Synergistic effect of Nod1 and Nod2 agonists with toll-like receptor agonists on human dendritic cells to generate interleukin-12 and T helper type 1 cells

Abstract

A synthetic Nod2 agonist, muramyldipeptide (MDP), and two Nod1 agonists, FK565 and FK156, mimic the bacterial peptidoglycan moiety and are powerful adjuvants that induce cell-mediated immunity, especially delayed-type hypersensitivity. In this study, we used human dendritic cell (DC) cultures to examine possible T helper type 1 (Th1) responses induced by MDP and FK565/156 in combination with various synthetic Toll-like receptor (TLR) agonists, including synthetic lipid A (TLR4 agonist), the synthetic triacyl lipopeptide Pam3CSSNA (TLR2 agonist), poly(I:C) (TLR3 agonist), and CpG DNA (TLR9 agonist). Immature DCs derived from human monocytes expressed mRNAs for Nod1, Nod2, TLR2, TLR3, TLR4, and TLR9. The stimulation of DCs with MDP and FK565 in combination with lipid A, poly(I:C), and CpG DNA, but not with Pam3CSSNA, synergistically induced interleukin-12 (IL-12) p70 and gamma interferon (IFN-gamma), but not IL-18, in culture supernatants and induced IL-15 on the cell surface. In correlation with the cytokine induction, an upregulation of the mRNA expression of these cytokine genes was observed. Notably, IL-12 p35 mRNA expression increased >1,000-fold upon stimulation with lipid A plus either MDP or FK565 compared with stimulation with each stimulant alone. In contrast, for the expression of CD83 and costimulatory molecules such as CD40, CD80, and CD86, no synergistic effects were observed upon stimulation with Nod plus TLR agonists. The culture supernatants of DCs stimulated with lipid A plus either MDP or FK565 activated human T cells to produce high levels of IFN-gamma, and the activity was attributable to DC-derived IL-12. These findings suggest that Nod1 and Nod2 agonists in combination with TLR3, TLR4, and TLR9 agonists synergistically induce IL-12 and IFN-gamma production in DCs to induce Th1-lineage immune responses.

FIG. 1.

Expression of Nods and TLRs in DCs derived from human monocytes. (A) Preparation of DCs from monocytes. Human monocytes were cultured in the presence of IL-4 (100 ng/ml) and GM-CSF (100 ng/ml) for 6 days. Cells were collected and stained with anti-HLA-DR (FITC) and anti-CD1a (PE) by flow cytometry to confirm the purity of DCs. (B) Phenotypes of DCs. The DCs were collected and stained with anti-CD1a, -HLA-DR, -CD83, -CD14, -CD40, -CD80, and -CD86 (solid lines) or matched isotype MAbs (dotted lines) and analyzed by flow cytometry. The results in panels A to C are representative of three independent experiments. The percentage of gated cells (A) and the mean fluorescence intensity (B) are indicated in the corner. (C) mRNA expression for Nods and TLRs in DCs. Total RNA was extracted from the cells and analyzed for Nod1, Nod2, TLR2, TLR3, TLR4, TLR9, and β-actin by RT-PCR.

FIG. 2.

Synergistic effect of MDP or FK565 with lipid A or LPS on induction of IL-12 p70 and IFN-γ, but not IL-8, and upregulation of CD83, CD80, CD86, and CD40 expression on DCs. DCs were stimulated for 72 h at 37°C with MDP (10 μg/ml), FK565 (10 μg/ml), MDP-LL (10 μg/ml), lipid A (10 ng/ml), and LPS (10 ng/ml). DCs were pretreated with an anti-IFN-γ MAb (10 μg/ml) or a matched isotype MAb (10 μg/ml) for 30 min at 37°C and then were stimulated with MDP, FK565, and LPS for 24 h (H). The amounts of IL-12 p70 (A and D), IFN-γ (B), and IL-8 (C) in the culture supernatants were analyzed by ELISA. Cells were stained with anti-CD83 (E), -CD80 (F), -CD86 (G), and -CD40 (H) or with matched isotype MAbs and analyzed by flow cytometry. The results indicate the mean fluorescence intensities (MFI). Data from ELISAs are expressed as mean values ± SD, and statistically significant differences are shown. ** and *, P < 0.01 and P < 0.05, respectively, versus medium-alone control; ## and #, P < 0.01 and P < 0.05, respectively, versus lipid A or LPS alone. The results are representative of three independent experiments.

FIG. 3.

Synergistic effect of MDP or FK565 with lipid A on induction of IL-12 p35, IL-12 p40, IFN-γ, and IL-15 mRNA expression in DCs. DCs were stimulated for 6 h (or 24 h for IL-15 mRNA) at 37°C with MDP (10 μg/ml), FK565 (10 μg/ml), and lipid A (10 ng/ml). Total RNA was extracted from the cells, and reverse transcription of the RNA samples to cDNAs was performed. Real-time PCR amplification was carried out using a LightCycler-FastStart DNA Master SYBR green I kit (Roche Diagnostics). The relative induction of IL-12 p35 (A), IL-12 p40 (B), IFN-γ (C), IL-15 (D), and IL-18 (E) mRNA expression was determined after normalization using β-actin. The results are representative of three independent experiments.

FIG. 4.

Synergistic effect of Nod agonists with TLR agonists on induction of IL-12 p70 and IFN-γ production and on upregulation of membrane-bound IL-15, CD40, CD80, and CD86 expression on DCs. DCs were stimulated for 72 h at 37°C with MDP (10 μg/ml), FK565 (10 μg/ml), lipid A (10 ng/ml), Pam3CSSNA (1 μM, 1.27 μg/ml), poly(I:C) (10 μg/ml), or CpG DNA (2 μM). The amounts of IL-12 p70 (A) and IFN-γ (B) in the culture supernatants were analyzed by ELISA. Data are expressed as mean values ± SD, and significant differences are shown. ** and *, P < 0.01 and P < 0.05, respectively, versus medium alone; ## and #, P < 0.01 and P < 0.05, respectively, versus the respective TLR agonist alone. Cells were stained with anti-human IL-15 (C), -CD40 (D), -CD80 (E), and -CD86 (F) or with matched isotype MAbs and analyzed by flow cytometry. The results indicate the mean fluorescence intensities (MFI). The results are representative of three independent experiments.

FIG. 5.

Combinatory stimulation of DCs by MDP or FK565 with lipid A induces IL-12, which in turn induces IFN-γ mRNA expression in T cells. (A) DCs were stimulated for 72 h at 37°C with MDP (10 μg/ml), FK565 (10 μg/ml), MDP-LL (10 μg/ml), lipid A (10 ng/ml), or various combinations of the compounds. After cultivation, the culture supernatants of DCs were collected. Human peripheral blood T cells were stimulated with the culture supernatants. (B) As a control experiment, T cells were also stimulated with the respective stimulants for 24 h at 37°C. (C) DC culture supernatants were pretreated with an anti-IL-12 MAb (10 μg/ml) or anti-IL-18 MAb (1 μg/ml) for 30 min at 37°C. The total RNA was then extracted from the T cells (A to C), and real-time PCR analyses were carried out. The relative induction of IFN-γ mRNA expression was determined after normalization using β-actin. (D) The amount of IFN-γ in the culture supernatants was analyzed by ELISA. The results are representative of three different experiments. Data are expressed as mean values ± SD, and statistically significant differences are shown. **, P < 0.01 versus medium alone.