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In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium‐sized nascent EVs of THP1 cells as well as of Optiprep‐purified platelets, and incubated them in EV‐depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein‐coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α‐chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF‐α, IL‐6, CD83, CD86 and HLA‐DR of human monocyte‐derived dendritic cells, EV‐free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein ‘contamination’ of EV preparations and may add a new perspective to EV research.

Summary Mesenchymal stromal cell-like (MSCl) cells generated from human embryonic stem cells are considered to be an eligible cell line to model the immunomodulatory behavior of mesenchymal stromal cells (MSCs) in vitro. Dendritic cells (DCs) are essential players in the maintenance and restoration of the sensitive balance between tolerance and immunity. Here, the effects of MSCl cells on the in vitro differentiation of human monocytes into DCs were investigated. MSCl cells promote the differentiation of CTLA-4 expressing DCs via the production of all-trans retinoic acid (ATRA) functioning as a ligand of RARα, a key nuclear receptor in DC development. These semi-matured DCs exhibit an ability to activate allogeneic, naive T cells and polarize them into IL-10 + IL-17 + double-positive T helper cells in a CTLA-4-dependent manner. Mapping the molecular mechanisms of MSC-mediated indirect modulation of DC differentiation may help to expand MSCs' clinical application in cell-free therapies., Graphical abstract, Highlights • Mesenchymal stromal cell-like cells alter moDC differentiation via RARα activation • Mesenchymal stromal cell-like cells express genes known to play role in ATRA synthesis • MoDCs, differentiated in the presence of MSCl-derived factors, express CTLA-4 • CTLA-4+ moDCs are able to induce polarization of IL-10- and IL-17-producing helper T cells, Molecular Biology; Immunology; Components of the Immune System; Stem Cells Research

Purpose. This study is aimed at investigating the phenotype, differentiation potential, immunomodulatory properties, and responsiveness of saphenous vein vessel wall-derived mesenchymal stromal cells (SV-MSCs) to various TLR ligands and proinflammatory cytokines, as well as comparing their features to those of their bone marrow-derived counterparts (BM-MSCs). Methods. SV-MSCs were isolated by enzymatic digestion of the saphenous vein vessel wall. Phenotype analysis was carried out by flow cytometry and microscopy, whereas adipogenic, chondrogenic, and osteogenic differentiation potentials were tested in in vitro assays. For comparative analysis, the expression of different stemness, proliferation, and differentiation-related genes was determined by Affymetrix gene array. To compare the immunomodulatory properties of SV-MSCs and BM-MSCs, mixed lymphocyte reaction was applied. To investigate their responses to various activating stimuli, MSCs were treated with TLR ligands (LPS, PolyI:C) or proinflammatory cytokines (TNFα, IL-1β, IFNγ), and the expression of various early innate immune response-related genes was assessed by qPCR, while secretion of selected cytokines and chemokines was measured by ELISA. Results. The isolated SV-MSCs were able to differentiate into bone, fat, and cartilage cells/direction in vitro. SV-MSCs expressed the most important MSC markers (CD29, CD44, CD73, CD90, and CD105) and shared almost identical phenotypic characteristics with BM-MSCs. Their gene expression pattern and activation pathways were close to those of BM-MSCs. SV-MSCs showed better immunosuppressive activity inhibiting phytohemagglutinin-induced T lymphocyte proliferation in vitro than BM-MSCs. Cellular responses to treatments mimicking inflammatory conditions were comparable in the bone marrow- and saphenous vein-derived MSCs. Namely, similar to BM-MSCs, SV-MSCs secreted increased amount of IL-6 and IL-8 after 12- or 24-hour treatment with LPS, PolyI:C, TNFα, or IL-1β, compared to untreated controls. Interestingly, a different CXCL-10/IP-10 secretion pattern could be observed under inflammatory conditions in the two types of MSCs. Conclusion. Based on our results, cells isolated from saphenous vein vessel wall fulfilled the ISCT’s (International Society for Cellular Therapy) criteria for multipotent mesenchymal stromal cells, and no significant differences in the phenotype, gene expression pattern, and responsiveness to inflammatory stimuli could be observed between BM-MSCs and SV-MSCs, while the latter cells have more potent immunosuppressive activity in vitro. Further functional assays have to be performed to reveal whether SV-MSCs could be useful for certain regenerative therapeutic applications or tissue engineering purposes.

Dendritic cells (DCs), as potent phagocytes engulf dead cells and present peptide fragments of tumor antigens or pathogens derived from infected cells to naive CD8+ T-lymphocytes. Dendritic cells can also induce apoptosis in target cells, thus getting an opportunity to sample their microenvironment. Here, we present that the supernatants of LPS- or CL075-activated DCs induced cell death in different cell lines, but during the differentiation to mature DCs, they lost their cytotoxic potential. Dexamethasone-pre-treated tolerogenic DCs induced less intensive death indicating that the tissue microenvironment can downregulate DC-mediated killing. Exploring the signaling of DC-induced cell death, we observed that the supernatant of activated DCs induced TNF-dependent cell death, since TNF antagonist blocked the cytotoxic activity of DCs, contrary to inhibitors of Fas and TRAIL receptors. We identified that the DC-induced killing is at least partially a RIPK1-dependent process, as RIPK1 positive target cells were more susceptible to DC-induced cell death than their RIPK1 deficient counterparts. Moreover, both the elevated phosphorylation of RIPK1 and the increase in RIPK1-caspase-8 interaction in target cells suggest that RIPK1-mediated signals contribute to DC supernatant-induced cell death. We also proved that the cytotoxic activity of DC-derived supernatant induced apoptosis in the target cells and not necroptosis, as it was completely abrogated with the pan caspase inhibitor (Z-VAD), while the necroptosis inhibitor (Nec-1) had no effect. Our work revealed that the supernatant of activated DCs induces the apoptosis of target cells in a RIPK1-dependent manner. This phenomenon could be relevant for the initiation of cross-presentation and may broaden the plethora of cytotoxic mechanisms acting against tumor cells.

Matrix metalloproteinases (MMPs) are endopeptidases with the potential to cleave extracellular matrix, support tissue renewal and regulate cell migration. Functional activities of MMPs are regulated by tissue inhibitors of MMPs (TIMPs) and disruption of the MMP-TIMP balance has pathological consequences. Here we studied the expression and secretion of MMPs and TIMPs in CD1a(-) and CD1a(+) monocyte-derived dendritic cell (DC) subpopulations. Our results showed that monocytes express TIMPs but lack MMPs, whereas upon differentiation to moDCs and in response to activation signals the expression of MMPs is increased and that of TIMPs is decreased. MMP-9 is expressed dominantly in the CD1a(-) subpopulation, while MMP-12 is preferentially expressed in CD1a(+) cells. Experiments performed with the synthetic MMP inhibitor GM6001 revealed that this drug efficiently inhibits the migration of moDCs through inactivation of MMPs. We conclude that modulation of MMP activity by GM6001 emerges as a novel approach to manipulate DC migration under inflammatory conditions.