Nikolett Marton

Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.

Background Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described. Aim Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). Methods and Results Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin. Conclusion Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

The emerging role of aryl hydrocarbon receptor in the activation and differentiation of Th17 cells

The aryl hydrocarbon receptor (AHR) is a cytoplasmic transcription factor, which plays an essential role in the xenobiotic metabolism in a wide variety of cells. The AHR gene is evolutionarily conserved and it has a central role not only in the differentiation and maturation of many tissues, but also in the toxicological metabolism of the cell by the activation of metabolizing enzymes. Several lines of evidence support that both AHR agonists and antagonists have profound immunological effects; and recently, the AHR has been implicated in antibacterial host defense. According to recent studies, the AHR is essential for the differentiation and activation of T helper 17 (Th17) cells. It is well known that Th17 cells have a central role in the development of inflammation, which is crucial in the defense against pathogens. In addition, Th17 cells play a major role in the pathogenesis of several autoimmune diseases such as rheumatoid arthritis. Therefore, the AHR may provide connection between the environmental chemicals, the immune regulation, and autoimmunity. In the present review, we summarize the role of the AHR in the Th17 cell functions.

The Emerging and Diverse Roles of Src-Like Adaptor Proteins in Health and Disease

Although Src-like adaptor proteins (SLAP-1 and SLAP-2) were mainly studied in lymphocytes, where they act as negative regulators and provide fine control of receptor signaling, recently, several other functions of these proteins were discovered. In addition to the well-characterized immunoregulatory functions, SLAP proteins appear to have an essential role in the pathogenesis of type I hypersensitivity, osteoporosis, and numerous malignant diseases. Both adaptor proteins are expressed in a wide variety of tissues, where they have mostly inhibitory effects on multiple intracellular signaling pathways. In this review, we summarize the diverse effects of SLAP proteins.

Distinct in vitro T-helper 17 differentiation capacity of peripheral naive T cells in rheumatoid and psoriatic arthritis

Background The T-helper 17 (Th17) cells have a prominent role in inflammation as well as in bone and join destruction in both rheumatoid and psoriatic arthritis (RA and PsA). Here, we studied Th17 cell differentiation in RA and PsA. Methods Blood samples from healthy donors, RA and PsA patients were collected. CD45RO− (naive) and CD45RO+ (memory) T cells were isolated from peripherial blood mononuclear cell by magnetic separation. Naive T cells were stimulated with anti-CD3, anti-CD28, and goat anti-mouse IgG antibodies and treated with transforming grow factor beta, interleukin (IL)-6, IL-1β, and IL-23 cytokines and also with anti-IL-4 antibody. IL-17A and IL-22 production were measured by enzyme linked immunosorbent assay, RORC, and T-box 21 (TBX21) expression were analyzed by quantitative polymerase chain reaction and flow cytometry. C-C chemokine receptor 6 (CCR6), CCR4, and C-X-C motif chemokine receptor 3 expression were determined by flow cytometry. Cell viability was monitored by impedance-based cell analyzer (CASY-TT). Results RORC, TBX21, CCR6, and CCR4 expression of memory T cells of healthy individuals (but not RA or PsA patients) were increased (p < 0.01; p < 0.001; p < 0.05; p < 0.05, respectively) compared to the naive cells. Cytokine-induced IL-17A production was different in both RA and PsA patients when compared to healthy donors (p = 0.0000026 and p = 0.0001047, respectively). By contrast, significant differences in IL-22 production were observed only between RA versus healthy or RA versus PsA patients (p = 0.000006; p = 0.0013454, respectively), but not between healthy donors versus PsA patients. Conclusion The naive CD4 T-lymphocytes are predisposed to differentiate into Th17 cells and the in vitro Th17 cell differentiation is profoundly altered in both RA and PsA.

08.06 Circulating exosomes play a role in the regulation of human in vitro osteoclastogenesis

Objectives Exosomes (EXOs) belong to the subcellular sized signalosomes called extracellular vesicles (EVs). EVs, which are present in blood and other bodily fluids, carry a broad spectrum of biomolecules that can influence cellular function. It is conceivable that the vesicle composition of EVs is altered in inflammatory diseases, and that this could contribute to disease pathogenesis. Bone tissue is the second biggest consumer of circulating EVs, but the effect of blood derived EXOs on bone homeostasis has not been described before. Our aim was to study the possible role of circulating EVs on the human in vitro osteoclastogenesis in healthy individuals and in those with active arthritis. Methods Blood samples of healthy volunteers, rheumatoid arthritis (RA, DAS: 4.79±1.96) according to the 2010 ACR/EULAR classification criteria and psoriatic arthritis (PsA, DAS: 2.79±0.72) patients with peripheral arthritis according to the classification criteria for psoriatic arthritis were collected. EXOs were isolated by gravity filtration and ultracentrifugation. To investigate the properties of EXO samples resistive pulse sensing technique, transmission electron microscopy, and western blot were performed. CD14+ cells were separated from PBMCs with positive selection (StemCell), and the cells were stimulated with 50 ng/ml recombinant human M-CSF, RANKL (PeproTech) and blood-derived EXOs. After 7 days, the cells were fixed and stained for tartrate resistant acid phosphatase (TRAP) (Sigma) and the TRAP positive multinucleated cells were counted. Student’s t test and one–way ANOVA with Tukey post hoc test were used as statistical analysis. Results EXO characterisation revealed the expression of classical exosome markers (i.e., CD9, CD63, flotilin-1, TSG-101) and an average diameter of 100 nm. Healthy (n=11) and RA derived (n=12) EXOs profoundly inhibited osteoclast differentiation (p<0.01), by contrast the PsA derived (n=10) EXOs had a stimulatory effect (p<0.05). In cross-treatment experiments where EXOs and CD14+ cells were interchanged between the 3 groups, healthy (n=5) and RA (n=5) derived EXOs inhibited (p<0.01) the generation of osteoclasts in all groups, while the PsA (n=7) derived EXOs did not have an inhibitory effect. Conclusions Our abovementioned data suggest that, in a disease-specific manner, circulating EXOs are novel regulators of the human osteoclastogenesis.

