Modulation of murine macrophage TLR7/8-mediated cytokine expression by mesenchymal stem cell-conditioned medium

Abstract

Increasing evidence suggests that mesenchymal stem cells (MSCs) play anti-inflammatory roles during innate immune responses. However, little is known about the effect of MSCs or their secretions on the ligand response of Toll-like receptor (TLR) 7 and TLR8, receptors that recognize viral single-stranded RNA (ssRNA). Macrophages play a critical role in the innate immune response to ssRNA virus infection; therefore, we investigated the effect of MSC-conditioned medium on cytokine expression in macrophages following stimulation with TLR7/8 ligands. After stimulation with TLR7/8 ligand, bone marrow-derived macrophages cultured with MSCs or in MSC-conditioned medium expressed lower levels of tumor necrosis factor (TNF) α and interleukin (IL) 6 and higher levels of IL-10 compared to macrophages cultured without MSCs or in control medium, respectively. The modulations of cytokine expression were associated with prostaglandin E2 (PGE2) secreted by the MSCs. PGE2 enhanced extracellular signal-related kinase (ERK) signaling and suppressed nuclear factor- κ B (NF- κ B) signaling. Enhanced ERK signaling contributed to enhanced IL-10 production, and suppression of NF- κ B signaling contributed to the low production of TNF- α . Collectively, these results indicate that MSCs and MSC-conditioned medium modulate the cytokine expression profile in macrophages following TLR7/8-mediated stimulation, which suggests that MSCs play an immunomodulatory role during ssRNA virus infection.

Figure 1

Modulation of cytokine expression by BMDMs cocultured with MSCs and then stimulated with TLR7/8 ligands. BMDMs (0.4 × 10⁶ cells/well) were incubated with or without MSCs (0.4 × 10⁶ cells/well) in 24-well plates. After 1 h, the cells were incubated with R848 (TLR7/8 ligand, 10 μg/mL) or loxoribine (TLR7 ligand, 300 μg/mL) for 24 h. The levels of murine TNF-α, IL-6, GM-CSF, IL-12 p70, and IL-10 in the culture supernatant were determined using ELISA. Data are expressed as the mean ± SEM. n = 3 in each group. *P < 0.05 and **P < 0.01, compared to the control group. N.D., not detected. Results are representative of 3 independent experiments.

Figure 2

Modulation of cytokine expression by BMDMs incubated in MSC-conditioned medium and then stimulated with TLR7/8 ligands. BMDMs were preincubated for 1 h in MSC-conditioned medium or control medium and then incubated with R848 (TLR7/8 ligand, 10 μg/mL) or loxoribine (TLR7 ligand, 300 μg/mL) for 24 h. (a–e) The levels of murine TNF-α (a), IL-6 (b), GM-CSF (c), IL-12 p70 (d), and IL-10 (e) in the culture supernatant were determined using ELISA. Data are expressed as the mean ± SEM. n = 3 in each group. *P < 0.05 and **P < 0.01, compared to the control medium group. N.D., not detected. (f–i) The levels of Il1b (f), Cxcl1 (g), Ccl3 (h), and Ccl5 (i) mRNA were determined using qRT-PCR. Data are expressed as fold increase over the level in the control medium group without TLR7/8 ligand stimulation (mean ± SEM). n = 3 in each group. *P < 0.05 and **P < 0.01, compared to the control medium group. Results are representative of 3-4 independent experiments.

Figure 3

Contribution of PGE₂/EP to the modulation of cytokine expression in BMDMs incubated in MSC-conditioned medium and then subjected to R848 stimulation. (a) PGE2 levels in MSC-conditioned medium or control medium were measured by ELISA. N.D., not detected. (b–d) BMDMs were incubated in MSC-conditioned medium or control medium with EP2 antagonist (AH6809, 10 μM) or EP4 antagonist (GW 627368X, 10 μM). DMSO was used as the vehicle for each EP antagonist. After 1 h, R848 (TLR7/8 ligand, 10 μg/mL) was added, and the BMDMs were incubated for an additional 24 h. The levels of TNF-α (b), IL-6 (c), and IL-10 (d) were determined using ELISA. *P < 0.05, compared to the control medium group. ^# P < 0.05, compared to the R848-stimulated cells (without EP antagonist). Data are expressed as the mean ± SEM. n = 3 in each group. Results are representative of 3 independent experiments.

Figure 4

Enhanced ERK and suppressed NF-κB signaling in BMDMs incubated in MSC-conditioned medium and then subjected to R848 stimulation. BMDMs were preincubated for 1 h in MSC-conditioned medium or control medium, after which R848 (TLR7/8 ligand, 10 μg/mL) was added. Cells were further incubated for the indicated times. (a) The levels of p-p38, p-JNK, and p-ERK were determined by immunoblotting. Beta-actin was used as a loading control. Relative band intensities ware quantified by densitometry analysis. (b) The data of (a) at 0.5 h after R848 stimulation obtained by densitometric analysis are shown as the mean ± SEM of 3 independent experiments. (c) The level of total IκB-α was determined by immunoblotting. Beta-actin was used as a loading control. Relative band intensities ware quantified by densitometry analysis. (d) The data of (c) at 1 h after R848 stimulation obtained by densitometric analysis are shown as the mean ± SEM of 3 independent experiments. *P < 0.05, compared to the control medium group.

Figure 5

ERK signaling is partially responsible for the modulated expression of IL-10, but not that of TNF-α or IL-6, in BMDMs preincubated in MSC-conditioned medium followed by R848 stimulation. (a) BMDMs were preincubated in MSC-conditioned medium or control medium with or without a cocktail consisting of an EP2 antagonist (AH6809, 10 μM) and an EP4 antagonist (GW 627368X, 10 μM). DMSO was used as a vehicle. After 1 h, R848 (TLR7/8 ligand, 10 μg/mL) was added, and BMDMs were incubated for an additional 0.5 h. The p-ERK level was determined by immunoblotting. Beta-actin was used as a loading control. Results are representative of 3 independent experiments. (b–d) BMDMs were preincubated in MSC-conditioned medium or control medium with or without MEK/ERK inhibitor (U-0126, 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 24 h. The levels of TNF-α (a), IL-6 (c), and IL-10 (d) were determined using ELISA. *P < 0.05 and **P < 0.01, compared to the control medium group. ^# P < 0.05, compared to the R848-stimulated cells (without MEK/ERK inhibitor). Data are expressed as the mean ± SEM. n = 3 in each group. Results are representative of 3 independent experiments.

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). ^# P < 0.05, 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. *P < 0.05, compared to the control medium group. ^# P < 0.05, compared to the control plasmid-transfected cells. Data are expressed as the mean ± SEM. n = 3 in each group. Results are representative of 3 independent experiments.