MDP-induced mRNA expression of NALP3 and caspase 1.

<p>RT-qPCR of mRNA of inflammasome member NALP3 (A) and caspase 1 (B). Arbitrary units. Isolated monocytes were stimulated for 24 hours. Stimulation with MDP increased the level of caspase 1 expression in control cells (p<0.03), whereas such an increase was not found in CD monocytes.</p

Kinetics of MDP-induced mRNA expression of TNF-α and IL-1β.

<p>RT-qPCR of mRNA of TNF-α (A) and IL-1β (B). Kinetic studies of mRNA-response (C and D). Isolated monocytes were stimulated for 5 minutes (0.08 hours), 4 hours and 24 hours. Stimulation with MDP increased the level of mRNA transcription in monocytes from control subjects (p<0.01 and p<0.02 for TNF-α and IL-1β, respectively), whereas an upregulation was not seen in monocytes isolated from CARD15 non-mutated CD patients. Arbitrary units. Due to high variability in IL1β expression in both control and CD patients, these expression data were normalised to expression values at t = 0 hours. *p<0.05; **p<0.01.</p

Expression of inflammasome related proteins.

<p>Immunoblotting of NALP3, ASC, and CARD8. Monocytes expressed all these proteins involved in caspase 1 maturation.</p

MDP-induced expression IL-1β and activation of p38 and IKKα/β.

<p>Immunoblotting of IL-1β (A), p38 (B), and IKKα/β (C). Quatitative IKKα/β phosphorylation, bars represent ranges (D). Monocytes were stimulated for 24 hours with MDP. IL-1β expression was increased in CD patients, but no response was seen to MDP. Monocytes stimulated with MDP had increased p38 phosphorylation, regardless of disease status. Contrary to the p38 response, reduced IKKα/β phosphorylation was seen in CD regardless of CARD15 status. Control monocytes did respond to MDP stimulation by increasing IKKα/β phosphorylation. CD: Crohn's disease. Crohn*: SNP8 homozygote.</p

Integrative Transcriptomic and Metabonomic Molecular Profiling of Colonic Mucosal Biopsies Indicates a Unique Molecular Phenotype for Ulcerative Colitis

Ulcerative colitis is the most prevailing entity of several disorders under the umbrella term inflammatory bowel disease, with potentially serious symptoms and devastating consequences for affected patients. The exact molecular etiology of ulcerative colitis is not yet revealed. In this study, we characterized the molecular phenotype of ulcerative colitis through transcriptomic and metabonomic profiling of colonic mucosal biopsies from patients and controls. We have characterized the extent to which metabonomic and transcriptomic molecular phenotypes are associated with ulcerative colitis versus controls and other disease-related phenotypes such as steroid dependency and age at diagnosis, to determine if there is evidence of enrichment of differential expression in candidate genes from genome-wide association studies and if there are particular pathways influenced by disease-associated genes. Both transcriptomic and metabonomic data have previously been shown to predict the clinical course of ulcerative colitis and related clinical phenotypes, indicating that molecular phenotypes reveal molecular changes associated with the disease. Our analyses indicate that variables of both transcriptomics and metabonomics are associated with disease case and control status, that a large proportion of transcripts are associated with at least one metabolite in mucosal colonic biopsies, and that multiple pathways are connected to disease-related metabolites and transcripts

Caspase 1 processing and IL-1β release.

<p>Immunoblotting of caspase 1 (A). CD patients had increased basal expression of pro-caspase 1 and active cleaved caspase 1. No response was seen to MDP stimulation. ELISA measurement of released active IL-1β (B). Media was taken from monocytes stimulated for 24 hours with MDP. No differences in IL-1β secretion were detected.</p

Clinical details.

<p>CD: Crohn's disease, CDAI: Crohn's Disease Activity Index.</p

Box plot of RT-PCR results.

<p>Real time RT-PCR quantification of genes differentially expressed in inflamed CD smokers and non-smokers. Transcript copy numbers for ring finger protein 138 (RFP138), phosphoglucomutase 2-like 1 (PGM2L1), metallothionein 2A (MT2A), STEAP family member 3 (STEAP3), and potassium inwardly-rectifying channel, subfamily J, member 2 (KCNJ2) were measured in RNA extracted from all inflamed CD biopsy samples. The copy numbers of ribosomal protein L10 were used for normalization between samples. The box border represents the interquartile range, and the horizontal line in the box is the median. *, significant difference between smokers and non-smokers.</p

Differential expressed genes on DNA micro-arrays in CD inflamed smokers vs. CD inflamed non-smokers.

<p>The significance level for the Wilcoxon's rank sum was set at 2×10⁻⁴.</p><p>The Q value is the minimum <i>false discovery rate</i> set at 0.15.</p><p>Fold Change: up-regulated (+), down-regulated (−) in the smokers.</p

Overrepresentation analysis for predicted transcription factor binding sites using Primo on the 1^st principal component.

<p>Overrepresentation analysis for predicted transcription factor binding sites using Primo on the 1^st principal component.</p