue damage in IgG4-RD.Further mechanism needs to be evaluated.