Isoflavones suppress LPS-induced cytokine secretion from DCs.

<p>Human MDDCs were activated with 100 ng/mL LPS or 100 ng/mL TNF-α +/− 100 µM genistein (G100) or daidzein (D100) for 18 h. TNF-α (A&E), IL-10 (B&F), IL-6 (C&G) and IL-12 (D&H) levels were measured in culture supernatants by cytometric bead arrays. Isoflavone-treated cells are shown in dark bars (bars 4&5). DMSO - vehicle control for genistein and daidzein (bar 3). The data pooled from at least 4 independent experiments from different cell donors for LPS stimulations and 4 experiments for TNF stimulations. Cytokine levels were measured in pooled culture supernatants from two replicate for each condition. Statistical significance is indicated by * (P≤0.05) (paired student t-test).</p

Suppression of LPS-induced DC marker expression by isoflavones.

<p>Human MDDCs were activated with 100 ng/mL LPS (A-C)- or 100 ng/mL TNF-α (D-F) +/− 100 µM genistein (G100) or daidzein (D100) for 18 h and stained with CD83 (A&D), CD80 (B&E) and CD86 (C&F). Isoflavone-treated cells are shown in dark bars (bars 4&5). DMSO - vehicle control for genistein and daidzein (bar 3). The percentage surface expression levels were calculated from the geometric mean fluorescent intensities where unstimulated controls (No LPS or No TNF, bar 1) were taken as 100%. The data pooled from at least 11 independent experiments from different cell donors for LPS stimulations and 7 experiments for TNF stimulations. At least two replicates were performed for each condition in every experiment. Statistical significance is indicated by ** (P≤0.01) or *** (P≤0.001) (paired student t-test).</p

Isoflavones, Genistein and Daidzein, Regulate Mucosal Immune Response by Suppressing Dendritic Cell Function

<div><p>Lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, has been shown to have a strong adjuvant effect towards inhaled antigens contributing to airway inflammation. Isoflavones are anti-inflammatory molecules present in abundant quantities in soybeans. We investigated the effect of isoflavones on human dendritic cell (DC) activation via LPS stimulation and subsequent DC-mediated effector cell function both <em>in vitro</em> and in a mouse model of upper airway inflammation. Human monocyte-derived DCs (MDDC) were matured with LPS (or TNF-α) +/− isoflavones (genistein or daidzein). The surface expression levels of DC activation markers were analyzed by flow cytometry. Mature DCs +/− isoflavones were washed and cultured with freshly-isolated allogenic naïve CD4⁺ T cells for 5 days or with autologous natural killer (NK) cells for 2 hours. The percentages of proliferating IFN-γ⁺ CD4⁺ T cells and cytokine levels in culture supernatants were assessed. NK cell degranulation and DC cytotoxicity were measured by flow cytometry. Isoflavones significantly suppressed the activation-induced expression of DC maturation markers (CD83, CD80, CD86) and MHC class I but not MHC class II molecules <em>in vitro</em>. Isoflavone treatment inhibited the ability of LPS-DCs to induce IFN-γ in CD4⁺ T cells. NK cell degranulation and the percentage of dead DCs were significantly increased in isoflavone-treated DC-NK co-culture experiments. Dietary isoflavones suppressed the mucosal immune response to intra-nasal sensitization of mice to ovalbumin. Similar results were obtained when isoflavones were co-administered during sensitization. These results demonstrate that soybean isoflavones suppress immune sensitization by suppressing DC-maturation and its subsequent DC-mediated effector cell functions.</p> </div

Isoflavones regulate DC-mediated T cell function.

<p>Human MDDCs were activated with 100 ng/mL LPS or 100 ng/mL TNF-α +/− 100 µM genistein (G100) or daidzein (D100) for 18 h, washed and incubated with allogenic CD4+ naïve T cells for 5 days. IFN-γ (A&D), TNF-α (B&E) and IL-10 (C&F) levels were measured by cytometric bead array in co-culture supernatants. Isoflavone-treated cells are shown in dark bars (bars 4&5). DMSO - vehicle control for genistein and daidzein (bar 3). The data pooled from at least 5 independent experiments from different cell donors for LPS stimulations and 2 experiments for TNF stimulations. Statistical significance is indicated by * (P≤0.05) (paired student t-test).</p

Effect of isoflavones on mucosal immune response.

<p>Balb/c mice (female, 5 per group) were fed either a soy-free or isoflavone-containing diet (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047979#s2" target="_blank">Materials and Methods</a>). Mice in each diet group were immunized with 50 µg OVA and 5 µg LPS as adjuvant +/− 25 µg isoflavones (genistein and daidzein). Serum samples were obtained after 8 weeks of sensitization and OVA-specific IgG1 (A) and IgG2a (B) levels were measured by ELISA. Statistical significance is indicated by * (P≤0.05) (Unpaired student t test with Welch's correction).</p

Examination of vancomycin resistance and VISA-like phenotypes in strains defective in FeS cluster assembly.

<p>Growth inhibition in the presence of vancomycin (Panel A), lysis of cells in the presence of lysostaphin (Panel B), and zymographic analyses of spent medium supernatant upon gels containing heat-killed WT cells as a substrate (Panel C) were assessed for the WT (JMB1100), Δ<i>sufT</i> (JMB1146), Δ<i>nfu</i> (JMB1165), Δ<i>nfu</i> Δ<i>sufT</i> (JMB2514), Δ<i>nfu</i> Δ<i>sufT</i> Δ<i>sufA</i> (JMB6835), Δ<i>sufA</i> (JMB2223), Δ<i>sufA ΔsufT</i> (JMB2224), Δ<i>nfu</i> Δ<i>sufA</i> (JMB6834) strains. The data in panels A and B represent the average value of two biological replicates. For data in Panel C spent medium supernatant from three biological replicates was standardized and combined prior to zymographic analyses. The arrows in Panel C point to bands that are reduced in intensity in strains lacking both Nfu and SufT.</p

Functionality of the <i>Mycobacterium tuberculosis</i> DUF59 protein Rv1466 in <i>S</i>. <i>aureus</i>.

