Matthew P. G. Barnett

The effects of dietary curcumin and rutin on colonic inflammation and gene expression in multidrug resistance gene-deficient (mdr1a-/-) mice, a model of inflammatory bowel diseases.

Damage of the intestinal epithelial barrier by xenobiotics or reactive oxygen species and a dysregulated immune response are both factors involved in the pathogenesis of inflammatory bowel diseases (IBD). Curcumin and rutin are polyphenolic compounds known to have antioxidant and anti-inflammatory activities, but their mechanism(s) of action are yet to be fully elucidated. Multidrug resistance gene-deficient (mdr1a-/- ) mice spontaneously develop intestinal inflammation, predominantly in the colon, with pathology similar to IBD, so this mouse model is relevant for studying diet-gene interactions and potential effects of foods on remission or development of IBD. The present study tested whether the addition of curcumin or rutin to the diet would alleviate colonic inflammation in mdr1a-/- mice. Using whole-genome microarrays, the effect of dietary curcumin on gene expression in colon tissue was also investigated. Twelve mice were randomly assigned to each of three diets (control (AIN-76A), control +0.2% curcumin or control +0.1% rutin) and monitored from the age of 7 to 24 weeks. Curcumin, but not rutin, significantly reduced histological signs of colonic inflammation in mdr1a-/- mice. Microarray and pathway analyses suggested that the effect of dietary curcumin on colon inflammation could be via an up-regulation of xenobiotic metabolism and a down-regulation of pro-inflammatory pathways, probably mediated by pregnane X receptor (Pxr) and peroxisome proliferator-activated receptor alpha (Ppara) activation of retinoid X receptor (Rxr). These results indicate the potential of global gene expression and pathway analyses to study and better understand the effect of foods in modulating colonic inflammation.

Alterations in the salivary proteome associated with periodontitis

AIM To identify changes in the salivary proteome associated with active periodontitis. MATERIALS AND METHODS Quantitative proteomics (two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis) was used to investigate whole saliva from individuals with severe periodontitis and their proteomic profiles before and after periodontal treatment were compared. RESULTS A comparison of 128 proteins across all saliva samples identified 15 protein spots with altered abundance. The predominant alteration observed was an increase in the abundance of the S100 proteins S100A8/A9/A6. Of the remaining proteins with altered abundance, haptoglobin, prolactin inducible protein and parotid secretory protein have previously been associated with host defence. CONCLUSION These results highlight the predominant involvement of S100 proteins in the host response during periodontitis, identify host defence components that have not been linked previously to this disease and suggest new potential biomarkers for monitoring disease activity in periodontitis.

The Role of Dietary Histone Deacetylases (HDACs) Inhibitors in Health and Disease

Modification of the histone proteins associated with DNA is an important process in the epigenetic regulation of DNA structure and function. There are several known modifications to histones, including methylation, acetylation, and phosphorylation, and a range of factors influence each of these. Histone deacetylases (HDACs) remove the acetyl group from lysine residues within a range of proteins, including transcription factors and histones. Whilst this means that their influence on cellular processes is more complex and far-reaching than histone modifications alone, their predominant function appears to relate to histones; through deacetylation of lysine residues they can influence expression of genes encoded by DNA linked to the histone molecule. HDAC inhibitors in turn regulate the activity of HDACs, and have been widely used as therapeutics in psychiatry and neurology, in which a number of adverse outcomes are associated with aberrant HDAC function. More recently, dietary HDAC inhibitors have been shown to have a regulatory effect similar to that of pharmacological HDAC inhibitors without the possible side-effects. Here, we discuss a number of dietary HDAC inhibitors, and how they may have therapeutic potential in the context of a whole food.

Genome-wide analysis of dietary eicosapentaenoic acid- and oleic acid-induced modulation of colon inflammation in interleukin-10 gene-deficient mice.

Background/Aims: Dietary n–3 polyunsaturated fatty acids can reduce inflammation via a range of mechanisms. This study tested the effect of dietary eicosapentaenoic acid (EPA) on intestinal inflammation using interleukin-10 gene-deficient (Il10–/–) mice. Methods: At 35 days of age, 12 weaned Il10–/– and 12 C57 mice were randomly assigned to one of two modified AIN-76A diets, supplemented with 3.7% purified ethyl esters of either EPA (n–3) or oleic acid (OA, control). To identify genes relevant to colon inflammation, transcription profiling (microarrays and qRT-PCR) and bioinformatic analyses were used. Results: In this study, dietary EPA reversed the decrease in colon fatty acid β-oxidation gene expression observed in OA-fed Il10–/– compared to C57 mice. Il10–/– mice fed the OA diet showed decreased expression of antioxidant enzyme genes, as well as those involved in detoxification of xenobiotics, compared to C57 mice on the same diet. In contrast, dietary EPA increased the expression of these genes in Il10–/– mice. Conclusions: These data indicate that dietary EPA-induced endogenous lipid oxidation which might have a potential anti-inflammatory effect on colon tissue. This is supported by the activation of the Ppara gene that regulates the expression of pro-inflammatory and immunomodulatory genes and proteins.

