Proteomics Profiling Of CD4~+T Cell-derived Exosomes From Patients With Rheumatoid Arthritis

Background:Rheumatoid arthritis(RA)is a common autoimmune disease that can lead to severe joint damage and disability.CD4+T cells play an important role in the development of RA.Exosomes are common membrane-fused nanoscale vesicles,30-150 nm in diameter,containing a variety of biomolecules,such as lipids,proteins and nucleic acids.Exosomes can mediate immune stimulation or suppression and drive inflammation,autoimmune responses and pathological processes of infectious diseases.Immune cell-derived exosomes can mediate communication between different cells of RA.Tandem mass tags(TMT)labeled quantification is a proteomics technique that utilizes isotope tags for the precise quantification of peptides and proteins.The TMT technique combined with liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS)method can effectively capture and quantify proteins and benefit the screening of biomarkers.This study aimed to perform proteomic analysis of CD4+T cell-derived exosomes of RA by TMT combined with LC-MS/MS technology to search for biomarkers and explore the potential pathogenesis of RA.Methods:ExoQuick kit was used to isolate exosomes from the supernatants of CD4+T cells.In this study,CD4+T cell-derived exosomes were collected from RA and control groups,while proteomic analysis of CD4+T cell-derived exosomes was performed by TMT combined with LC-MS/MS technique.Differentially expressed proteins were screened according to fold change(FC).FC≥1.1 and P<0.05 were differentially expressed proteins upregulated in the RA group,whereas FC<1/1.1 and P<0.05 were differentially expressed proteins downregulated in the RA group.Volcano plots and clustered heat maps were used to show the differential expression of proteins between the RA and control groups.We conducted gene ontology(GO)enrichment analysis,kyoto encyclopedia of genes and genomes(KEGG)analysis and gene set enrichment analysis(GSEA)on proteins,and the most significantly up-regulated and down-regulated proteins were further validated by enzyme linked immunosorbent assay(ELISA)and western blot(WB).Results:The exosomes we obtained had a typical vesicle-like structure with a diameter between 60-120 nm and express CD81 and TSG101,which were classical markers of exosomes.Compared to the control group,the proteomic results showed that there were 3 up-regulated differentially expressed proteins and 31 down-regulated differentially expressed proteins in the RA group.It indicated that dihydropyrimidinaserelated protein 3(DPYSL3)and APCS(serum amyloid p-component)were significantly increased in CD4+T cell-derived exosomes,whereas proteasome activator complex subunit 1(PSME1)and proteasome activator complex subunit 2(PSME2)were significantly decreased in the RA group.GO analysis showed that the differentially expressed proteins were mainly associated with the pathways of "positive regulation of gene expression" and "positive regulation of smooth muscle cell proliferation".Analysis of the KEGG pathway indicated that the differential expressed proteins were mainly associated with the "processing and presentation of antigen"pathway.GSEA results showed that the proteins were connected with the "acute phase response" and "PI3K-AKT" signaling pathways.ELISA results showed that compared to the control group,DPYSL3 was significantly up-regulated in CD4+T cell-derived exosomes in the RA group,whereas PSME1 was down-regulated in CD4+T cell-derived exosomes,which was consistent with the results of proteomics profiling.Conclusion:The results of this study indicated that the proteins in CD4+T cellderived exosomes differed between the RA and control groups.CD4+T cell-derivedexosomes may be involved in the pathogenesis of RA through signaling pathways such as "smooth muscle cell proliferation","antigen processing and presentation" and"PI3K-AKT".DPYSL3 and PSME1 may become useful biomarkers for RA.It may help to clarify the diagnosis of RA and to explore the pathogenesis of RA further.