03.10 Regulation of the th17 cell differentiation in rheumatoid arthritis

Background Th17 cells have a central role in the inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)−17A, −21,–22, and tumour necrosis factor-α. We studied in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA). Materials and methods Mononuclear cells (PBMC) were isolated from peripheral blood of healthy volunteers and rheumatoid arthritis (RA) patients. CD45RO- (naive) and CD45RO+ (memory) T cells were isolated from PBMC with a two-step negative magnetic separation method. They were activated with anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml) and with crosslinking (1 µg/ml) antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with anti-IL-4 (10 µg/ml) blocking antibody. After 5 and 10 days the IL-17 and IL-22 production were measured by ELISA and the RORc and Tbet expression were measured by qPCR. The expression of CCR6, CCR4 and CXCR3 of the cells were determined by flow cytometric analysis. Cell viability was monitored by impendance-based cell analyzer (CASY-TT). Results In healthy donors the memory T cells were characterised by higher RORc (p<0.05) and Tbet (p<0.05) expression levels compared to the naive T cells. This difference between the naive and memory T cells was not detected in RA patients (p=0.5625). The naive T cells from RA patients expressed more RORc (p<0.05), but not Tbet than the healthy donor derived naive cells. The CCR6 and CCR4 expression were higher in memory T cells of healthy donors than those of the naive T-cells (p<0.05) however this difference between T cell subpopulations of RA patients was not significant (p=0.06). The IL17 and IL22 production was increased by IL1+IL23+IL6+anti-IL4 treatment in healthy donors and RA patients (p<0.05) as well. Interestingly in RA the IL1+IL-23+anti-IL4 treatment also promoted the IL17 and IL22 production (p<0.05), suggesting that in vitro IL6 treatment is not required for the IL-17 and IL-22 production. Conclusions Our data suggest that the increased baseline RORc expression of the CD45RO- cells may contribute to the accelerated Th17 differentiation in RA. The IL-17 and IL-22 production are differently regulated in RA compared to the healthy donors.

A7.21 The effect of extracellular vesicles on human in vitro osteoclastogenesis

Introduction Extracellular vesicles (EV) are subcellular sized intercellular messengers, which are present in various biological fluids. EVs carry a wide variety of biomolecules and they may alter the recipient cells’ functions. The microvesicles are plasma membrane derived EVs; the exosomes, the smallest vesicles, originate from the endosome. Our objectives were to investigate the effect of EVs on the human in vitro osteoclastogenesis, and to characterise the serum EV profile of healthy donors, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients. Methods Blood samples of healthy volunteers, RA patients and PsA patients with peripheral arthritis were collected into acid citrate dextrose vacutainer tubes. EVs were isolated by filtration and centrifugation. EV samples were stained with fluorescent anti-CD3, CD14, CD15, CD19, CD42b, CD235a antibodies (BioLegend) and detected by flow cytometry. CD14+ cells were extracted from PBMCs by using positive selection method (StemCell), and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 24 hrs (PeproTech). Then the samples were treated with 50–50 ng/ml recombinant human M-CSF and RANKL (PeproTech) and with or without blood derived microvesicles or exosomes. After 7 days, the cells were fixed and stained for tartrate resistant acid phosphatase (TRAP) using a commercially available kit (Sigma). TRAP-positive cells with at least 3 nuclei were considered as osteoclasts and counted by using the ImageJ software. Results RA and PsA EV samples showed increased expression of lymphocyte markers (CD3, CD19) compared to healthy donor-derived EVs (n = 6). Microvesicles did not alter the number of mature osteoclasts. By contrast, exosomes significantly (p < 0.01) inhibited the osteoclast differentiation of the healthy (n = 10) and RA (n = 10) donor-derived monocytes, but not that of the PsA patients’ monocytes (n = 7). Conclusion Our present data suggest that the plasma profile of EVs might be altered in active arthritis compared to physiological condition, and exosomes or their cargo can regulate the human osteoclastogenesis.

