IntroductionOsteoarthritis (OA) is an age-related musculoskeletal disease and the most common form of arthritis characterised by low grade synovial inflammation and articular cartilage degeneration. OA has many risk factors such as genetic predisposition, age and gender, but also joint specific (e.g. trauma) and systemic (obesity, hormones). Also many different cell types have been implicated in the pathogenesis of OA and interplay between these cells contribute to the dynamic events associated with disease. Recently, extracellular vesicles (EVs) have emerged as mediators of intercellular communication. There is however very little known about the pathophysiological role of EVs in musculoskeletal diseases. In this study, we investigate whether plasma EVs from OA patients can drive chondrocyte terminal differentiation, a pathophysiological process observed in affected joints and a hallmark of OA.ObjectivesStudy the effect of plasma-derived EVs from OA patients during chondrogenenic differentiation of mesenchymal stem cells.MethodsPlasma-derived extracellular vesicles (pEVs) were isolated from plasma of OA patients and age-matched healthy controls using size-exclusion chromatography. EV containing fractions were characterised according to ISEV guidelines.1 Pelleted MSCs were stimulated with TGF-β and BMP-2 to induce chondrogenic differentiation, either in the presence of pEVs isolated from OA patients or healthy controls. After 8 days, RNA was isolated and RT-qPCR was performed to determine the gene expression profiles.ResultsNo significant difference was observed in particle concentration, size or protein concentration between OA patients and age-matched controls. In the presence of pEVs from OA patients MSC-derived chondrocytes showed a significant increase in the expression of MMP13 (6.1-fold), RUNX2 (1.9-fold) and RANKL (2.3-fold), compared to pEVs from healthy controls. A trend towards higher ADAMTS5 expression (2.5-fold, p= 0.0685) with OA pEVs was also observed. Additionally, we found significantly higher expression of WISP1 (24-fold), suggesting activation of Wnt signalling. All other proinflammatory genes tested were not significantly different between the two groups.ConclusionsHere, we show direct evidence that circulating pEVs from OA patients can enhance OA-related genes, like MMP13, in MSC-derived chondrocytes. The expression profile found suggest the presence of Wnt-proteins on pEVs from OA patients, which are known to be involved in cartilage development and can induce chondrocyte hypertrophy, and we previously have shown that WISP-1 expression is a feature of experimental and human OA.2 We showed that pEVs can transfer disease-related phenotypic changes and this might implicate them in the pathophysiological process of OA.References. Lötvall J, et al. Journal of Extracellular Vesicles2014.. Blom AB, et al. Arthritis & Rheumatology2009.Disclosure of interestNone declared

BackgroundExtracellular vesicles (EVs) play a role in cell-cell communication and contain numerous signalling molecules inside and on their cell membrane. Although their function remains to be elucidated, evidence accumulates that EVs play a regulatory role in immunity during health and disease. They contain numerous proteins, lipids, RNA, DNA and sometimes cell organelles such as mitochondria. In a recent study was shown that Immunoglobuline M-Rheumatoid factor (IgM-RF) is present on milk derived exosomes.1 High IgM-RF levels in Rheumatoid Arthritis (RA) predict a more severe disease and comorbidities, probably due to their involvement in immune complex formation and activation of complement (crucial mediators of the effector phase of inflammation in the pathogenesis of RA).ObjectivesIn this study we investigate whether RF +EVs are detectable in the circulation of RA patients and if this relates to parameters of disease activity.MethodsEVs were isolated from platelet-free plasma of 38 RA patients and from age and sex-matched 24 healthy controls (HC) by size exclusion chromatography. EV markers (tetraspanins) were detected by Western blot and miRNA content by RT-qPCR. Particle size and concentration were measured by