The human leukemia monocytic cell line, THP-1, was purchased from American Type Culture Collections (ATCC) (Rockville, MD, USA). RPMI-1640, L-glutamine, HEPES, sodium pyruvate, glucose, 2-mercaptoethanol, penicillin/streptomycin mixture, 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), phosphate buffered saline solution (PBS), Sirtinol and other chemicals of analytical grade were from Sigma (Milan, Italy). Lipopolysaccharide (LPS) was from InvivoGen (San Diego, CA, USA). Fetal bovine serum (FBS), as well as TRIzol for RNA extraction were from Invitrogen Life Technologies (Milan, Italy). High-capacity cDNA archive kit, TaqMan Gene Expression Mastermix, TaqMan Gene Expression assays (Assays-on-Demand) for human 18S mRNA (ID: Hs99999901_s1), TNF-α (ID: Hs00174128_m1), IL-6 (ID: Hs00985639_m1), IL-1β, (ID: Hs01555410_m1) were from Applied Biosystems (Applera Corp., Milan, Italy). Assay location (midposition of fluorogenic probe), reference sequences and other relevant information are published online by Applied Biosystems (Foster City, CA).

Instant ELISA kits for the quantitative detection of human IL-1β, IL-6 and TNF-α were from eBioscience (Vienna, Austria). Nuclear Extraction Kit and Electrophoretic Mobility Shift Assay (EMSA) for identifying NF-κB were supplied by Panomics (Santa Clara, CA, USA). Protein G PLUS-Agarose Immunoprecipitation reagent were from Santa Cruz Biotechnology (Dallas, TX, USA) and the antibodies rabbit anti-p65, sheep anti-acetylated Lysine and rabbit anti-sheep IgG (HRP) were from Abcam (Cambridge, UK). ECL Plus detection system were from GE Healthcare Bio-Sciences (Pittsburgh, PA, USA).

The flavonoid fraction of bergamot juice (BJe) has been provided by the company “Agrumaria Corleone” (Palermo, Italy). The fruits of Citrus bergamia were coming from crops located in the province of Reggio Calabria (Italy). The extract was centrifuged at 6000 rpm/min for 15 minutes to remove any impurities and successively transformed into a dry powder by the method of spray drying. Small aliquots of BJe were stored at −20°C. Finally, the drug was defrosted, diluted in culture media, pH adjusted to 7.4 and filtered just prior to use.

Chemical composition of BJe was investigated as previously described [32]. Briefly, BJe was solubilized in methanol to a concentration of 1 mg/mL, ultrasonicated and filtered by a 0.2 µm nylon membrane (Millipore, Milan, Italy). Qualitative and quantitative determination of flavonoids in BJe was performed using a UHPLC coupled online to an LCMS–IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). Flavonoids were identified on the basis of diode array spectra, MS molecular ions and MS/MS fragmentation patterns. Data obtained were compared with those available in scientific literature. Molecular formula was calculated by the Formula Predictor software (Shimadzu).

THP-1 cells were maintained in RPMI 1640 supplemented with L-glutamine (2 mM), HEPES (10 mM), sodium pyruvate (1 mM), glucose (2.5 g/l), 2-mercaptoethanol (0,05 mM), 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, at 37°C in a 5% CO_2/95% air humidified atmosphere. Medium was renewed every 2 days and split performed when cells reached maximum density (1×10⁶ cells/ml). In our experimental conditions, THP-1 cells were seeded at a density of 5×10⁵ cells/ml into culture plates in RPMI complete medium plus 10% FBS and incubated at 37°C with lipopolysaccharide (LPS; 500 ng/ml) for 3 hs, in the presence or absence of BJe (0.05–0.1–0.5 mg/ml) and/or Sirtinol (1–5–10 µM), which were added to the culture medium 30 min prior to LPS treatment. In all experiments, equal volumes of PBS or DMSO were added to the medium of control cultures (controls were performed using non-stimulated cell). Either concentrations of LPS, BJe and Sirtinol were chosen according to our preliminary optimization studies. After incubation, cells were harvested by centrifugation to assess cellular viability, gene expression, activation of transcription factor NF-κB and acetylation status of p65. Media were collected in order to evaluate cytochine release.

To assess either LPS and BJe adverse effects on cell viability, we evaluated the mitochondrial activity of living cells by a MTT quantitative colorimetric assay. After treatment, THP-1 cells were harvested by centrifugation and, after counting, they were incubated in 96-well culture plates at a density of 5×10⁴ cells/well with fresh red-phenol free medium containing MTT (0.5 mg/mL) at 37°C for 4 hs. Then, insoluble formazan crystals were dissolved in 100 µL of a 0.04 N HCl/isopropanol solution for 1 h. The optical density in each well was evaluated by spectrophotometrical measurement. Absorbance was determined at 570 nm using a microplate reader (Tecan Italia, Cologno Monzese, Italy). All experiments were performed in eightplicate and repeated three times.

After RNA isolation with TRIzol reagent, RNA (3 µg) was reverse transcribed with High- Capacity cDNA Archive kit according to the manufacturer's instructions. Then, mRNA levels of IL-6, IL-1β, TNF-α were analyzed by real-time PCR using TaqMan gene expression assays according to the manufacturer's instructions. 18S mRNA was used as endogenous controls. Quantitative PCR reactions were set up in triplicate in a 96-well plate and were carried out in 10 µl reactions containing 1× TaqMan Gene Expression Mastermix, 1× TaqMan-specific assay and 20 ng RNA converted into cDNA. qPCR was performed in a 7900HT Fast Real-Time PCR System with the following profile: one cycle at 50°C for 2 min, then 95°C for 10 min, followed by 50 cycles at 95°C for 15 s and 60°C for 1 min. Data were collected and analyzed using SDS 2.3 and RQ manager 1.2 software (Applied Biosystems, Foster City, CA) using the 2^(−ΔΔCt) relative quantification method. Values are presented as fold change relative to unstimulated cells.

In order to detect human IL-6, IL-1β and TNF-α, an enzyme-linked immunosorbent assay was performed in cell-free culture supernatants of THP-1 monocytes, using Instant ELISA Kits. Before detection, supernatants recovered from treated and untreated cells were concentrated 10-fold by freeze-drying. All freeze-dried samples were reconstituted by the addition of distilled water. Briefly, according to the manufacturer's guidelines, 50 µl of standards or samples (supernatants recovered from treated and untreated cells) were incubated in 96-well plates at room temperature for 3 hs with shaking. After washing 5 times with 400 µl of wash buffer, 100 µl of the provided substrate solution were added to each well and the plates were incubated in the dark for 10 min. The enzyme reaction was then stopped by pipetting 100 µl of stop solution into each well and the absorbance was determined at 450 nm using a microplate reader (Tecan, Italy). All experiments were performed in triplicate.

At the end of the treatment, THP-1 cells were harvested by centrifugation. After washing twice with cold PBS, the isolation of nuclear cell proteins was performed using a commercial nuclear extraction kit following the manufacturer's guidelines. Protein concentrations were determined using a Bradford method. The presence of NF-κB DNA binding activity in cellular nuclear extracts of LPS-treated and control cells was evaluated by subsequent electrophoretic mobility shift assay, using Affymetrix EMSA Kits according to the manufacturer's instructions. Briefly, nuclear extracts were incubated with the biotin-labeled NF-κB probe and then the protein/DNA complexes were separated on a non-denaturing 6% polyacrylamide gel. After transferring onto nylon membranes bound complexes were detected via streptavidin-HRP and a chemiluminescent substrate and visualized on Kodak film. The bands were scanned and quantified by densitometric analysis with ImageJ 1.47, an open source software freely downloadable from the US National Institute of Health website (http://imagej.nih.gov/ij/).

For each sample, 50 µg of nuclear extract were incubated with rabbit anti-p65 for 1 h at 4°C on a rotator. Negative control was set by incubating nuclear proteins under similar conditions but without the immunoprecipitating antibody. Afterwards, 35 µl of resuspended Protein G PLUS-Agarose beads were added to each tube and the samples were incubated at 4°C overnight on a rocker platform. The agarose beads were extensively washed the next day and pellets were resuspended in 40 µl of 1x Laemmli buffer, boiled for 5 min and resolved by SDS-PAGE. Proteins were then transferred onto nitrocellulose membrane and non-specific binding sites were pre-blocked by incubation with 5% non-fat dry milk in Tris-buffered saline containing 0.15% Tween 20 for 1 h at room temperature. The blot was probed overnight at 4°C with primary antibody anti-acetylated Lysine (from sheep, diluted 1∶1000), followed by incubation for 2 hs with horseradish peroxidase-conjugated anti-sheep secondary antibody (diluted 1∶3000). Final detection was performed by using ECL chemiluminescence system; then, bands were scanned and quantified by densitometric analysis with ImageJ software.

Data obtained from three separate experiments were expressed as mean ± SEM, and analyzed by one-way analysis of variance (ANOVA) and the Student-Newman Keuls test using GraphPad Prism software (San Diego, CA). p values lower than 0.05 were considered significant.

A chromatogram of the BJe composition is shown in figure 1. Peaks 1–20 correspond to identified flavonoid present in the extract and their amounts are showed in table 1. The main flavonoids identified (mg/g) were Neohesperidin (105.27), Naringin (101.88), Melitidin (75.89), Neoeriocitrin (56.61) and Hesperetin (55.65). Quantitative values were obtained as the average of quintuplicate analyses and were in accordance with tipical BJ flavonoid profile reported in the literature [27], [32].

In order to assess BJe toxicity in cell cultures, preliminary experiments were carried out using MTT test. Exposure of THP-1 monocytes to different BJe concentrations (in a range 0.05–0.5 mg/ml) for 3.5 hs didn't show any significant reduction of cell viability when BJe was added in presence or absence of LPS, as shown by MTT data (Fig. 2).

Based on preliminary experiments we used LPS at the concentration of 500 ng/ml that was able to trigger the release of pro-inflammatory factors in THP-1 cells within three hours. Under these conditions, the Real-time PCR analysis showed that LPS stimulation of THP-1 monocytes resulted in a dramatic increase of mRNA of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α), relative to endogenous 18S mRNA levels. In particular, IL-6 mRNA increase was higher than those observed for IL-1β and TNF-α. The IL-6 mRNA levels in LPS-treated cells was almost one hundred-fold higher than those found in untreated cells (Fig. 3A). These effects were reduced in presence of BJe. Specifically, LPS-induced IL-6 up-regulation was strongly reduced in presence of BJe 0.05 mg/ml (by ∼70%), with a maximum reduction of ∼80% with 0.1 mg/ml of BJe (p<0.001). Even though to a lesser extent, also BJe 0.5 mg/ml was able to reduce IL-6 mRNA levels (∼60%; p<0.001). In parallel, the LPS-induced increase in IL-1β gene expression (36-fold higher than controls) was significantly reduced by all BJe concentrations used (from 70 to 80%; p<0.001; Fig. 3B). In a similar way, stimulation of THP-1 monocytes with LPS caused a 5-fold increase of TNF-α mRNA levels in comparison with untreated cells, while a significant down-regulation of LPS-stimulated TNF-α gene expression was observed in presence of BJe treatment (in the range 0.05–0.5 mg/ml), resulting in mRNA levels similar to those found in control cells (Fig. 3C).

In order to confirm the LPS-induced up-regulation of pro-inflammatory genes, we assessed the release of the analyzed cytokines by detection in the supernatants collected at the end of the treatment. As shown in figure 4, in comparison to untreated cells, 3 hs of LPS stimulation was able to trigger the secretion of significant amount of IL-1β and especially of TNF-α (4,7 and 37 fold increases, respectively) in THP-1 monocytes, but not of IL-6. These LPS-induced releases were reduced by BJe treatment. In particular, both BJe 0.1 and 0.5 mg/ml decreased significantly the amount of IL-1β secreted by 27 and 25%, respectively (p<0.05 vs LPS-treated cultures; Fig 4B), while all BJe concentrations used affected the release of TNF-α between 17 and 50% (p<0.05 and p<0.001 vs LPS-treated cells; Fig 4C). Interestingly, the most effective concentration of BJe able to diminish the protein levels of both IL-1β and TNF-α was 0.1 mg/ml.

Given the reported pivotal role of NF-κB in inflammatory response induced by various stimuli, we also investigated its role in THP-1 cell response to LPS-induced injury, in presence of the most effective concentration of BJe (0.1 mg/ml). EMSA analysis of nuclear fractions showed that nuclear translocation and specific DNA binding activity of NF-κB increased in THP-1 cell cultures, after 3 hs of incubation with LPS (Fig. 5). Interenstingly, BJe 0.1 mg/ml was able to inhibit the LPS-induced NF-κB activation.

Since it is known that SIRT1 may suppress inflammation by deacetylation of NF-κB subunit, in a subset of experiments we also investigated its regulatory effects on NF-κB activation. As shown in fig. 6, the inhibitory effect of 0.1 mg/ml BJe on the LPS-induced activation of NF-κB was counteracted at highest dose of Sirtinol, the well known inhibitor of SIRT1. In particular, 10 µM of Sirtinol reverted the NF-κB inhibition due to BJe treatment, suggesting a role of SIRT1 in the modulatory effect of BJe on NF-κB signaling.

In order to clarify the involvement of SIRT1's deacetylating activity and the molecular mechanism underlying the BJe-mediated inhibition of NF-κB, an immunoprecipitation assay was carried out to evaluate the acetylation status of RelA/p65 subunit. As shown in Fig.7, BJe 0.1 mg/ml treatment reverted the LPS-enhanced acetylation of p65 in THP-1 monocytes. In addition, increasing concentrations of Sirtinol were able to suppress the inhibitory effect of BJe on the activation of NF-κB, via p65 acetylation, underscoring that NF-κB–mediated inflammatory cytokine production may be directly linked to SIRT1 activity.