Effect of increasing concentrations of natural or synthetic simplexide on IL-6 (panel A) and CXCL8 (panel B) release from monocytes.

<p>Polystyrene plates were coated with indicated concentrations of glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addiction of cells. After 24 hours of incubation, the supernatant was collected and centrifuged (1000×<i>g</i>, 4°C, 5 min). Cytokines and chemokines were determined by ELISA. The values are expressed as ng of IL-6 or CXCL8 per mg of total proteins. Data are the mean ± SEM of six experiments. * p<0.05 <i>vs</i>. control.</p

Kinetics of IL-6, CXCL8, TNF-α and IL-10 release from monocytes induced by simplexide.

<p>The cells were incubated (37°C, 3–24 h) with simplexide (10 µM). At the end of incubations, the supernatant was collected and centrifuged (1000×<i>g</i>, 4°C, 5 min). Cytokines and chemokines were determined by ELISA. The values are expressed as ng of IL-6, CXCL8, TNF-α or IL-10 per mg of total proteins. Data are the mean ± SEM of five experiments.</p

Effect of CD1d silencing on simplexide-induced release of CXCL8.

<p>Silencing of CD1d was performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111326#s2" target="_blank">Methods</a>”. (A) Western blot of monocytes transfected with two siRNA oligonucleotides (S1 and S5) or an irrelevant oligonucleotide (Sham). The immunoblot shown is representative of three different experiments. (B) Monocytes non-transfected (WT) or transfected with siRNA oligonucleotides (S1 and S5) or Sham were incubated (37°C, 24 h) with X-VIVO alone (unstimulated) or simplexide (10 µM). At the end of incubation, the supernatant was collected and centrifuged (1000×<i>g</i>, 4°C, 5 min). CXCL8 was determined by ELISA. The values are expressed as ng of CXCL8 per mg of total proteins. Data are the mean ± SEM of four experiments. * p<0.05 <i>vs</i>. the respective unstimulated. § p<0.05 <i>vs</i>. Sham.</p

Effect of polymyxin B on simplexide- and LPS-induced release of IL-6, CXCL8 and TNF-α from monocytes.

<p>Monocytes were incubated for 8 h (TNF-α) or 24 h (IL-6 and CXCL8) with simplexide (10 µM) or LPS (100 ng/ml) either in the absence (Control) or the presence of polymyxin B (50 µg/ml). Data are the mean ± SE of three experiments.</p><p>* p<0.05 vs. respective untreated.</p>†<p>p<0.05 vs. respective control.</p><p>Effect of polymyxin B on simplexide- and LPS-induced release of IL-6, CXCL8 and TNF-α from monocytes.</p

Effect of simplexide on C1R and C1R-CD1d cell lines.

<p>C1R and C1R-CD1d were incubated (37°C, 24 h) with X-VIVO alone (unstimulated), simplexide (10 µM), KRN7000 (1 µM) or PMA (100 ng/ml). At the end of incubation, the supernatant was collected and centrifuged (1000×<i>g</i>, 4°C, 5 min). CXCL8 was determined by ELISA. The values are expressed as pg of CXCL8 per mg of total proteins. Data are the mean ± SEM of six experiments. * p<0.05 <i>vs</i>. the respective unstimulated.</p

Effect of DPPE-PEG on simplexide-induced IL-6 (panel A) and CXCL8 (panel B) release from monocytes.

<p>Monocytes were preincubated (37°C, 5 min) with X-VIVO alone, or with the indicated concentrations of DPPE-PEG (1–30 µg/ml) and then stimulated (37°C, 24 h) with simplexide (•; 10 µM), α-GalCer (▪; 50 µM) or LPS (♦; 1 µg/ml). At the end of incubation, the supernatant was collected and centrifuged (1000×<i>g</i>, 4°C, 5 min). Cytokines and chemokines were determined by ELISA. The values are expressed as ng of IL-6 or CXCL8 per mg of total proteins. Data are expressed as percent inhibition of the maximum response induced by simplexide or LPS alone calculated as (R−R_b)/(R_max−R_b) ×100, where R is the release in samples treated with the agonists plus DPPE-PEG, R_b is the release in unstimulated samples and R_max is the release in samples stimulated with agonists alone. Data are the mean ± SEM of five experiments. The lines represent the best fit for inhibition of simplexide, α-GalCer or LPS.</p

Chemical structure of simplexide.

<p>Simplexide is a glycolipid composed of long-chain secondary alcohols glycosylated by a disaccharide chain containing α-glucose and α-galactose. Natural simplexide is a mixture of homologues, with alkyl chains of different length (R) linked to the central CHOH group. In addition, a significant part of the alkyl chains has a methyl branch in the second-to-last or third-to-last carbon atoms. The percentage of each molecular species detected in natural simplexide is shown on the right. Synthetic simplexide was prepared as a chemically homogeneous compound.</p

Effect of simplexide on IL-6, CXCL8, IL-10 and TNF-α mRNA expression in monocytes.

<p>The cells were incubated (37°C) for different time periods (3, 6, or 12 h) with simplexide (10 µM) or vehicle alone. mRNA levels for <i>IL6</i>, <i>CXCL8</i>, <i>IL10</i> and <i>TNFα</i> were quantitated by real-time PCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111326#s2" target="_blank">Methods</a>). Expression of <i>IL6</i>, <i>CXCL8</i>, <i>IL10</i>, and <i>TNFα</i> (normalized for <i>GAPDH</i>) was expressed as fold changes <i>vs.</i> untreated cells. Data are the mean ± SE of five experiments. <b>*</b>p<0.05 vs. untreated.</p

Effect of increasing concentrations of simplexide on IL-6 (panel A), CXCL8 (panel B), TNF-α (panel C) and IL-10 (panel D) release from monocytes.

<p>Monocytes were incubated (37°C, 8 h for TNF-α and IL-10 or 24 h for IL-6 and IL-8) with X-VIVO alone (control) or with the indicated concentrations of simplexide or LPS. At the end of incubation, the supernatant was collected and centrifuged (1000×<i>g</i>, 4°C, 5 min). Cytokines and chemokines were determined by ELISA. The values are expressed as ng of IL-6, CXCL8, TNF-α or IL-10 per mg of total proteins. Data are representative of ten independent experiments.</p

In vitro expansion of human iNKT cells.

<p>Human PBMC were stimulated with α-GalCer (100 nM) or simplexide (100 nM) for 7 days. iNKT expansion has been determined as percentage of Vα24⁺ cells among CD3⁺ lymphocytes. (A) Representative contour plots are shown. (B) Data are the mean ± SEM of four experiments. * p<0.05 <i>vs</i>. unstimulated.</p