electron microscopy and nanosight tracking analysis. Protein concentration was determined by micro-BCA. RF levels were measured using a commercial ELISA. The percentage of RF +EVs was determined by measuring bound and unbound PHK labelled EVs to Protein L magnetic beads in a fluorometer.ResultsMean EV particle size, concentration and protein content were not different between RA patients and HC. 27 of the 38 RA patients were classified as RF+ (>10 IU/ml) and of the clinical parameters studied only their erythrocyte sedimentation rate (ESR) was higher (31 vs 14 mm/hr). In 14 RF +patients, RF was detectable on a small portion of EVs not exceeding 4% of the total number of circulating EVs. Interestingly, RA patients with RF +EVs showed higher disease activity as assessed by patient global health assessment using a visual analogue scale (63 vs 31), blood C-reactive protein (22 vs 9 mg/L) and ESR (43 vs 19 mm/hr) levels, than RA patients with undetectable RF +EVs.ConclusionsThis study shows for the first time that in a subpopulation of RA patients RF is present on EVs, which might originate from their B-cells. The higher disease activity in RA patients expressing RF on their EVs suggests that RF +EVs are involved in RA pathogenesis.Reference[1] Yang M, et al. Comparative proteomic analysis ofmilk-derived exosomes in human and bovine colostrumandmature milk samples by iTRAQ-coupled LC-MS/MS. Food Research International2017;92(2017):17–25.Disclosure of InterestNone declared

Development of rheumatoid arthritis (RA) is associated with environmental factors and several studies show a connetion with diet. Recently, exosomes are identified in both bovine and human breast milk. Exosomes are small (30–200 nm) membrane-derived extracellular vesicles that carry immunoregulatory microRNAs, proteins and lipids, and mediate intercellular communication. In this study, we investigated the effect of oral intake of bovine milk-derived extracellular vesicles (BMEVs) on spontaneous polyarthritis in IL-1Ra deficient mice and on collagen-induced arthritis (CIA) in DBA1/J mice. BMEVs were isolated from skimmed milk by ultracentrifugation and characterised by nanoparticle tracking analysis, electron microscopy, anti-CD63 staining, and PCR. BMEVs were labelled with PKH-67 to determined the uptake by cells using FACS and confocal microscopy. TGFb levels were measured with a CAGA-fLuc reporter construct. Naïve T cells were cultured for 5 days with an inflammatory cocktail in the presence of milk exosomes, to induce Th17 differentiation as assessed by RORgT and IL-17 mRNA expression. In IL-1Ra-/- mice, BMEVs were administered daily by oral gavage starting at week 5 till 15 after birth and arthritis was scored macroscopically. In CIA, mice received BMEVs via their drinking water starting 1 week before immunisation till day 40. Arthritis was scored macroscopically and on histology. To determine the effect on immunity, serum IgG levels were measured by ELISA, T-cell specific gene expression (T-bet, RORgT, GATA-3) in LPS stimulated splenocytes by Luminex and RT-qPCR. The BMEV particle size was around 120 nm and expressed the exosome markers tetraspanin CD63, miRNA’s (miR-let-7a, -16, -30a, -92a, -223), and milk specific beta-casein and beta-lactoglobulin mRNA. Acidification, up to gastric acid level (pH 2) left the BMEVs intact, and did not alter their inhibitory effect on NFkB-activation. Active TGFb was detected on BMEVs and incubation of naïve T cells with BMEVs induced significant Th17 differentiation to a similar extent as TGFb. However, BMEVs treatment of mice showed a delayed onset of arthritis in both the IL-1Ra-/- and CIA model. Diminished cartilage pathology and bone marrow inflammation was observed in both models. BMEVs also reduced the circulation levels of MCP1 and IL-6 levels and their production by splenic cells. In BMEV treated CIA, circulating anti-collagen type II IgGa level was reduced and this was accompanied by reduced splenic Th1 and Th17 numbers. Bovine milk-derived extracellular vesicles are bioactive, probably can withstand the harsh conditions of the gut, and oral delivery ameliorates disease in two RA models.