The effects of erythrocyte-derived microvesicles on pro-inflammatory cytokine (TNFα and IL-8) release in monocytic cells (THP-1) stimulated with Lipopolysaccharide (LPS)
Cardiff Metropolitan University
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AIM: To determine whether erythrocyte-derived microvesicles (EMVs) can activate monocytic cells (THP-1 cells) and enhance an inflammatory response, as assessed by the release of inflammatory cytokines: Tumour necrosis factor-alpha (TNFα) and Interleukin-8 (IL-8) with and without stimulation from endotoxin Lipopolysaccharide (LPS). MATERIALS & METHODS: Optimisation experiments were carried out to determine the ideal concentrations of E. coli derived LPS, extracellular vesicles (EVs) and ideal techniques to separate the red blood cell (RBC) EVs. The protocol was constructed based around these trials. EVs were derived from samples of whole and packed blood which were spun at 17,000g and 100,000g to render the samples acellular and isolate the EVs. The isolated EV samples were analysed using Accuri C6 flow cytometry (BD) and the NanoSight™ LM10 system to determine correct isolation and characterisation of the EVs as MVs. The finding from the flow cytometry was used to determine the EV concentrations for incubation with the Human monocytic cell line THP-1 cells (ECACC – Public Health England) both with and without the presence of LPS to stimulate a pro-inflammatory condition. An enzyme-linked immunosorbent assays (ELISA) analysis was performed for both IL-8 and TNFα to determine cytokine release with and without LPS stimulation and with increasing EV concentrations. The data from the ELISA testing was statistically analysed with a one-way ANOVA test to determine significance (P value >0.05). RESULTS: The flow cytometry and nanosight analysis confirmed the EVs were MVs within the 200nm size range, they were identified as largely of RBC origin as denoted by the CD235a labelling and confirmed successful isolation technique. A homogenous sized population was identified for the SAG-M samples and a heterogeneous-sized population for WB by the nanosight analysis from a contribution of different cell types. Both results confirmed the presence of MVs and some exosomes whilst importantly excluding the presence of apoptotic cells. The ELISA analysis showed a strong response with the presence of LPS. EV-induced activity was dose-dependent. The 100,000g isolation method proved to be more successful at separating EVs and the results clearly show inflammatory responses occurring. However, further investigations will need to take place, due to the overstimulation of control THP-1 cells with and without LPS to draw valid 4 conclusions from the results. The data suggests that an increased number of EVs can lead to the activation of monocytes and subsequent release of pro-inflammatory cytokine TNFα and chemokine IL-8 leading to an exacerbation of an inflammatory response. It is possible that smaller particles such as exosomes may also be playing a role with inducing cytokine activity, which needs to be investigated further. CONCLUSION: Cytokine activity increased with the presence of LPS suggesting that there is a potential for individual with underlying inflammatory conditions receiving blood transfusions with an increased EV concentration to exacerbate their condition and lead to potential negative clinical outcomes. However, due to various limitations further research would need to be undertaken.
BSc. Biomedical Science (Hons) course
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