fig4

Figure 4. UCMSC-EVs suppress inflammatory cytokines in both LPS-induced THP-1 and PHA-induced PBMC inflammatory models. (A) The gene expression of inflammatory cytokines (IL-1β, IL-6, TNF-α) was quantified by RT-qPCR. The relative expression of each gene was first quantified from fold change (2^-ΔΔCt) and then normalized in between LPS control (PC: 100%) and naïve state (NC: 0%). The dose-response curve shows percent cytokine gene expression at different UCMSC-EV concentrations compared to maximum cytokine levels (LPS alone without UCMSC-EVs; 100%). Data are presented as the mean ± SD, based on two independent biological replicates for the sample groups and three independent biological replicates for the control groups; (B) The bar graph shows that PHA markedly increased IFN-γ secretion in PBMCs (46-fold differences between positive and negative controls, ^****P < 0.0001, one-way ANOVA with Dunnett’s post hoc test, n = 3 replicates). Treatment with UCMSC-EVs significantly reduced IFN-γ concentrations compared with PC groups (35%-42% reduction with EV treatment, ^**P < 0.01, one-way ANOVA with Dunnett’s post hoc test, n = 3 replicates); (C) The dot plot showed the negative correlation between PEDF concentration of UCMSC-EVs quantified by ELISA and the IFN-γ concentrations measured from the UCMSC-EV treatments in the PHA-induced PBMC model. The r = -0.724, P = 0.005, n = 11 UCMSC-EV production lots. IL-1β: Interleukin-1 beta; IL-6: interleukin-6; IFN-γ: interferon gamma; LPS: lipopolysaccharide; NC: negative control; PBMCs: peripheral blood mononuclear cells; PC: positive control; PEDF: pigment epithelium-derived factor; PHA: phytohemagglutinin; RT-qPCR: reverse transcription quantitative polymerase chain reaction; THP-1: human monocytic leukemia cell line THP-1; TNF-α: tumor necrosis factor-alpha; UCMSC-EVs: umbilical cord mesenchymal stem cell-derived extracellular vesicles; SD: standard deviation; ANOVA: analysis of variance; ELISA: enzyme-linked immunosorbent assay.