<i>Il10^−/−</i> mice exhibit accelerated tumor progression.

<p>A. Percent of tumor-bearing WT and <i>Il10^−/−</i> mice with high-grade or invasive carcinoma (upper panel). Representative low-grade adenoma observed in WT mice (middle panel). Representative invasive carcinoma observed in <i>Il10^−/−</i> mice (lower panel).</p

<i>Il10^−/−</i>; <i>Myd88^−/−</i> mice show decreased tumor multiplicity and expression of <i>Il12p40</i> and <i>Tnfα</i> mRNA.

<p>A. Representative histology observed in <i>Il10^−/−</i> and <i>Il10^−/−</i>; <i>Myd88^−/−</i> mice treated with AOM. B. Tumor multiplicity in <i>Il10^−/−</i> and <i>Il10^−/−</i>; <i>Myd88^−/−</i> mice treated with AOM. C. Relative expression of <i>Il12p40</i> and <i>Tnfα</i> mRNA in the distal colons of <i>Il10^−/−</i> and <i>Il10^−/−</i>; <i>Myd88^−/−</i> mice.</p

Modulation of microbiota-dependent colitis directly affects tumor development.

<p>A. Comparison of inflammation scores for <i>Il10^−/−</i> mice under SPF, <i>B. vulgatus</i> mono-associated and germ free conditions. B. Tumor multiplicity in <i>Il10^−/−</i> mice under SPF, <i>B. vulgatus</i> mono-associated and germ free conditions.</p

TLE exacerbates DSS-induced histological colitis and increased inflammatory gene expression.

<p>(A) Colonic histological sections were scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004562#s2" target="_blank">Materials and Methods</a>. Data were analyzed by ANOVA and comparison between groups performed using the Mann-Whitney U test. Values are mean±SEM; n = 6 in each group. Results are representative of 3 independent experiments. (B) IL-6, TNFα and MCP-1 mRNA accumulation from total colonic samples was determined using RT-PCR. GAPDH was used as an internal control. Representative results of 3 independent experiments are shown. C, AIN76A diet.</p

TLE inhibits LPS-induced IL-12p40 gene expression in murine splenocytes.

<p>(A) LPS-induced IL-12p40 mRNA accumulation following TLE treatment and LPS stimulation was measured using an ABI 7900HT Fast Real-Time PCR System. GAPDH was used as an internal control. Data are expressed as fold of the control (B) IL-12p40 secretion was measured by ELISA. Values are mean±SEM. All data were analyzed by Student's <i>t</i> test. Experiments were performed in triplicate and repeated three times.</p

Analysis of tumor induction and inflammation.

<p>A. Representative examples of WT (left panel) and <i>Il10^−/−</i> colons (middle and right panels) from AOM-treated mice 16 weeks after transfer to SPF conditions. B. Tumor penetrance and multiplicity in WT and <i>Il10^−/−</i> mice (left and right panels) (p = 0.034). C. WT and <i>Il10^−/−</i> colon inflammation scores, distal colon <i>Il12p40</i> and <i>Tnfα</i> mRNA levels after 18–20 weeks under SPF conditions (p = 0.026).</p

<i>Il10^−/−</i> mice develop inflammation displaying active Rel A (NFkB) signaling.

<p>A. Phosphorylated-Rel A immunostaining of normal <i>Il10^−/−</i> colon tissue. B. Enlargement of Rel A positive immune infiltrate in A. C. Phosphorylated Rel A immunostaining in <i>Il10^−/−</i> adenoma. D. Enlargement of adenoma in C. E. Rabbit IgG negative control.</p

Experimental timeline for AOM-induced colitis-associated colon tumorigenesis and analysis.

<p>Germ free mice were transferred to SPF conditions and allowed to acclimate for 5 weeks. Mice were given AOM injections once a week for 6 weeks (black arrowheads). Development of colitis and tumor formation was monitored by colonoscopy from weeks 4 to 18 after transfer from germ free to SPF conditions (white arrowheads). Mice were sacrificed between 18 and 20 weeks and tissues processed for histological and mRNA expression analysis.</p

TLE worsens DSS-induced experimental colitis.

<p>(A) Weight lost in TLE or control diet-fed mice exposed to 3% DSS. Values are mean±SEM; n = 6 in each group. Data were analyzed by ANOVA and comparison between groups performed using the Mann-Whitney U test. Results are representative of 3 independent experiments. (B). Colonic inflammation visualized by colonoscopy after 4 d of 3% DSS-exposure. Representative images of 6 different mice are shown. (C) The Colons of NF-κB^EGFP mice were imaged using an EGFP macroimaging system. Photomicrographs are shown on the left. Representative images of 6 different mice are shown. C, AIN76A diet.</p

Increased apoptosis in TLE-fed, DSS-exposed mice.

<p>(A) Numbers of TUNEL positive cells in C, DSS+C and DSS+TLE (n = 3/group). Bars show the mean±SEM of the number of apoptotic cells per 100 total nuclei. Data were analyzed by ANOVA and comparison between groups performed using the Mann-Whitney U test. (B) Apoptotic cells in colon crypt epithelial cells of TLE-fed and control diet-fed mice were determined by fluorometric TUNEL assay. Representative sections were taken on 3 d of DSS-exposure. Representative results of 3 independent experiments are shown. C, AIN76A diet.</p