Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic ß-catenin.

Although the role of the Wnt pathway in colon carcinogenesis has been described previously, it has been recently demonstrated that Wnt signaling originates from highly dynamic nano-assemblies at the plasma membrane. However, little is known regarding the role of oncogenic APC in reshaping Wnt nanodomains. This is noteworthy, because oncogenic APC does not act autonomously and requires activation of Wnt effectors upstream of APC to drive aberrant Wnt signaling. Here, we demonstrate the role of oncogenic APC in increasing plasma membrane free cholesterol and rigidity, thereby modulating Wnt signaling hubs. This results in an overactivation of Wnt signaling in the colon. Finally, using the Drosophila sterol auxotroph model, we demonstrate the unique ability of exogenous free cholesterol to disrupt plasma membrane homeostasis and drive Wnt signaling in a wildtype APC background. Collectively, these findings provide a link between oncogenic APC, loss of plasma membrane homeostasis and CRC development.

Exfoliated epithelial cell transcriptome reflects both small and large intestinal cell signatures in piglets.

Assessing intestinal development and host-microbe interactions in healthy human infants requires noninvasive approaches. We have shown that the transcriptome of exfoliated epithelial cells in feces can differentiate breast-fed and formula-fed infants and term and preterm infants. However, it is not fully understood which regions of the intestine that the exfoliated cells represent. Herein, the transcriptional profiles of exfoliated cells with that of the ileal and colonic mucosa were compared. We hypothesized that exfoliated cells in the distal colon would reflect mucosal signatures of more proximal regions of the gut. Two-day-old piglets (n = 8) were fed formulas for 20 days. Luminal contents and mucosa were collected from ileum (IL), ascending colon (AC), and descending (DC) colon, and mRNA was extracted and sequenced. On average, ∼13,000 genes were mapped in mucosal tissues and ∼10,000 in luminal contents. The intersection of detected genes between three mucosa regions and DC exfoliome indicated an approximately 99% overlap. On average, 49% of the genes in IL, AC, and DC mucosa were present in the AC and DC exfoliome. Genes expressed predominantly in specific anatomic sites (stomach, pancreas, small intestine, colon) were detectable in exfoliated cells. In addition, gene markers for all intestinal epithelial cell types were expressed in the exfoliome representing a diverse array of cell types arising from both the small and large intestine. Genes were mapped to nutrient absorption and transport and immune function. Thus, the exfoliome represents a robust reservoir of information in which to assess intestinal development and responses to dietary interventions.NEW & NOTEWORTHY The transcriptome of exfoliated epithelial cells in stool contain gene signatures from both small and large intestinal mucosa affording a noninvasive approach to assess gut health and function.

Assessing the Multivariate Relationship between the Human Infant Intestinal Exfoliated Cell Transcriptome (Exfoliome) and Microbiome in Response to Diet.

Gut microbiota and the host exist in a mutualistic relationship, with the functional composition of the microbiota strongly influencing the health and well-being of the host. In addition to the standard differential expression analysis of host genes to assess the complex cross-talk between environment (diet), microbiome, and host intestinal physiology, data-driven integrative approaches are needed to identify potential biomarkers of both host genes and microbial communities that characterize these interactions. Our findings demonstrate that the complementary application of univariate differential gene expression analysis and multivariate approaches such as sparse Canonical Correlation Analysis (sCCA) and sparse Principal Components Analysis (sPCA) can be used to integrate data from both the healthy infant gut microbial community and host transcriptome (exfoliome) using stool derived exfoliated cells shed from the gut. These approaches reveal host genes and microbial functional categories related to the feeding phenotype of the infants. Our findings also confirm that combinatorial noninvasive -omic approaches provide an integrative genomics-based perspective of neonatal host-gut microbiome interactions.

Loss of aryl hydrocarbon receptor potentiates FoxM1 signaling to enhance self-renewal of colonic stem and progenitor cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that senses xenobiotics, diet, and gut microbial-derived metabolites, is increasingly recognized as a key regulator of intestinal biology. However, its effects on the function of colonic stem and progenitor cells remain largely unexplored. Here, we observed that inducible deletion of AhR in Lgr5+ stem cells increases the percentage of colonic stem cells and enhances organoid initiating capacity and growth of sorted stem and progenitor cells, while AhR activation has the opposite effect. Moreover, intestinal-specific AhR knockout increases basal stem cell and crypt injury-induced cell proliferation and promotes colon tumorigenesis in a preclinical colitis-associated tumor model by upregulating FoxM1 signaling. Mechanistically, AhR transcriptionally suppresses FoxM1 expression. Activation of AhR in human organoids recapitulates phenotypes observed in mice, such as reduction in the percentage of colonic stem cells, promotion of stem cell differentiation, and attenuation of FoxM1 signaling. These findings indicate that the AhR-FoxM1 axis, at least in part, mediates colonic stem/progenitor cell behavior.

