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.

Keywords: extracellular vesicles; miRNA; rheumatoid arthritis; synovial fluid.

Figure 1

Assessment of synovial fluid EV enrichments prepared using size-exclusion chromatography. (A) Synovial fluid EV enrichments prepared by SEC were assessed for the presence of the canonical EV markers (HSP70, syntenin, FLOT1, Rab 27b, TSG101 and annexin 1) via Western blotting. (B) The quality of EV enrichments was further assessed by transmission electron microscopy (scale bars = 200 nm). (C) miRNA concentration per mL of rheumatoid arthritis synovial fluid in joints with high- or low-grade joint inflammation. Group means are indicated. Data analysed with Student’s i-test. Error bars = SEM. ** denotes p-value < 0.01.

Figure 2

Biological processes associated with experimentally validated targets of highly ranked SF EV miRNA. Pathway analysis was performed on experimentally validated gene targets of the 10 highest-ranked miRNAs. The percentage of target genes associated with each biological process are indicated.

Figure 3

SF EV miRNAs have diverse immunoregulatory capacities. (A) MA plot of miRNA counts per million reads (CPM) vs. fold change. Differentially expressed miRNAs with an adjusted p-value < 0.05 are highlighted in blue. miRNAs with an adjusted p-value < 0.05 and a log2 fold change >3 are labelled. (B,C) Biological processes associated with target genes of the miRNAs significantly increased in joints with (B) high-, and (C) low-grade inflammation. The percentage of target genes associated with each biological process is indicated.