{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T22:24:57Z","timestamp":1785450297300,"version":"3.56.0"},"reference-count":36,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,5,6]],"date-time":"2021-05-06T00:00:00Z","timestamp":1620259200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Royal Children\u2019s Hospital Foundation","award":["-"],"award-info":[{"award-number":["-"]}]},{"name":"Reid Family Trusts","award":["-"],"award-info":[{"award-number":["-"]}]},{"name":"National Health and Medical Research Council of Australia","award":["1154325; 1113577"],"award-info":[{"award-number":["1154325; 1113577"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/ijms22094910","type":"journal-article","created":{"date-parts":[[2021,5,6]],"date-time":"2021-05-06T11:10:27Z","timestamp":1620299427000},"page":"4910","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":31,"title":["Extracellular Vesicles in Synovial Fluid from Rheumatoid Arthritis Patients Contain miRNAs with Capacity to Modulate Inflammation"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5036-7906","authenticated-orcid":false,"given":"Andrew D.","family":"Foers","sequence":"first","affiliation":[{"name":"The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia"},{"name":"Department of Medical Biology, University of Melbourne, Parkville 3052, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra L.","family":"Garnham","sequence":"additional","affiliation":[{"name":"The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia"},{"name":"Department of Medical Biology, University of Melbourne, Parkville 3052, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon","family":"Chatfield","sequence":"additional","affiliation":[{"name":"The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia"},{"name":"Department of Medical Biology, University of Melbourne, Parkville 3052, Australia"},{"name":"Department of Rheumatology, Royal Melbourne Hospital, Parkville 3050, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9221-2892","authenticated-orcid":false,"given":"Gordon K.","family":"Smyth","sequence":"additional","affiliation":[{"name":"The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia"},{"name":"School of Mathematics &amp; Statistics, University of Melbourne, Parkville 3010, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lesley","family":"Cheng","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5581-2354","authenticated-orcid":false,"given":"Andrew F.","family":"Hill","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ian P.","family":"Wicks","sequence":"additional","affiliation":[{"name":"The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia"},{"name":"Department of Medical Biology, University of Melbourne, Parkville 3052, Australia"},{"name":"Department of Rheumatology, Royal Melbourne Hospital, Parkville 3050, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6881-775X","authenticated-orcid":false,"given":"Ken C.","family":"Pang","sequence":"additional","affiliation":[{"name":"The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia"},{"name":"Murdoch Children\u2019s Research Institute, Parkville 3052, Australia"},{"name":"Department of Paediatrics, University of Melbourne, Parkville 3052, Australia"},{"name":"Department of Adolescent Medicine, Royal Children\u2019s Hospital, Parkville 3052, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,5,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"654","DOI":"10.1038\/ncb1596","article-title":"Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells","volume":"9","author":"Valadi","year":"2007","journal-title":"Nat. Cell Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1350","DOI":"10.1002\/art.40076","article-title":"Review: Extracellular Vesicles in Joint Inflammation","volume":"69","author":"Foers","year":"2017","journal-title":"Arthritis Rheumatol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1038\/nrrheum.2015.170","article-title":"Extracellular vesicles\u2014New tool for joint repair and regeneration","volume":"12","author":"Malda","year":"2016","journal-title":"Nat. Rev. Rheumatol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"402","DOI":"10.3389\/fendo.2018.00402","article-title":"Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation","volume":"9","author":"Hayder","year":"2018","journal-title":"Front. Endocrinol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/ar3013","article-title":"Plasma and synovial fluid microRNAs as potential biomarkers of rheumatoid arthritis and osteoarthritis","volume":"12","author":"Murata","year":"2010","journal-title":"Arthritis Res. Ther."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"R81","DOI":"10.1186\/ar3006","article-title":"Altered microRNA expression profile with miR-146a upregulation in CD4+ T cells from patients with rheumatoid arthritis","volume":"12","author":"Li","year":"2010","journal-title":"Arthritis Res. Ther."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/j.immuni.2014.05.019","article-title":"MicroRNA-containing T-regulatory-cell-derived exosomes suppress pathogenic T helper 1 cells","volume":"41","author":"Okoye","year":"2014","journal-title":"Immunity"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"578335","DOI":"10.3389\/fgene.2020.578335","article-title":"Epigenetic Mechanisms in Immune Disease: The Significance of Toll-Like Receptor-Binding Extracellular Vesicle-Encapsulated microRNA","volume":"11","author":"Bosch","year":"2020","journal-title":"Front. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Bernard, M.A., Zhao, H., Yue, S.C., Anandaiah, A., Koziel, H., and Tachado, S.D. (2014). Novel HIV-1 MiRNAs Stimulate TNF\u03b1 Release in Human Macrophages via TLR8 Signaling Pathway. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0106006"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1490145","DOI":"10.1080\/20013078.2018.1490145","article-title":"Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography","volume":"7","author":"Foers","year":"2018","journal-title":"J. Extracell. Vesicles"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e1185","DOI":"10.1002\/cti2.1185","article-title":"Proteomic analysis of extracellular vesicles reveals an immunogenic cargo in rheumatoid arthritis synovial fluid","volume":"9","author":"Foers","year":"2020","journal-title":"Clin. Transl. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2158","DOI":"10.1016\/j.bbadis.2017.06.002","article-title":"IGF-1R signalling contributes to IL-6 production and T cell dependent inflammation in rheumatoid arthritis","volume":"1863","author":"Erlandsson","year":"2017","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1080\/08916934.2017.1344970","article-title":"Kinase inhibitors of the IGF-1R as a potential therapeutic agent for rheumatoid arthritis","volume":"50","author":"Tsushima","year":"2017","journal-title":"Autoimmunity"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-017-11233-w","article-title":"Stat3 as a potential therapeutic target for rheumatoid arthritis","volume":"7","author":"Oike","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"374","DOI":"10.1038\/ni.3691","article-title":"Mechanisms and consequences of Jak\u2013STAT signaling in the immune system","volume":"18","author":"Villarino","year":"2017","journal-title":"Nat. Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"12731","DOI":"10.18632\/oncotarget.7262","article-title":"miR-451a is underexpressed and targets AKT\/mTOR pathway in papillary thyroid carcinoma","volume":"7","author":"Minna","year":"2016","journal-title":"Oncotarget"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5965","DOI":"10.4049\/jimmunol.1201437","article-title":"miR-451 Regulates Dendritic Cell Cytokine Responses to Influenza Infection","volume":"189","author":"Rosenberger","year":"2012","journal-title":"J. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"6004","DOI":"10.1074\/jbc.M116.765107","article-title":"Down-regulation of microRNA-451a facilitates the activation and proliferation of CD4+ T cells by targeting Myc in patients with dilated cardiomyopathy","volume":"292","author":"Zeng","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"996","DOI":"10.1002\/jcb.21335","article-title":"MicroRNA-223 is a key factor in osteoclast differentiation","volume":"101","author":"Sugatani","year":"2007","journal-title":"J. Cell. Biochem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4667","DOI":"10.1074\/jbc.M805777200","article-title":"Impaired Micro-RNA Pathways Diminish Osteoclast Differentiation and Function","volume":"284","author":"Sugatani","year":"2009","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3240","DOI":"10.1002\/art.34550","article-title":"Brief Report: Amelioration of collagen-induced arthritis in mice by lentivirus-mediated silencing of microRNA-223","volume":"64","author":"Li","year":"2012","journal-title":"Arthritis Rheum."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Chen, Q., Wang, H., Liu, Y., Song, Y., Lai, L., Han, Q., Cao, X., and Wang, Q. (2012). Inducible MicroRNA-223 Down-Regulation Promotes TLR-Triggered IL-6 and IL-1\u03b2 Production in Macrophages by Targeting STAT3. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0042971"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1125","DOI":"10.1038\/nature06607","article-title":"Regulation of progenitor cell proliferation and granulocyte function by microRNA-223","volume":"451","author":"Johnnidis","year":"2008","journal-title":"Nature"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.cell.2018.03.006","article-title":"Metazoan MicroRNAs","volume":"173","author":"Bartel","year":"2018","journal-title":"Cell"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"E2110","DOI":"10.1073\/pnas.1209414109","article-title":"MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response","volume":"109","author":"Fabbri","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.clim.2016.12.005","article-title":"Estrogen-regulated STAT1 activation promotes TLR8 expression to facilitate signaling via microRNA-21 in systemic lupus erythematosus","volume":"176","author":"Young","year":"2017","journal-title":"Clin. Immunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1038\/nn.3113","article-title":"An unconventional role for miRNA: Let-7 activates Toll-like receptor 7 and causes neurodegeneration","volume":"15","author":"Lehmann","year":"2012","journal-title":"Nat. Neurosci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1002\/art.39544","article-title":"Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation","volume":"68","author":"Kim","year":"2016","journal-title":"Arthritis Rheumatol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2106","DOI":"10.4049\/jimmunol.1700730","article-title":"Extracellular MicroRNAs Induce Potent Innate Immune Responses via TLR7\/MyD88-Dependent Mechanisms","volume":"199","author":"Feng","year":"2017","journal-title":"J. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1038\/nmeth.4185","article-title":"EV-TRACK: Transparent reporting and centralizing knowledge in extracellular vesicle research","volume":"14","author":"Mestdagh","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1009","DOI":"10.1001\/jama.1990.03450080095039","article-title":"Synovial fluid tests. What should be ordered?","volume":"264","author":"Shmerling","year":"1990","journal-title":"JAMA"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e108","DOI":"10.1093\/nar\/gkt214","article-title":"The Subread aligner: Fast, accurate and scalable read mapping by seed-and-vote","volume":"41","author":"Liao","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1093\/bioinformatics\/btt656","article-title":"featureCounts: An efficient general purpose program for assigning sequence reads to genomic features","volume":"30","author":"Liao","year":"2013","journal-title":"Bioinformatics"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1093\/bioinformatics\/btp616","article-title":"edgeR: A Bioconductor package for differential expression analysis of digital gene expression data","volume":"26","author":"Robinson","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2597","DOI":"10.1002\/pmic.201400515","article-title":"FunRich: An open access standalone functional enrichment and interaction network analysis tool","volume":"15","author":"Pathan","year":"2015","journal-title":"Proteomics"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"D296","DOI":"10.1093\/nar\/gkx1067","article-title":"miRTarBase update 2018: A resource for experimentally validated microRNA-target interactions","volume":"46","author":"Chou","year":"2018","journal-title":"Nucleic Acids Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/9\/4910\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:57:27Z","timestamp":1760162247000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/9\/4910"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,5,6]]},"references-count":36,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2021,5]]}},"alternative-id":["ijms22094910"],"URL":"https:\/\/doi.org\/10.3390\/ijms22094910","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,5,6]]}}}