I3C in the diet licenses oral tolerance.

<p>Mice were fed a placebo (black bars) or I3C containing diet (white bars) and were either not tolerized (PBS = control) or tolerized with OVA (dotted bars). Mice were immunized and then boosted 2 (A), 3 (B), and 4 weeks (C) later. Antibody titers were determined at the day of each boost and one week after the third boost (D). Antibody titers are depicted as % of placebo/PBS-treated animals that were set to 100%. Data are pooled from three independent experiments. N = 12–13 mice/group. Mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test. *≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.</p

Quantification of I3C and metabolites in mouse serum after oral uptake.

<p>Mice were fed for up to 7 days with chow containing 2 g/kg I3C. For the 15 minute value, mice received 5 mg/I3C per mouse diluted in 10%DMSO/olive oil by gavage. Serum samples were prepared and analyzed for the presence of I3C, DIM, and ICZ. Sera were spiked with 4-methoxyindole as the internal standard to calibrate for possible differences in extraction efficiency. Note that I3C is detectable only after the bolus feeding, but not when I3C was given in the chow, and is thus eaten in smaller amounts over the entire day. Y-axis: arbitrary units.</p

Quantification of I3C metabolites in serum.

<p>Quantification of I3C metabolites in serum.</p

Increase of DC in lamina propria and increased expression of <i>aldh</i> in the small intestine.

<p>A-B: <i>aldh1</i> expression after continuous feeding of I3C. Mice were fed for 49 days with normal or I3C enriched chow and expression of retinaldehyde dehydrogenase <i>aldh1a1</i> (A) and <i>aldh1a3</i> (B), analyzed by quantitative PCR. Fold change compared to housekeeping gene GAPDH is shown. Shown is the mean ± SEM of pooled data from two independent experiments. C-D: mice were fed continuously for 28 days normal chow (“placebo”) or I3C-enriched chow before tolerance induction. Samples were taken on day 21, i.e., 49 days after start of dietary intervention. For gating strategy see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180321#sec002" target="_blank">Material and methods</a> C: Percentage of CD11c⁺ (MHCII positive or negative) in living lamina propria cells of the small intestine (left), and the CD103+ subpopulation in CD11c+MHCII (right) DC in the lamina propria. D. typical histograms N = 9. Mean ± SEM. Unpaired Student’s t-test. *≤0.05, ** ≤0.01, *** ≤0.001.</p

I3C feeding dampens parameters of peanut allergy in mice.

<p>Mice were fed with normal (NC) I3C-enriched chow (I3C), sensitized to peanut extract, and then challenged by an i.p. injection with peanut extract (PE). A: IgG1 B: IgE antibody titers. C: rectal temperatures were taken after peanut injection. Mean rectal body temperature for placebo control was 38.3°C. The number of mice with a drop in temperature of >2% (considered a symptom of allergy), i.e., with a temperature below 37.4°C, were counted at 10, 30, 50, 70, 90, and 110 minutes. D: Anaphylactic scores. 0 = no symptoms; (N = 4 for control groups, N = 5 for PE groups. Mean± SEM is shown; A, B, C. statistical significance calculated with One-way ANOVA followed by Tukey’s multiple comparisons test: *≤0,05, **≤0,01, ***≤0,001,. NC = normal chow; I3C = 2 g/kg I3C supplemented chow graph.</p

CYP induction in small intestine.

<p>A-D: Mice were fed by gavage with TCDD, I3C or DMSO (as solvent for TCDD) at the indicated doses. Shown is <i>cyp1a1</i> expression relative to the housekeeping gene <i>rps6</i> in the three sections of the small intestine 4 hours after gavage (A, B), or 24 hours after gavage (C, D). Note the scale differences for TCDD and I3C. N = 3; Mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparison test. *≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001. E: Mice were fed for 49 days with chow containing 2 g/kg I3C. On day 49, RNA was isolated from small intestine and <i>cyp1a1</i> expression was analyzed by quantitative PCR. The results of two pooled independent experiments are shown. N = 11–15. Mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparison test. *≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.</p