Metabolomic analysis identifies inflammatory and noninflammatory metabolic effects of genetic modification in a mouse model of Crohn's disease.

Interleukin-10 is an immunosuppressive cytokine involved in the regulation of gastrointestinal mucosal immunity toward intestinal microbiota. Interleukin-10-deficient (IL10(-/-)) mice develop Crohns disease-like colitis unless raised in germ-free conditions. Previous gas chromatography-mass spectrometry (GC-MS) metabolomic analysis revealed urinary metabolite differences between IL10(-/-) and wildtype C57BL/6 mice. To determine which of these differences were specifically associated with intestinal inflammation arising from IL10-deficiency, urine samples from IL10(-/-) and wildtype mice, housed in either conventional or specific pathogen-free conditions, were subjected to GC-MS metabolomic analysis. Fifteen metabolite differences, including fucose, xanthurenic acid, and 5-aminovaleric acid, were associated with intestinal inflammation. Elevated urinary levels of xanthurenic acid in IL10(-/-) mice were attributed to increased production of kynurenine metabolites that may induce T-cell tolerance toward intestinal microbiota. Liquid chromatography-mass spectrometry analysis confirmed that plasma levels of kynurenine and 3-hydroxykynurenine were elevated in IL10(-/-) mice. Eleven metabolite differences, including glutaric acid, 2-hydroxyglutaric acid, and 2-hydroxyadipic acid, were unaffected by the severity of inflammation. These metabolite differences may be associated with residual genes from the embryonic stem cells of the 129P2 mouse strain that were used to create the IL10(-/-) mouse, or may indicate novel functions of IL10 unrelated to inflammation.

Changes in colon gene expression associated with increased colon inflammation in interleukin-10 gene-deficient mice inoculated with Enterococcus species.

BackgroundInappropriate responses to normal intestinal bacteria may be involved in the development of Inflammatory Bowel Diseases (IBD, e.g. Crohns Disease (CD), Ulcerative Colitis (UC)) and variations in the host genome may mediate this process. IL-10 gene-deficient (Il10-/-) mice develop CD-like colitis mainly in the colon, in part due to inappropriate responses to normal intestinal bacteria including Enterococcus strains, and have therefore been used as an animal model of CD. Comprehensive characterization of changes in cecum gene expression levels associated with inflammation in the Il10-/-mouse model has recently been reported. Our aim was to characterize changes in colonic gene expression levels in Il10-/-and C57BL/6J (C57; control) mice resulting from oral bacterial inoculation with 12 Enterococcus faecalis and faecium (EF) strains isolated from calves or poultry, complex intestinal flora (CIF) collected from healthy control mice, or a mixture of the two (EF·CIF). We investigated two hypotheses: (1) that oral inoculation of Il10-/-mice would result in greater and more consistent intestinal inflammation than that observed in Il10-/-mice not receiving this inoculation, and (2) that this inflammation would be associated with changes in colon gene expression levels similar to those previously observed in human studies, and these mice would therefore be an appropriate model for human CD.ResultsAt 12 weeks of age, total RNA extracted from intact colon was hybridized to Agilent 44 k mouse arrays. Differentially expressed genes were identified using linear models for microarray analysis (Bioconductor), and these genes were clustered using GeneSpring GX and Ingenuity Pathways Analysis software. Intestinal inflammation was increased in Il10-/-mice as a result of inoculation, with the strongest effect being in the EF and EF·CIF groups. Genes differentially expressed in Il10-/-mice as a result of EF or EF·CIF inoculation were associated with the following pathways: inflammatory disease (111 genes differentially expressed), immune response (209 genes), antigen presentation (11 genes, particularly major histocompatability complex Class II), fatty acid metabolism (30 genes) and detoxification (31 genes).ConclusionsOur results suggest that colonic inflammation in Il10-/-mice inoculated with solutions containing Enterococcus strains is associated with gene expression changes similar to those of human IBD, specifically CD, and that with the EF·CIF inoculum in particular this is an appropriate model to investigate food-gene interactions relevant to human CD.

