Modulation of Murine Macrophage TLR7/8-Mediated Cytokine Expression by Mesenchymal Stem Cell-Conditioned Medium
Figure 6
NF-κB signaling is partially responsible for the modulated expression of TNF-α, but not that of IL-6 or IL-10, in BMDMs preincubated in MSC-conditioned medium and then subjected to R848 stimulation. (a) BMDMs were incubated in MSC-conditioned medium or control medium with or without a cocktail consisting of an EP2 antagonist (AH6809, 10 μM) and EP4 antagonist (GW 627368X, 10 μM). DMSO was used as the vehicle. After 1 h, R848 (TLR7/8 ligand, 10 μg/mL) was added, and BMDMs were incubated for an additional 1 h. The level of total IκB-α was determined by immunoblotting. Beta-actin was used as a loading control. Results are representative of 3 independent experiments. (b–e) BMDMs were transfected with RelA cFlag pcDNA3 plasmid or pcDNA3.1 plasmid (as a control) using FuGENE6 Transfection Reagent. After 24 h, the cells were incubated for 1 h in either MSC-conditioned medium or control medium, after which they were stimulated with TLR7/8 ligand (R848) or vehicle for 24 h in either MSC-conditioned medium or control medium. The level of Rela mRNA was determined using qRT-PCR (b). Data are expressed as the fold increase over the level of Rela in control plasmid-transfected cells cultured in control medium without R848 stimulation (mean ± SEM). , compared to the control plasmid-transfected cells. The levels of TNF-α (c), IL-6 (d), and IL-10 (e) in the supernatant were determined using ELISA. , compared to the control medium group. , compared to the control plasmid-transfected cells. Data are expressed as the mean ± SEM. in each group. Results are representative of 3 independent experiments.
