VSL#3 inhibits LPS-induced phosphorylation of STAT-1.

<p>(<b>A</b>) LPS-induced genes, significantly suppressed by VSL#3, were analyzed by using Metacore Network analysis (Transcription factor). STAT-1 was predicted to be a key driver of this cluster. DC were stimulated for 3 hours, and phosphorylation of NF-κBp65 (<b>B</b>) and STAT-1 (<b>C</b>) was quantified in nuclear extracts using the Trans-am assay. Absorbance values (mean ± SD) of 5 individual donors are shown. Rank-Wilcoxon paired test: * p<0.05, ** p<0.01.</p

mRNA expression levels of 84 chemokines and cytokines after 4 hours of stimulation with LPS, VSL#3 or a combination of both, as determined by qPCR arrays.

<p>mRNA expression levels of 84 chemokines and cytokines after 4 hours of stimulation with LPS, VSL#3 or a combination of both, as determined by qPCR arrays.</p

Temporal Colonic Gene Expression Profiling in the Recurrent Colitis Model Identifies Early and Chronic Inflammatory Processes

<div><p>The recurrent TNBS-colitis model in BALB/c mice has been proposed as a model of Inflammatory Bowel Disease with a shifting pattern of local cytokines with the expression of Th1 cytokines during the early phase, Th17 cytokines during the intermediate phase and Th2 cytokines during late fibrotic stages. In this study, we evaluated the development of pathology in time–in conjunction with genome-wide gene expression in the colons–in response to three weekly intrarectal instillations of TNBS. During this time-frame mice develop colitis with extensive cellular infiltration of (sub)mucosa and mildly to moderately affected crypt architecture. These pathological processes were sensitive to local treatment with budesonide. Gene expression profiling confirmed an acute phase response after each intrarectal TNBS-challenge. In addition, a chronic inflammatory process developed over time particularly evident from a gradual increase in expression of mast cell related genes. The changes in pathological hallmarks were consistent with a temporal expression of mRNA encoding a selection of chemokines. In conclusion, the early stages of the recurrent TNBS-colitis model reflect several aspects of inflammatory bowel disease which are sensitive to immunomodulation.</p> </div

Disease characteristics of the recurrent TNBS model and efficacy of budesonide treatment.

<p>The development of colitis induction is evident from periods of loss of body weight following each rectal instillation of TNBS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050388#pone-0050388-g001" target="_blank">Figure 1A</a>; AUC p<0.005). At end-point, inflamed colons show significantly increased weight/length ratios and colon thickness (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050388#pone-0050388-g001" target="_blank">Figure 1B</a>); both aspects are suppressed by budesonide. TNBS colitis induction is associated with a significantly increased histological score (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050388#pone-0050388-g001" target="_blank">Figure 1C</a>); this score is significantly lower in budesonide treated animals. Bars represent group mean values of 10–12 mice/group, error bars represent SEM. Mann-Whitney U-test; *p<0.05, **p<0.01, ***p<0.001.</p

Relative circulating cytokine concentrations in TNBS-induced colitis^a.

a<p>In two independent studies, serum concentrations of 23 cytokines were determined by multiplex technology on day 28. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050388#pone.0050388.s002" target="_blank">Table S2</a> concentrations are presented as pg/ml ± SEM. In each study, values were normalized and expressed as a percentage of the mean concentration of the corresponding healthy control mice. Relative concentrations and shown as % ±SEM.</p>b<p>significantly (p<0.05) different relative serum concentration from that in healthy mice.</p>c<p>significantly (p<0.05) different relative serum concentration from that in vehicle treated mice.</p

Master regulators in colitis development.

a<p>Transcription factors were identified in biological networks generated using MetaCore™ software. Transcription factors represented in this table were identified in significant networks containing a minimal of 8 nodes.</p>b<p>Level of significance of the transcription factor network is classified based on z-scores: +, z-score between 50 and 100; ++, z-score above 100. No score means that the networks for these transcription factors were not identified at the time-point or had a z-score below 50.</p

Colitis induction is associated with increased numbers of innate and adaptive immune cells.

<p>The composition of the cellular infiltrate and the effect of budesonide treatment were analyzed by immunohistochemistry. (<b>A</b>) Enumeration of the cells showed significantly increased cell numbers per mm² tissue due to TNBS colitis induction. Budesonide treatment significantly reduced the number of these infiltrating cells. Bars represent group mean values of 10–12 mice/group, error bars represent SEM. Mann-Whitney U-test; *p<0.05, **p<0.01, ***p<0.001. (<b>B</b>) Double staining of CD11b+ and F480+ cells using fluorescence microscopy confirmed co-expression of these two cell surface markers.</p

Effect of recurrent TNBS administrations on cell adhesion molecules and chemokines.

<p>(<b>A</b>) Local mRNA expression of cell adhesion molecules and chemokines was determined within the time-course microarray experiment. The size of the effect of TNBS-induced colitis on gene expression in comparison to healthy colons is indicated in a heatmap. These differences are based on mean expression levels of 5 individual animals per time-point. (<b>B</b>) Circulating serum chemokines were analyzed by multiplex technology. Relative serum concentrations of concentrations of GM-CSF, MCP-1 (CCL2), MIP-1α (CCL3), and MIP-1β (CCL4) are graphically depicted; significant differences are indicated by #.</p

Transcriptome analysis in the recurrent TNBS-induced colitis model.

<p>To determine temporal patterns of gene expression levels that were affected by the repeated TNBS instillations, microarray analysis was performed on RNA isolated from colon tissue of colitic mice at day 9, 14, 16, 21, 23, and 28 (n = 5/group) (<b>A</b>) A heat-map of the genes with significantly modified gene expression revealed multiple clusters with different temporal expression patterns. (<b>B</b>) Bar graph indicates the number of up regulated (red) and down-regulated genes during colitis development at each time point. Genes were considered significant with a False discovery rate (FDR) p value <0.05. FDR was used to correct for multiple comparisons.</p

TNBS –induced colitis is associated with increased numbers of mast cells and α-defensins.

<p>(<b>A</b>) mRNA expression of selected mast cell specific genes in affected colon tissue (n = 5/group). Heatmap represents fold changes in TNBS colitis mice as compared to healthy control mice. (<b>B</b>) Quantification of toluidine blue positive cells confirmed TNBS induced up-regulation of mast cells. (<b>C</b>) mRNA expression of α- and β-defensin genes in affected colon tissue.</p