Chylomicron formation promotes intestinal OVA absorption.

<p>Fasted mice were gavaged with a dispersion of 0.05 ml ¹²⁵I-labeled OVA (black bars) in PBS plus 0.15 ml of either LCT, MCT, or LCT plus 6 µl of Pluronic L-81 (Pl-81). Radioactivity in the entire plasma per mouse (top panel) and in pooled MLN per mouse (bottom panel) was measured 90 minutes later. Another group of mice was gavaged with identical solutions, except that ¹²⁵I-OVA was replaced with [³H]-retinol (white bars). Shown are averages±S.D. of 4 mice per experimental group; * indicates statistically significant differences between feeding groups (P<0.05; ANOVA, Bonferroni's posthoc analysis). The figure shows a representative outcome of two repeats.</p

Chylomicron formation promotes intestinal absorption of full-length, antigenic OVA.

<p>Fasted mice were gavaged with 0.2 ml emulsions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008442#pone-0008442-g001" target="_blank">Figure 1</a>, except that ¹²⁵I-OVA was replaced with 25 mg OVA. Blood samples were obtained from the submandibular vein at indicated time points and analyzed for OVA by Western blotting.</p

Chylomicron formation promotes systemic dissemination and antigen presentation of dietary antigen.

<p>Naïve BALB/C mice were injected with 2.5×10⁶ CFSE labeled T cells from DO11.10 TCR transgenic mice. After 24 h, the mice were fasted (4 h) and gavaged with OVA (25 mg) in 0.2 ml PBS or 25 mg OVA in 0.05 ml PBS+0.15 ml of either MCT, LCT, or LCT plus Pl-81. Mice were then fasted for an additional 6 h. Three days later, inguinal LN cells were isolated, stained with anti-CD4 and KJ1-26 (TCR clonotypic antibody), and analyzed by flow cytometry. Histograms show representative CFSE dilution profiles of gated CD4+, KJ1-26+ T cells as a measure of cell division. The % of cells under markers M1 and M2 represent cells which have not or have undergone cell division respectively. Each panel represents a typical result of three experimental repeats.</p

Plasma chylomicrons transport dietary OVA.

<p>Fasted mice were gavaged with 0.2 ml LCT-containing emulsions also containing 25 mg OVA. Plasma was isolated 1 h later, and 55 µl were fractionated via FPLC. The grey line of the chromatogram shows the elution profile of a mouse injected i.p. with Poloxamer P-407 1 h prior to gavage to inhibit chylomicron clearance, which caused a milky plasma appearance (inset) and a greatly increased first peak. The solid line shows the elution profile of a mouse not previously injected with Poloxamer P-407. The fractions of this mouse, indicated by the vertical separators, were subjected to immunoprecipitation for detection of OVA (lower panel).The experiment was repeated three times with similar outcomes.</p

Uptake and secretion of OVA in association with chylomicrons by intestinal epithelial cells.

<p>(A) CaCo-2 cells were incubated with 20 µg/ml Alexa-red OVA for 1 h at 37°C. Nuclei were stained with DAPI (blue). (B) CaCo-2 cells on Transwell filters were incubated overnight at the apical side with 0.1 mg/ml OVA, washed from both sides, then incubated apically with 1.6 mM oleic acid (OA), butyric acid(BA), or oleic acid plus Pl-81 (2 µl/ml). Basolateral medium was collected 16 h later, and OVA was detected by immunoblotting. OVA was immunoprecipitated from the basolateral medium with anti-OVA coupled to protein A-Sepharose (or protein A-Sepharose only), followed by Western blotting of unwashed precipitate for detection of Apo-B. As shown in (C), ApoB-48 co-precipitated with OVA.</p

CD4 T-cell accumulation in mesenteric adipose tissue of OVA-fed, OVA sensitized mice.

<p>Naïve or OVA-sensitized C57Bl/6 mice were fed 1% OVA-containing high-fat diets for 14 weeks and SVF cells from their mesenteric adipose tissue were analyzed by flow cytometry. Two representative scatter plots are shown (A). The bar graph (B) shows the average percentage CD4 or CD8 T-lymphocytes ± S.D. (n = 5 mice per group). The asterisk (*) indicates statistically significant differences (P<0.05; Student's T-test).</p

Expression of CD3 and osteopontin in mesenteric fat in response to dietary antigen.

<p>Naïve mice (A–C) or OVA-sensitized mice (D–F) were fed 1% egg-white diets for two weeks, and mesenteric adipose tissue was stained for CD3 (Panels A,B, D, E; red signal) or osteopontin (C, F; brown signal). Nuclei in A,B,D and E were stained blue with DAPI.</p

Inflammatory immune responses to gut antigen in mesenteric adipose tissue impair glucose tolerance.

<p>OVA-sensitized (solid lines and symbols) or naïve (dashed lines, open symbols) mice were put on 1% OVA-containing high-fat diets for the indicated duration, and a glucose tolerance test was then performed on fasted mice. Glucose clearance was significantly impaired in sensitized BALB/c mice after 10 and 14 weeks (linear mixed model test for identical trajectories) and trended to significantly decrease in C57Bl/6 mice after 14 weeks.</p

Inflammatory gene expression in mesenteric and subcutaneous fat in response to gut antigen.

<p>OVA-sensitized BALB/c mice (black bars) or naïve BALB/c mice (white bars) were fed 1% egg-white-containing low- or high- fat diets for 2 or 10 weeks, and expression of OPN, IFNγ and FOXP3 were measured in mesenteric and subcutaneous adipose tissue. Groups contained five to six mice, and were compared by two-way ANOVA after log-transformation of the data. Groups not sharing the same letter were considered statistically significantly different by Bonferroni-adjusted post-hoc tests.</p

No significant effect of immune responses to gut antigen on body weight or fat mass.

<p>Groups of naïve or sensitized BALB/c mice (n = 6 per group) were fed 1% OVA containing low- or high- fat diets. Whereas mice on high-fat diets gained more weight than mice on low-fat diets, there was no significant difference between naïve and sensitized mice within each diet group. LF  =  low-fat diet, HF  =  high fat diet, N =  naïve, S =  sensitized. (B) Weight gain of body fat (in grams) of naïve (white bars) or sensitized (black bars) mice (n = 6 per group) on 1% OVA diets for 2 weeks (B) or 10 weeks (C). Bonferroni-adjusted post-hoc tests following two-way ANOVA revealed statistically significant differences between groups not sharing the same letter.</p