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

Abstract

In rheumatoid arthritis (RA), extracellular vesicles (EVs) are associated with both the propagation and attenuation of joint inflammation and destruction. However, the specific EV content responsible for these processes is largely unknown. Investigations into identifying EV content are confounded by the challenges in obtaining high-quality EV preparations from synovial fluid. Implementing a size exclusion chromatography-based method of EV isolation, coupled with small RNA sequencing, we accurately characterised EV miRNAs in synovial fluid obtained from RA patients and investigated the differences between joints with high- and low-grade inflammation. Synovial fluid was obtained from the joints of 12 RA patients and, based on leukocyte counts, classified as either high (n = 7)- or low (n = 5)-grade inflammation. Using size exclusion chromatography, EVs were purified and small RNA was extracted and sequenced on a NextSeq 500. Sequencing reads were aligned to miRBase v21, and differences in miRNA profiles between RA patients with high- and low-grade joint inflammation were analysed. In total, 1972 distinct miRNAs were identified from RA synovial fluid EVs. miRNAs with less than five reads in fewer than five patients were filtered out, leaving 318 miRNAs for analysis. Analysis of the most abundant miRNAs suggested that they negatively regulate multiple genes relevant to inflammation, including signal transducer and activator of transcription 3 (STAT3), which lies downstream of IL-6 and has a pro-inflammatory role in RA. Synovial fluid from joints with high-grade inflammation contained 3.5-fold more EV miRNA per mL of synovial fluid (p = 0.0017). Seventy-eight EV miRNAs were differentially expressed between RA joints with high- and low-grade inflammation, and pathway analysis revealed that their target genes were commonly involved a variety of processes, including cellular apoptosis, proliferation and migration. Of the 49 miRNAs that were elevated in joints with high-grade inflammation, pathway analysis revealed that genes involved in cytokine-mediated signalling pathways were significantly enriched targets. In contrast, genes associated with reactive oxygen species signalling were significantly enriched as targets of the 29 miRNAs elevated in joints with low-grade inflammation. Our study identified an abundance of EV miRNAs from the synovial fluid of RA patients with the potential to modulate inflammation. In doing so, we defined potential mechanisms by which synovial fluid EVs may contribute to RA pathophysiology.

TMT-Based Quantitative Proteomics Analysis of Synovial Fluid-Derived Exosomes in Inflammatory Arthritis

“…Analyzing the composition of SF is helpful to clarify the pathogenesis and distinguishing biomarkers of IA. Recently, the function of exosomes in SF was established ( 15 , 16 ). Exosomes in SF can lead to inflammation, degeneration of cartilage, and destruction of joints ( 10 ).…”

Technological Approaches in the Analysis of Extracellular Vesicle Nucleotide Sequences

“…Urinary EVs’ isomiRs of miR-21, miR-204, and miR-375 are highly differentially expressed in prostate cancer individuals ( Koppers-Lalic et al, 2016 ), while urinary EV-increased expressions of miR-29a-3p and miR-200a-3p are altered in patients with nephropathy in Fabry disease ( Levstek et al, 2021 ). The altered EV miRNA levels are also reported in autoimmune diseases such as systemic lupus erythematosus, Sjogren’s syndrome, and type 1 diabetes ( Michael et al, 2010 ; Katsiougiannis et al, 2019 ; Tesovnik et al, 2020 ; Foers et al, 2021 ).…”

Made by cells for cells – extracellular vesicles as next-generation mainstream medicines

“…Because of their complex composition, EVs offer unique opportunities not only for early and precise detection of a disease, but also to obtain insights into the disease stage and its pathogenesis (Bongiovanni et al, 2021). For instance, a recent study has shown that EV miRNAs in synovial fluid are different in the joints of rheumatoid arthritis patients with high-and low-grade inflammation (Foers et al, 2021). Moreover, there is an upregulation of diseaseassociated miRNAs in brain-derived EVs in Alzheimer's patients, which has been exploited to establish the first early diagnostic blood test for a neurodegenerative disease (Cheng et al, 2020).…”