Soybean isoflavones regulate dendritic cell function and suppress allergic sensitization to peanut Madhan Masilamani, PhD, John Wei, BA, Shiven Bhatt,

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Soybean isoflavones regulate dendritic cell function and suppress allergic sensitization to peanut Madhan Masilamani, PhD, John Wei, BA, Shiven Bhatt, BA, Misu Paul, BS, Stefanie Yakir, Hugh A. Sampson, MD Journal of Allergy and Clinical Immunology Volume 128, Issue 6, Pages 1242-1250.e1 (December 2011) DOI: 10.1016/j.jaci.2011.05.009 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 A, Chemical structure of the soybean isoflavones genistein, daidzein, and glycitein. B-E, Isoflavone profile of soybean and animal diets: powdered defatted soybeans (Fig 1, B), peanut (Fig 1, C), soy-free diet (Fig 1, D), and isoflavone diet (Fig 1, E) were extracted with acetonitrile, and HPLC was performed with a C-18 column. Peanut and the soy-free diet were completely devoid of isoflavones (Fig 1, C and D). Two distinct peaks representative of genistein and daidzein were seen in the isoflavone diet (Fig 1, E). Journal of Allergy and Clinical Immunology 2011 128, 1242-1250.e1DOI: (10.1016/j.jaci.2011.05.009) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 A, Murine sensitization protocol (see the Methods section). B, Mice fed with either the soy-free diet (n = 12) or the diet containing genistein and daidzein (gen&daid; 1500 ppm each; n = 12) for at least 2 weeks before the experiment were sensitized and challenged with peanut extract as shown in the Methods section and compared with naive unsensitized mice (n = 9). The clinical symptoms of anaphylaxis were given a score of between 0 and 5. Data from 3 independent experiments are shown (∗∗∗P < .001, 1-way ANOVA). C, Rectal temperatures of mice receiving the soy-free diet (n = 8) or the genistein and daidzein diet (n = 4) were measured at 30 minutes after 2 successive challenges (∗P < .05, t test). Data from 2 independent experiments are shown. Journal of Allergy and Clinical Immunology 2011 128, 1242-1250.e1DOI: (10.1016/j.jaci.2011.05.009) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 A, The percentage of mast cells in ear sections from mice receiving the soy-free diet (n = 12) or the genistein and daidzein (gen&daid) diet (n = 12) that are degranulated in each group and compared with naive unsensitized mice (n = 9). Data from 3 independent experiments are shown as box-and-whisker Tukey plots (∗∗∗P < .001, 1-way ANOVA). B, Plasma MMCP-1 levels were tested in blood obtained within 60 minutes of challenge by means of ELISA. Data from 3 independent experiments are shown as box-and-whisker Tukey plots (∗P < .05, 1-way ANOVA). Journal of Allergy and Clinical Immunology 2011 128, 1242-1250.e1DOI: (10.1016/j.jaci.2011.05.009) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Peanut-specific serum IgE (A), IgG2a (B), and IgG1 (C) levels were estimated by means of ELISA from murine blood collected on weeks 3, 6, and 8 and plotted as line graphs. Data from 5 to 10 mice from each group from 2 independent experiments are shown (∗∗P < .01, and ∗P < .05, t test). gen&daid, Genistein and daidzein. Journal of Allergy and Clinical Immunology 2011 128, 1242-1250.e1DOI: (10.1016/j.jaci.2011.05.009) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Mice were maintained on either a soy-free or isoflavone diet for at least 2 weeks and administered 20 μg of CT/100 μL of PBS by means of intragastric gavage. Single-cell preparations of the MLNs isolated after 18 hours of CT feeding were stained with the following fluorescent-conjugated antibodies followed by flow cytometry: CD11c, MHC-II (A/E), CD103, CD80, CD83, and CD86. The histograms for CD86, CD80, and CD83 stainings of MLN DCs falling in CD103+CD11c+MHC-IIhi gates are shown. The histograms shown are from a representative mouse from each group from 3 independent experiments. PBS- and CT-treated mice are indicated as dotted gray and solid black lines, respectively (∗P < .05, t test). gen&daid, Genistein and daidzein. Journal of Allergy and Clinical Immunology 2011 128, 1242-1250.e1DOI: (10.1016/j.jaci.2011.05.009) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Human MDDCs were activated with 1 μg/mL CT with or without 100 μmol/L isoflavones or dimethyl sulfoxide (dmso; vehicle control) for 18 hours and stained with CD83 (A), CD80 (B), and CD86 (C). Unstimulated controls (No CT) or CT- or CT & isoflavone-treated cells were shown in solid gray, solid black, and shaded histograms, respectively. The percentage surface expression levels were calculated from the geometric mean fluorescent intensities, in which unstimulated controls (No CT) were taken as 100%, and shown as bar graphs. Data from a total of at least 4 experiments are shown (∗P < .05 and ∗∗∗P < .001, 1-way ANOVA). daid, Daidzein; gen, genistein. Journal of Allergy and Clinical Immunology 2011 128, 1242-1250.e1DOI: (10.1016/j.jaci.2011.05.009) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Human MDDCs were activated with 1 μg/mL CT with or without isoflavones (100 μmol/L) or dimethyl sulfoxide (dmso; vehicle control) for 18 hours. IL-6 (A) and IL-8 (B) levels were measured in DC supernatants by using the cytometric bead array. The data from 7 individual experiments are shown as box-and-whisker Tukey plots. Activated DCs were washed and incubated with allogeneic CD4+ naive T cells for 5 days. IL-13 (C), IL-5 (D), and IL-9 (E) levels were measured by using the cytometric bead array in supernatants of DC–T-cell cultures. Data from 4 individual experiments are shown as box-and-whisker Tukey plots (∗P < .05, 1-way ANOVA). Journal of Allergy and Clinical Immunology 2011 128, 1242-1250.e1DOI: (10.1016/j.jaci.2011.05.009) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Isoflavone titration. Human MDDCs were activated with 1 μg/mL CT in the presence or absence of titrated amounts of genistein or daidzein. The concentration of isoflavones ranged from 6.25 to 200 μmol/L. The percentage surface expression levels of CD83 (top panel), CD80 (middle panel), and CD86 (bottom panel) were calculated from the geometric mean fluorescent intensities, in which the unstimulated control (No CT) was taken as 100%, and shown as bar graphs. Journal of Allergy and Clinical Immunology 2011 128, 1242-1250.e1DOI: (10.1016/j.jaci.2011.05.009) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions