All chemical reagents, and LPS from P. gingivalis were purchased from Sigma-Aldrich Co. (St. Louis, MO). Cell culture reagents were purchased from Gibco/Invitrogen Corp. (Carlsbad, CA). SAGE (GM − 1111) was obtained from GlycoMira, LLC (Salt Lake City, UT).

Human peripheral blood mononuclear cells (PBMC) were isolated and purified from Leukocyte Concentrate, which was purchased from Long Island Blood Bank (Melville, NY). The project (2007–6548) was approved for exemption by Stony Brook University Committees on Research Involving Human Subjects (CORIHS). Human PBMC were isolated by density gradient centrifugation as described by us previously [19]. PMBC were cultured in serum-free macrophage media (Invitrogen Corp, Carlsbad, CA) for 18 h. LPS from P. gingivalis (50 ng/mL) or vehicle were added before the incubation. SAGE was added at final concentrations of 25 to 200 μg/ml. Conditioned media (CM) were analyzed for the cytokines and pro-inflammatory mediators, Tumor necrosis factor – alpha (TNF-α), Interleukin − 1 beta (IL-1β), Interleukin − 6 (IL-6), and Prostaglandin E2 (PGE2), by enzyme-linked immunosorbent assay (ELISA) as described by us previously [20].

In separate assays, PBMC were cultured for 7 days to allow for maturation to macrophage. Macrophages were then cultured for 18 h with or without LPS (50 ng/mL). SAGE at different concentrations were also added to the culture at the beginning of the experiment. The CM were analyzed for 1. TNF-α, IL-1β, IL-6 and PGE2 by ELISA; 2. MMPs by gelatin zymography and Western blot; and 3. TLR-2 and TLR-4 expression by Western blot. Gelatin zymography and Western blot assays were described below.

Human PBMC (1 × 106 cells/well) were cultured in serum-free media (37 °C, 5% CO2/95%O2 18 h) with LPS (50 ng/mL), SAGE, or vehicle alone. The CellTiter 96® AQueous One Solution Reagent contains a tetrazolium compound was added to the culture and incubated for 2 h. The formazan product was quantified by measuring the absorbance at 490 nm. The MTS assay kit was purchased from Promega Corp. (Madison, WI).

Cell culture CM collected from the above assays were analyzed for pro-inflammatory cytokines: TNF-α, IL-1β, IL-6 and PGE2. ELISA kits for TNF-α, IL-1β, IL-6 and PGE2, were purchased from R&D Systems, Inc. (Minneapolis, MN). ELISA assays were performed and followed manufacturer’s protocol.

The gelatin zymography system were purchased from Invitrogen Corp. (Carlsbad, CA). MMP-2 and MMP-9 standards were purchased from R&D Systems, Inc. (Minneapolis, MN). CM collected from the above cell culture assays were analyzed by gelatin zymography as described by us previously [20]. SDS-PAGE gels with 1 mg/ml gelatin were used. After electrophoresis, the gels were washed with 2.5% Triton X-100 and incubated at 37 °C overnight in calcium assay buffer (40 mM Tris/HCl, 200 mM NaCl, 10 mM CaCl2, pH 7.5), then stained with Coomassie Brilliant Blue R-250. As described by us earlier [19], clear zones of lysis against a blue background indicate gelatinolytic activity and levels of MMP-9 and MMP-2 were quantified by measuring band intensity using Image J analysis software [21].

CM collected from human macrophage cell culture were analyzed for TLR-2, TLR-4, and MMP-9 by Western blot analysis. All Western blot procedures were performed as described by us previously [22]. Prestained molecular weight markers were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). CM samples were electrophoresed on SDS-PAGE (8% separating and 4% stacking gels). Proteins were transferred to nitrocellulose membranes, and incubated with primary antibodies (TLR-2: Abcam, Cambridge, MA; TLR-4: Life Technologies, Carlsbad, CA; MMP-9: Cell Signaling Technology, Danvers, MA). Excess primary antibodies were removed before incubation with the secondary antibodies conjugated to horseradish peroxidase (Goat anti-rabbit antibodies, Cell Signaling Co., Danvers, MA). Detection of the protein bands was carried out by scanning the membranes with Invitrogen™ iBright™ FL1000 Western Blot Imaging Systems (Thermo Fisher Scientific, Inc., USA) and quantified by measuring band intensity using Image J analysis software. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Cytokine, MMP and receptor levels between groups were analyzed by analysis of variance (ANOVA), with P ≤ 0.05 taken as statistically significant. Normal versus LPS is represented by #; LPS versus SAGE treatment is represented by *.