Global DNA methylation measurement by HPLC using low amounts of DNA.

Epigenetic changes in chromatin structure can influence gene expression without affecting the DNA sequence. The most commonly studied epigenetic modification, DNA methylation, has been implicated in normal tissue development and disease progression, and can be influenced by diet and other environmental factors. Current HPLC methods of determining DNA methylation may require relatively large amounts of DNA (50 μg); as many tissues have low DNA yields, this can be hard to achieve. We isolated DNA from mouse colon and liver in a study investigating post-natal supplementation with selenium and folic acid. After optimizing the methods to account for lower initial DNA amounts, we digested 3 μg of DNA to deoxynucleotide monophosphates, then purified and quantified it. Samples were analyzed by reversed-phase HPLC to determine global DNA methylation levels using commercial nucleotide standards. The HPLC column was cooled to 6(C (reducing run time), and detection was at 280 nm (UV). We showed that methylated cytosine can be accurately and reproducibly measured in as little as 3 μg of DNA using this HPLC analysis method (within-assay CV <2%). We also used this method to detect reduced DNA methylation in liver (P = 0.009) in response to post-natal supplementation with selenium and folate.

Dietary arachidonic acid‐mediated effects on colon inflammation using transcriptome analysis

Increased levels of n-6 arachidonic acid (AA), a precursor of pro-inflammatory eicosanoids, have been found in the colon mucosa of inflammatory bowel disease patients when compared with healthy subjects. The hypothesis was that dietary AA would aggravate colon inflammation by changing expression of genes in inflammatory signaling pathways. AA-enriched diet was fed to IL10 gene-deficient (Il10-/-) mice, model of a inflammatory bowel disease, and compared with Il10-/- mice fed an oleic acid control diet. Effects of AA on gene expression profiles during colitis were examined using whole genome microarray analysis. Dietary AA decreased the expression levels of some colonic genes in ER stress, complement system, nuclear respiratory factor 2-mediated oxidative stress and positive acute phase response pathways compared with Il10-/- mice fed an oleic acid diet. AA increased the expression levels of fatty acid catabolism genes, but decreased that of lipid synthesis genes during colitis, likely by sterol regulatory element binding transcription factor 1 and target gene regulation. A link has been suggested between AA and reduction of intestinal fibrosis by down-regulating the expression levels of pro-inflammatory and fibrotic marker genes. Contrary to the hypothesis, these findings suggest that dietary AA, in the present experimental conditions, is not pro-inflammatory, reduces ER stress and protects colonocytes from oxidative stress in Il10-/- mice.

Investigating micronutrients and epigenetic mechanisms in relation to inflammatory bowel disease

Epigenomic regulation, via DNA methylation, histone modification and non-coding RNA, is increasingly recognised as having a key role in normal development and function of an organism, acting to control cellular and tissue growth and differentiation. It is also thought to be involved in many complex diseases now common in the Western world, including cardiovascular disease, type 2 diabetes, obesity and inflammatory bowel disease (IBD). There is a range of evidence to suggest that nutrition plays a vital role in the protection from such diseases. However, there is little information about the role of nutrition on the epigenetic regulation of IBD. This review aims to elucidate the interactions of nutrients and the epigenome in IBD. More specifically, the plasticity of epigenetic modifications that occur due to low selenium and folate levels in the diet during gestation and lactation will be discussed. A better understanding of this plasticity, and of nutrient-epigenome interactions, will have important implications for enhancing human health through foods.

Dietary A1 β-casein affects gastrointestinal transit time, dipeptidyl peptidase-4 activity, and inflammatory status relative to A2 β-casein in Wistar rats.

Abstract We compared the gastrointestinal effects of milk-based diets in which the β-casein component was either the A1 or A2 type in male Wistar rats fed the experimental diets for 36 or 84 h. Gastrointestinal transit time was significantly greater in the A1 group, as measured by titanium dioxide recovery in the last 24 h of feeding. Co-administration of naloxone decreased gastrointestinal transit time in the A1 diet group but not in the A2 diet group. Colonic myeloperoxidase and jejunal dipeptidyl peptidase (DPP)-4 activities were greater in the A1 group than in the A2 group. Naloxone attenuated the increase in myeloperoxidase activity but not that in DPP-4 activity in the A1 group. Naloxone did not affect myeloperoxidase activity or DPP-4 activity in the A2 group. These results confirm that A1 β-casein consumption has direct effects on gastrointestinal function via opioid-dependent (gastrointestinal transit and myeloperoxidase activity) and opioid-independent (DPP-4 activity) pathways.