<p>Growth of the WT and Δ<i>sufT</i> strains carrying empty vector (pCM28) and the Δ<i>sufT</i> strain carrying pCM28_<i>rv1466</i> or pCM28_trunc_<i>rv1466</i> in 18AA glutamate medium (Panel A) or 20AA glucose medium (Panel B) are shown. The data represent the average of two biological replicates. Error bars signify standard deviations.</p

Isoflavones regulate DC-mediated NK cell degranulation and cytotoxicity.

<p>Flow cytometric gating strategy for DC-NK cell co-culture: Human MDDCs were activated with 100 ng/mL LPS, 1 µg/ml CT, or 100 ng/mL TNF-α +/− 100 µM genistein (G100) or daidzein (D100) for 18 h, washed and incubated with allogenic NK cells for 2 h in culture medium with FITC conjugated anti-human LAMP-1 and LAMP-2 Abs (2.5 µg/ml). The cells were then stained for CD83, CD56, CD69 and live/dead discriminator and analyzed by flow cytometry (A). Effector autologous NK cells were incubated with stimulant +/− isoflavone-treated target DCs (as above) in ratios ranging from 20∶1 to 0.63∶1 as indicated and stained as above. The percentage of LAMP-1/2^hi cells were plotted for each E:T ratio and stimulation condition as line graph (B). Effector allogenic NK cells were incubated with stimulant +/− isoflavones treated target DCs (as above) in 1∶1 ratio and stained as above (C & D). The percentage of LAMP-1/2^hi cells (C) and the percentage of DC cell death (D) are plotted for all the conditions and shown as bar graphs. DMSO - vehicle control for genistein and daidzein. The data pooled from 7 independent experiments from different cell donors for LPS stimulation, 5 experiments for CT stimulations and 2 experiments for TNF stimulations. DMSO : vehicle control. Statistical significance is indicated by * (P≤0.05) or ** (P≤0.01) (paired student t test).</p

Examining the need for SufT upon exposure of fermenting cells to dioxygen.

<p>Panel A: Transcription of genes utilized for FeS assembly are increased when fermenting cells are exposed to dioxygen (reaeration). The WT strain (JMB1100) was cultured fermentatively for 4.5 hours before one set of cultures was exposed to dioxygen while the control cultures experienced continuous anaerobic growth. mRNA abundances corresponding to the <i>sufT</i> and <i>sufC</i> genes were assessed using quantitative RT-PCR. Data are presented as a ratio of transcript abundance upon dioxygen exposure to the abundance upon continued anaerobic incubation. The gene transcription profiles for the <i>sufC</i> gene upon reaeration were previously published [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006233#pgen.1006233.ref004" target="_blank">4</a>]. Panel B: AcnA activity is decreased in the Δ<i>sufT</i> strain upon reaeration. The <i>acnA</i>::<i>TN</i> (JMB3537; parent) and the <i>acnA</i>::<i>TN</i> Δ<i>sufT</i> (JMB3539) strains containing p<i>acnA</i>, which contains <i>acnA</i> under the transcriptional control of a xylose inducible promoter, were cultured anaerobically before one set of cultures was exposed to dioxygen, while the control cultures experienced continuous anaerobic growth. The data in both panels represent the average of three biological replicates. Error bars signify standard deviations.</p

Analyses of Aconitase function in a Δ<i>sufT</i> strain.

<p>Panel A: <i>S</i>. <i>aureus</i> Δ<i>nfu</i> and Δ<i>sufT</i> mutant strains are defective for growth with glutamate as a carbon source. Growth traces of the WT (JMB1100), Δ<i>sufT</i> (JMB1146), and Δ<i>nfu</i> (JMB1165) strains in defined minimal medium containing the canonical 20 amino acids and glutamate as a carbon source (20AA glutamate medium). Panel B: Aconitase (AcnA) activity is decreased in strains lacking SufT or Nfu. Culture optical densities, as well as AcnA activities were assessed for the WT (JMB1100), Δ<i>sufT</i> (JMB1146), and Δ<i>nfu</i> (JMB1165) strains over the course of aerobic growth. Panel C: AcnA activity is decreased in a <i>sufT</i> mutant independent of <i>acnA</i> transcription and AcnA abundance. AcnA activity was assessed from the <i>acnA</i>::<i>TN</i> (JMB4432), <i>acnA</i>::<i>TN</i> Δ<i>sufT</i> (JMB4374), and the genetically complemented <i>acnA</i>::<i>TN</i> Δ<i>sufT sufT</i>⁺ (JMB4373) strains. All strains contained the p<i>acnA</i> plasmid, which contains <i>acnA</i> under the transcriptional control of a xylose inducible promoter. Top: Western blot analyses of the AcnA_FLAG displaying AcnA protein abundance in each strain, determined in duplicate. Panel D: Transcriptional activity of <i>sufC</i> is not decreased in Δ<i>nfu</i> and Δ<i>sufT</i> mutant strains. Transcriptional activity of <i>sufC</i> was assessed in the WT (JMB1100), Δ<i>sufT</i> (JMB1146), and Δ<i>nfu</i> (JMB1165) strains. The data represent the average of four (Panel A) or three (Panel B, C, and D) biological replicates and error bars represent standard deviations. Error bars are shown in all figures but may not be visible where error is low.</p