A2.39 Cytokine-induced regulation of human TH17 differentiation

Background and objectives Th17 cells have a central role in the inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)-17A, -17F, -21, -22, and tumour necrosis factor-α. We studied in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA). Materials and methods CD45RO- and CD45RO+ cells were isolated from peripheral blood mononuclear cells with a two-step negative magnetic separation method. The cells were activated with anti-CD3 and anti-CD28 antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with anti-IL-4 (10 μg/ml) blocking antibody. After 5 and 10 days the IL-17 and IL-22 production were measured by ELISPOT and ELISA; the RORc and Tbet expression were measured by quantitative real-time PCR. Cell viability was monitored by impendance-based cell analyzer (CASY-TT). Results Although the anti-CD3/CD28 activation increased the IL-17 and IL-22 production; it did not alter the RORc expression of the CD45RO- cells of healthy donors. The IL-22 production and the Tbet expression of the RORc producing cells were inhibited by TGFβ and stimulated by IL-23 treatment. The CD45RO+ cells of healthy donors expressed higher level of RORc and T-bet without stimulation or cytokine treatment and also higher amount of IL-17 compared to the CD45RO- cells. By contrast, there was no difference between the RORc expression of the freshly separated CD45RO+ and CD45RO- cells of RA patients. The production of IL-17 and IL-22 of the differentiated CD45RO- cells from RA patients were both increased by IL-23 treatment. Conclusions The IL-17 and IL-22 production are differently regulated during the human Th17 differentiation. Furthermore, our present data suggest that the increased baseline RORc expression of the CD45RO- cells may contribute to the accelerated Th17 differentiation in RA.

AB0023 The Regulation of Human In Vitro Th17 Cell Differentiation by Cytokines

Background Th17 cells have a central role in inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)-17A, -17F, -21, -22, tumor necrosis factor-α and the expression of master regulator transcription factor RORc. The Th17 cells have essential role in many autoimmune diseases via induction of the development of inflammation. Objectives We studied the in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA). Methods CD45RO- and CD45RO+ cells were isolated from peripheral blood mononuclear cells with a two-step negative magnetic separation. The cells were activated with anti-CD3 and anti-CD28 antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with an anti-IL-4 (10 μg/ml) blocking antibody. After 5 and 10 days IL-17 and IL-22 productions were measured by ELISPOT and ELISA; the RORc and Tbet expression were measured by quantitative real-time PCR methods. Cell viability was monitored by impendance-based cell analyzer (CASY-TT). Results Although anti-CD3/CD28 activation increased the IL-17 and IL-22 productions, it did not alter the RORc expression of the CD45RO- cells of healthy donors. The IL-22 production and the Tbet expression of the RORc producing cells were inhibited by TGFβ and stimulated by IL-23 treatment. The CD45RO+ cells of healthy donors expressed higher level of RORc, T-bet and IL17 without stimulation or cytokine treatment compared to the CD45RO- cells. However, there was no difference between the RORc and Tbet expression of the freshly separated CD45RO+ and CD45RO- cells of RA patients. IL-23 treatment increased both of IL-17 and IL-22 cytokine production of the differentiated CD45RO- cells. Conclusions IL-17 and IL-22 production are differently regulated during human Th17 differentiation. Our data suggest that the increased baseline RORc expression of CD45RO- cells may contribute to the accelerated Th17 differentiation in RA. Disclosure of Interest None declared

AB0074 The Effect of Extracellular Vesicles on Human in Vitro Osteoclastogenesis in Health and in Arthritis

Background Extracellular vesicles (EVs) like microvesicles (MVs) and exosomes (EXOs) are released in an evolutionary conserved manner by cells. EVs mediate intercellular communication with the transmission of molecules from their parent cell to their targets. Objectives Our objective was to investigate the effect of EVs on human in vitro osteoclastogenesis in healthy donors, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients. Methods Blood samples of healthy volunteers, RA patients and PsA patients with peripheral arthritis were collected. EVs were isolated by filtration and differential centrifugation. CD14+ cells were isolated from PBMCs by using positive selection, and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 24 hrs. Then the samples were treated with 50 ng/ml recombinant human RANKL and blood-derived microvesicles or exosomes. After 7 days, the cells were fixed and stained for tartarate resistant acid phosphatase (TRAP). TRAP-positive cells with at least 3 nuclei were considered as osteoclasts and counted by using the ImageJ software. Results Healthy and RA-derived exosomes significantly (p<0.01) inhibited osteoclast differentiation of the CD14+ cells from the same donors, while PsA-derived exosomes had a stimulatory effect (p<0.05) on osteoclastogenesis. In cross-induction experiments where EXOs and cells were interchanged between the 3 groups healthy (n=5) and RA (n=5) derived EXOs inhibited (p<0.01) the generation of osteoclasts in both groups but PsA (n=7) derived EXOs did not have an inhibitory effect. Microvesicles did not alter the number of mature osteoclasts in any groups. Conclusions Our data suggest that EXOs profoundly regulate human osteoclastogenesis. PsA-derived EXOs stimulate, while healthy and RA-derived EXOs inhibit the osteoclast differentiation. Disclosure of Interest None declared