We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesicle (EV) enrichment using standard methods. In this study of human SF, a size exclusion chromatography (SEC)-based method of EV enrichment is shown to deplete contaminants that remain after standard ultracentrifugation-based enrichment methods. Specifically, considerable levels of serum albumin, the high-density lipoprotein marker, apolipoprotein A-I, fibronectin and other extracellular proteins and debris are present in EVs prepared by differential ultracentrifugation. While the addition of a sucrose density gradient purification step improved purification quality, some contamination remained. In contrast, using a SEC-based approach, SF EVs were efficiently separated from serum albumin, apolipoprotein A-I and additional contaminating proteins that co-purified with high-speed centrifugation. Finally, using high-resolution mass spectrometry analysis, we found that residual contaminants which remain after SEC, such as fibronectin and other extracellular proteins, can be successfully depleted by proteinase K. Taken together, our results highlight the limitations of ultracentrifugation-based methods of EV isolation from complex biological fluids and suggest that SEC can be used to obtain higher purity EV samples. In this way, SEC-based methods are likely to be useful for identifying EV-enriched components and improving understanding of EV function in disease.

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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.

In coeliac disease (CeD), immune‐mediated small intestinal damage is precipitated by gluten, leading to variable symptoms and complications, occasionally including aggressive T‐cell lymphoma. Diagnosis, based primarily on histopathological examination of duodenal biopsies, is confounded by poor concordance between pathologists and minimal histological abnormality if insufficient gluten is consumed. CeD pathogenesis involves both CD4+ T‐cell‐mediated gluten recognition and CD8+ and γδ T‐cell‐mediated inflammation, with a previous study demonstrating a permanent change in γδ T‐cell populations in CeD. We leveraged this understanding and explored the diagnostic utility of bulk T‐cell receptor (TCR) sequencing in assessing duodenal biopsies in CeD. Genomic DNA extracted from duodenal biopsies underwent sequencing for TCR‐δ (TRD) (CeD, n = 11; non‐CeD, n = 11) and TCR‐γ (TRG) (CeD, n = 33; non‐CeD, n = 21). We developed a novel machine learning‐based analysis of the TCR repertoire, clustering samples by diagnosis. Leave‐one‐out cross‐validation (LOOCV) was performed to validate the classification algorithm. Using TRD repertoire, 100% (22/22) of duodenal biopsies were correctly classified, with a LOOCV accuracy of 91%. Using TCR‐γ (TRG) repertoire, 94.4% (51/54) of duodenal biopsies were correctly classified, with LOOCV of 87%. Duodenal biopsy TRG repertoire analysis permitted accurate classification of biopsies from patients with CeD following a strict gluten‐free diet for at least 6 months, who would be misclassified by current tests. This result reflects permanent changes to the duodenal γδ TCR repertoire in CeD, even in the absence of gluten consumption. Our method could complement or replace histopathological diagnosis in CeD and might have particular clinical utility in the diagnostic testing of patients unable to tolerate dietary gluten, and for assessing duodenal biopsies with equivocal features. This approach is generalisable to any TCR/BCR locus and any sequencing platform, with potential to predict diagnosis or prognosis in conditions mediated or modulated by the adaptive immune response. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

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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.

The uveal tract consists of the iris, the ciliary body and the choroid; these three distinct tissues form a continuous layer within the eye. Uveitis refers to inflammation of any region of the uveal tract. Despite being grouped together anatomically, the iris, ciliary body and choroid are distinct functionally, and inflammatory diseases may affect only one part and not the others. Cellular structure of tissues direct their function, and understanding the cellular basis of the immune environment of a tissue in health, the “steady state” on which the perturbations of disease are superimposed, is vital to understanding the pathogenesis of those diseases. A contemporary understanding of the immune system accepts that haematopoietic and yolk sac derived leukocytes, though vital, are not the only players of importance. An array of stromal cells, connective tissue cells such as fibroblasts and endothelial cells, may also have a role in the inflammatory reaction seen in several immune-mediated diseases. In this review we summarise what is known about the cellular composition of the uveal tract and the roles these disparate cell types have to play in immune homeostasis. We also discuss some unanswered questions surrounding the constituents of the resident leukocyte population of the different uveal tissues, and we look ahead to the new understanding that modern investigative techniques such as single cell transcriptomics, multi-omic data integration and highly-multiplexed imaging techniques may bring to the study of the uvea and uveitis, as they already have to other immune mediated inflammatory diseases.