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 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

Effect of isoflavones on LPS-induced MHC expression on DCs.

<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 HLA-ABC (A&D), HLA-E (B&E) and HLA-DR (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 10 independent experiments from different cell donors for LPS stimulations and 5 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 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

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

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

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

Offspring’s response to peanut sensitization based on maternal feeding of peanut.

<p>(A) Experimental protocol. Serum antibodies (B-E) and an anaphylactic response to the peanut challenge as indicated by the elevated score during the oral (PO) challenge (F) (baseline symptom score 0 in all the mice), the lowered body temperature during the intraperitoneal (IP) challenge (G), and after the peanut sensitization were assessed in offspring born to mothers exposed to peanut preconceptionally, who either continued to feed peanut during pregnancy and lactation (PC+PG+LC) and those who did not (PC). Offspring born to mothers never exposed to peanut served as controls (None). Naïve, non-sensitized mice served as controls. Shown is a mean with SEM. >15 mice were used per group.</p

OVA epicutaneous sensitization after feeding OVA with or without murine milk.

<p>(A) Experimental protocol. Offspring come from naïve mothers. Offspring’s response to OVA challenge (B-C) and antibody responses to OVA sensitization (D-F). Mice post-weaning at 3–4 weeks of age were fed OVA +/- OVA-immune or naïve milk for 5 days followed by cutaneous sensitization with OVA. Mice immunized without prior OVA feeding (Unfed) were used as controls. X axis for D-F shows the dilution factor for the serum. Each group has 5 mice. Shown is a mean with SEM. **, p<0.01, ***, p<0.001, ****, p<0.00001 when compared to unfed.</p

Intestinal peanut uptake to the Peyer’s patches.

<p>Mice were gavage-fed FITC-labeled CPE in the presence of naïve murine milk or immune milk. Peyer’s patches were collected 10 min later. A-B show representative sections of immunofluorescence. FAE, follicle associated epithelium; SED, subepithelial dome. FITC, CPE; Magenta, CD11c+. C) Number of DCs staining positive for CPE per patch. **, p<0.01 when compared to immune milk.</p