Proteomic analysis of extracellular vesicles reveals an immunogenic cargo in rheumatoid arthritis synovial fluid

• Published in: Clinical & translational immunology 2020, Vol.9 (11), p.e1185-n/a
• Main Authors: Foers, Andrew D, Dagley, Laura F, Chatfield, Simon, Webb, Andrew I, Cheng, Lesley, Hill, Andrew F, Wicks, Ian P, Pang, Ken C
• Format: Article
• Language: English
• Subjects: Chromatography; citrullination; Citrulline; Datasets; Degranulation; Extracellular vesicles; Fibroblasts; Immunogenicity; Inflammation; Joint diseases; Mass spectroscopy; Nanoparticles; Neutrophils; Original; Osteoarthritis; Patients; Peptides; Proteins; Proteomes; Proteomics; Rheumatoid arthritis; Synovial fluid; Transmission electron microscopy
• ISSN: 2050-0068; 2050-0068
• DOI: 10.1002/cti2.1185

Objectives Extracellular vesicles (EVs) from rheumatoid arthritis (RA) synovial fluid (SF) have been reported to stimulate the release of pro‐inflammatory mediators from recipient cells. We recently developed a size exclusion chromatography (SEC)‐based method for EV isolation capable of high‐quality enrichments from human SF. Here, we employed this method to accurately characterise the SF EV proteome and investigate potential contributions to inflammatory pathways in RA. Methods Using our SEC‐based approach, SF EVs were purified from the joints of RA patients classified as having high‐level (n = 7) or low‐level inflammation (n = 5), and from osteoarthritis (OA) patients (n = 5). Protein profiles were characterised by mass spectrometry. Potential contributions of EV proteins to pathological pathways and differences in protein expression between disease groups were investigated. Results Synovial fluid EVs were present at higher concentrations in RA joints with high‐level inflammation (P‐value = 0.004) but were smaller in diameter (P‐value = 0.03) than in low‐level inflammation. In total, 1058 SF EV proteins were identified by mass spectrometry analysis. Neutrophil and fibroblast markers were overrepresented in all disease groups. Numerous proteins with potential to modulate inflammatory and immunological processes were detected, including nine citrullinated peptides. Forty‐five and 135 EV‐associated proteins were significantly elevated in RA joints with high‐level inflammation than in RA joints with low‐level inflammation and OA joints, respectively. Gene ontology analysis revealed significant enrichment for proteins associated with ‘neutrophil degranulation’ within SF EVs from RA joints with high‐level inflammation. Conclusion Our results provide new information about SF EVs and insight into how EVs might contribute to the perpetuation of RA. Here, we apply a stringent size‐exclusion chromatography‐based method in combination with quantitative proteomics to accurately profile protein content within synovial fluid extracellular vesicles from a cohort of arthritis patients. Our results show large numbers of extracellular vesicles from neutrophil and fibroblast origin are present in rheumatoid arthritis synovial fluid, and these contain a protein cargo with capacity to stimulate immune responses. Our results provide new information about synovial fluid extracellular vesicles and insight into how extracellular vesicles might contribute to the perpetuation of rh