Y; 2The Blood Cell Analysis Group, Division of Medical Biochemistry, Oslo G Protein-Coupled Receptor Kinase 6 (GRK6) Proteins Biological Activity University Hospital, Ullev , Norway; 3Oslo University Hospital-The Norwegian Radium Hospital, Oslo, NorwayIntroduction: Extracellular vesicles (EV) represent a crucial mode of intercellular communication by serving as cars for molecular transfer involving cells. The specific functions of EV on target cells depend on the capacity of EV to interact with recipient cells, delivery of their precise contents and initiating downstream signaling. The present study has investigated if THP1- and SW480-derived microvesicles (MV) and exosomes (EXO) are in a position to enter and activate an inflammatory response in human main monocytes. Techniques: Collection and isolation of EV: THP-1 (human leukemia monocytic) as well as the SW480 (human colon adenocarcinoma) cells have been cultured at 37 , 5 CO2 in serum-free RPMI media for 24 hours. Subpopulations of EV had been obtained from sequential centrifugation from the 4500xg supernatant; in certain MV have been pelleted by 17000xg, 30 min and EXO obtained by filtration with the 17000xg supernatant having a 0.22mm filter (Millex GV) and concentrated by a 100kDa Centricon filter (Amicon ltra-4). Particle size and concentration of EV had been analyzed by NTA. Functionality of EV in human primary monocytes: Elutriation-purified, cryopreserved monocytes (1.5 x 105 in150 mL) from healthful donors had been thawed and re-suspended in 10 (v/v) FCS-RPMI. MV and EXO (1010-108) (derived from THP-1 and SW480 cells) fluorescently labeled with PKH67 (Sigma Aldrich) were incubated with monocytes for 4 hours at 37 , 5 CO2. Subsequently, the supernatants were harvested and stored at -80 until the secretion of IL1-b, IL6, IL8, TNF-a, MCP-1, MIP-1b and IP10 proteins (Luminex) have been analyzed. The uptake of EV in monocytes was analyzed by flow cytometry (BD Accuri C6) and fluorescence microscopy/live imaging (Nikon Eclipse Ti). Benefits: THP-1 and SW480 derived MV and EXO were all internalized by human principal monocytes within a dose-dependent manner. The exposure of EV induced a dose-dependent secretion of IL1-b, IL6, IL8, TNFa, MCP-1, MIP-1b and IP10 in the monocytes. Our data show that MV and EXO derived from various cell lines impact the secretion of inflammatory molecules to different extents. Summary/Conclusion: Extracellular vesicles derived from THP-1 and SW480 cells are internalized and induce inflammatory responses in human principal monocytes. Funding: Regional Investigation Network on Extracellular Vesicles, SouthEastern Norway Regional Wellness AuthorityIntroduction: Mutual interplay among Kupffer cells (KCs) and hepatocytes plays a role in the improvement of non-alcoholic liver steatosis and steatohepatitis. Excessively activated by lipid accumulation Kupffer cells (KCs) release a large amount of pro-inflammatory cytokines, which are dangerous to hepatic cells. Other way around, hepatocytes secrete numerous components with prospective influence on KCs. The aim of our study was to assess exosomal miRNA cargo of hepatic cells primed in vitro by inflammatory stimuli to be able to determine miRNA, which potentially could in response regulate expression of transcripts involved in KCs. Also, in this setting we assessed the action of sylimarin the compound with recognized mild hepatoprotective action. Approaches: We have performed sequencing of exosomal miRNA from Hep2G cells treated with: Angiotensinogen Proteins supplier TNF-alpha, INF-gamma, silimarin. We’ve got used EdgeR’ to detect transcripts differentially regulate.
