TLR2, a receptor for the recognition of PGN, has been reported to induce the production of type I interferon responses [8,9]. In this study, we tested the effect of Nod2, another receptor of PGN, on the production of interferon-α and β with a specific ligand MDP in human aortic endothelial cells (HAEC). The results showed that the activation of NOD2 with MDP induced a dose dependent production of the gene transcripts of interferons (Figure 1a α and β induced by MDP with the concentrations from 0.5 to 10 µg/mL was significant (P < 0.05, Figure 1b,c). When the HAECs were treated with 10 µg/mL MDP for various hours, we found that the induction of interferons showed two peaks with one in 1 h and another in 24 h (Figure 1d–f). These results suggested that HAECs is a source of interferons by the activation of NOD2 with MDP.

The expression of NOD2 can be up-regulated through TLR4 in LPS-treated macrophages [10]. In our studies, we detected the effect of NOD2 ligand MDP on the expression of NOD1 and NOD2. As shown in Figure 2a the treatment of HAECs with the various concentrations of MDP up-regulated the gene expression of NOD2, but down-regulated the gene expression of NOD1, a receptor for another breakdown product of PGN [11]. qRT-PCR results showed that the regulations induced by MDP were significant with the concentration of MDP from 1 to 10 µg/mL (Figure 2b). These results suggested that MDP has a positive feedback regulation for NOD2 expression.

Tumor necrosis factor (TNF) has been reported to induce the expression of interferon β through the activation of interferon-regulatory factor 1 (IRF1) pathway [12]. To confirm whether the up-regulation of interferons induced by MDP is dependent on the TNF pathway, we first detected the effect of TNFα on the gene expression of type I interferons in HAECs. As expected, the treatment of HAECs with the various concentrations of TNFα induced a dose-dependent up-regulation of interferon α and β (Figure 3a,b). However, when we tested the effect of MDP on TNFα expression, the results showed that no gene expression of TNFα was found within 24 h (Figure 3c. Furthermore, when HAECs were pre-treated with TNFα neutralizing antibody, the induction of IFNs induced by MDP re-stimulation was not decreased (Figure 3d). These results suggested that the up-regulation of interferons induced by MDP is not dependent on the TNF signaling.

The expression of type I interferons is regulated by the interferon regulatory factors (IRFs) in response to various factors [13,14,15]. To confirm which IRF is responsible for the induction of interferon α and β induced by MDP in HAECs, the IRF expression was first tested by RT-PCR. The results showed that HAECs expressed IRF1, 2, 3, 9, but not IRF4, 5, 6, 7, 8 (Figure 4a). Then we measured the phosphorylation of IRF3, which is the common regulator for interferon induction [13,14], and found that MDP induced the activation of IRF3 at time points 5, 10 and 30 minutes (Figure 4b). The semi-quantitation data showed the significant up-regulation of phosphorylated IRF3 compared with the non-treated group (Figure 4c). This data suggested that MDP may induce the up-regulation of type I interferons via IRF3 signaling pathway.

The activation of NOD2 by MDP induces the production of pro-inflammatory cytokines [16,17], and functions to regulate the innate immunity in response to infection [18]. The down-regulation of NOD2 reduces the MDP-induced cytokine production [19]. In our study, we also detected the effect of MDP on the pro-inflammatory cytokine expression in HAECs. The results showed that MDP induced dose-dependent up-regulation of pro-inflammatory cytokines, including IL-1β, IL-8 and MCP-1 (Figure 5a,b), suggesting that HAECs may also play roles in the induction of the innate immunity through the activation of NOD2.

Primary human aortic endothelial cells (HAECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and maintained in endothelial culture medium (ScienCell) supplemented with 5% FCS, 1% endothelial cell growth supplement, and antibiotics. All cells were cultured in a humidified atmosphere with 5% CO₂ at 37 °C. Rabbit anti-human phosphorylated and total IRF3 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Muramyldipeptide (MDP), the minimal bioactive peptidoglycan motif and recognized by NOD2, was purchased from InvivoGen (San Diego, CA, USA). Recombinant human TNFα was purchased from ABGAB (Chicago, IL, USA).

Total RNA was extracted from 2 to 5 × 10⁶ cells using TRIzol (Invitrogen, Carlsbad, CA, USA), as described by the manufacturer. mRNA was reverse transcribed with RevertAid (MBI Fermentas, Burlington Ontario, Canada) at 42 °C for 60 min, and the resulting cDNA was subjected to PCR (95 °C for 1 min followed by 25–30 cycles at 95 °C for 30 s, 60 °C for 30 s, and 68 °C for 1 min and an extension for 10 min at 68 °C). PCR products were separated on 1.0% agarose gels and visualized with ethidium bromide. Forward and reverse primer pairs are listed (5´to 3´) as follows:

Endothelial cells (1–2 × 10⁶) were lysed in 200 mL lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 mg/mL leupeptin). The cell lysate was centrifuged at 12,000 g at 4 °C for 5 min. Total proteins were electrophoresed on 10% SDS-PAGE gels, and transferred onto Immobilon P membranes (Millipore, Billerica, MA, USA). The membranes were blocked by incubation in 3% nonfat dry milk for 1 h at room temperature and then incubated with primary antibodies in PBS containing 0.01% Tween 20 overnight at 4 °C. After incubation with a horseradish peroxidase-conjugated secondary antibody, the protein bands were detected with SuperSignal chemiluminescent substrate-stable peroxide solution (Pierce Rockford, IL, USA) and BIOMAX-MR film (Eastman Kodak Co., Rochester, NY, USA). When necessary, the membranes were stripped with Restore Western blot stripping buffer (Pierce) and re-probed with antibodies against various cellular proteins.

The qRT-PCR was performed as described by Sun et al. [20]. Briefly, total RNA from cells was isolated and reverse transcribed as above. The cDNA was amplified using TaqMan Universal PCR master mix (Applied Biosystems, Foster City, CA, USA) and an ABI Prism 7500 sequence detection system (Applied Biosystems). Amplification of the target genes was normalized using the amplification levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control. The efficiency of the PCR was tested by amplification of the target from serially diluted cDNA generated from the reverse transcription of a stock set of human RNA. Data analysis and calculations were performed using the 2^−ΔΔCT comparative method, as described by the manufacturer. Gene expression is shown as the fold inductions of a gene measured in MDP-treated samples, relative to samples cultured with medium.

All experiments were performed at least three times, and the representative results are shown. Results are expressed as the mean ± S.D. Differences between groups were examined for statistical significance using Student’s t test, and P values equal to or less than 0.05 were considered statistically significant.