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

Abstract

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 (Pvalue = 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.

Extracellular Vesicles in Synovial Fluid from Rheumatoid Arthritis Patients Contain miRNAs with Capacity to Modulate Inflammation

“…These observations are consistent with our previous report detailing SEC as a method for obtaining high-quality EV enrichments from SF [ 10 ]. Previously, we demonstrated that RA patients with high-grade synovial inflammation have increased concentrations of SF EVs [ 11 ]. Consistent with this, we detected a 3.5-fold increase in the EV miRNA concentration per mL of SF in joints with high-grade inflammation ( p -value = 0.0017; Figure 1 C), suggesting the potential involvement of SF EV miRNAs in inflammatory processes in RA.…”

“…For example, PTEN is targeted by multiple miRNAs enriched in high- and low-grade inflammation, as well as four of the 10 most prevalent miRNAs across all patients. Considering our previous observation of significant increases in total SF EV numbers within RA joints with high-grade inflammation when compared to those with low-grade inflammation [ 11 ]—combined with the significant enrichment in total EV miRNA concentration from joints with high-grade inflammation seen here (3.5-fold; p -value = 0.0017)—EV miRNA levels within RA joints could have a profound effect on inflammatory activity, whereas differences in EV miRNA profiles between joints with high- and low-grade inflammation might make more subtle contributions.…”

“…SF was obtained from RA patients undergoing arthrocentesis, as previously described [ 10 , 11 ], and used with informed consent and the approval of the Melbourne Health Human Research Ethics Committee (projects 2005.056 and 2010.293). Following needle aspiration, SF was centrifuged at 2000× g for 20 min to remove cells, then aliquoted and stored at −80 °C until the time of experiment.…”

“…Following needle aspiration, SF was centrifuged at 2000× g for 20 min to remove cells, then aliquoted and stored at −80 °C until the time of experiment. SF was classified as originating from joints with either high- or low-grade inflammation, as reported previously [ 11 ]. Specifically, the inflammatory status of the index aspirated joint was assessed based on the presence of SF white cell counts of either greater or less than 2000 cells/µL, which is a cut-off previously assessed as showing reasonable performance in distinguishing between inflammatory and non-inflammatory disease (sensitivity: 0.84; specificity: 0.84) [ 31 ].…”

Stromal cell–derived DEL-1 inhibits Tfh cell activation and inflammatory arthritis

“…EDIL3, the DEL-1-encoding gene, is a susceptibility locus in ankylosing spondylitis, a type of inflammatory arthritis that affects the vertebrae (65). Moreover, the DEL-1 protein is enriched in the synovial fluid of RA patients with low-level inflammation as compared to RA patients with high-level inflammation (66), suggesting that DEL-1 might play a protective role in human RA. These reports and our findings from the present study, therefore, suggest that DEL-1 may be a promising novel therapeutic for the treatment of inflammatory arthritis.…”