Non-invasive evaluation of the equine gastrointestinal mucosal transcriptome.

Evaluating the health and function of the gastrointestinal tract can be challenging in all species, but is especially difficult in horses due to their size and length of the gastrointestinal (GI) tract. Isolation of mRNA of cells exfoliated from the GI mucosa into feces (i.e., the exfoliome) offers a novel means of non-invasively examining the gene expression profile of the GI mucosa. This approach has been utilized in people with colorectal cancer. Moreover, we have utilized this approach in a murine model of GI inflammation and demonstrated that the exfoliome reflects the tissue transcriptome. The ability of the equine exfoliome to provide non-invasive information regarding the health and function of the GI tract is not known. The objective of this study was to characterize the gene expression profile found in exfoliated intestinal epithelial cells from normal horses and compare the exfoliome data with the tissue mucosal transcriptome. Mucosal samples were collected from standardized locations along the GI tract (i.e. ileum, cecum, right dorsal colon, and rectum) from four healthy horses immediately following euthanasia. Voided feces were also collected. RNA isolation, library preparation, and RNA sequencing was performed on fecal and intestinal mucosal samples. Comparison of gene expression profiles from the tissue and exfoliome revealed correlation of gene expression. Moreover, the exfoliome contained reads representing the diverse array of cell types found in the GI mucosa suggesting the equine exfoliome serves as a non-invasive means of examining the global gene expression pattern of the equine GI tract.

Colonic mucosal and exfoliome transcriptomic profiling and fecal microbiome response to a flaxseed lignan extract intervention in humans.

BACKGROUND: Microbial metabolism of lignans from high-fiber plant foods produces bioactive enterolignans, such as enterolactone (ENL) and enterodiol (END). Enterolignan exposure influences cellular pathways important to cancer risk and is associated with reduced colon tumorigenesis in animal models and lower colorectal cancer risk in humans. OBJECTIVES: The aim of this study was to test the effects of a flaxseed lignan supplement (50 mg secoisolariciresinol diglucoside/d) compared with placebo on host gene expression in colon biopsies and exfoliated colonocyte RNA in feces and fecal microbial community composition, and to compare responses in relation to ENL excretion. METHODS: We conducted a 2-period randomized, crossover intervention in 42 healthy men and women (20-45 y). We used RNA-seq to measure differentially expressed (DE) genes in colonic mucosa and fecal exfoliated cells through the use of edgeR and functional analysis with Ingenuity Pathway Analysis. We used 16S ribosomal RNA gene (V1-V3) analysis to characterize the fecal microbiome, and measured END and ENL in 24-h urine samples by gas chromatography-mass spectrometry. RESULTS: We detected 32 DE genes (false discovery rate <0.05) in the exfoliome, but none in the mucosal biopsies, in response to 60 d of lignan supplement compared with placebo. Statistically significant associations were detected between ENL excretion and fecal microbiome measured at baseline and at the end of the intervention periods. Further, we detected DE genes in colonic mucosa and exfoliome between low- and high-ENL excreters. Analysis of biopsy samples indicated that several anti-inflammatory upstream regulators, including transforming growth factor ß and interleukin 10 receptor, were suppressed in low-ENL excreters. Complementary analyses in exfoliated cells also suggested that low-ENL excreters may be predisposed to proinflammatory cellular events due to upregulation of nuclear transcription factor κB and NOS2, and an inhibition of the peroxisome proliferator-activated receptor γ network. CONCLUSIONS: These results suggest that ENL or other activities of the associated gut microbial consortia may modulate response to a dietary lignan intervention. This has important implications for dietary recommendations and chemoprevention strategies. This study was registered at clinicaltrials.gov as NCT01619020.

Dietary fat and fiber interact to uniquely modify global histone post-translational epigenetic programming in a rat colon cancer progression model.

Dietary fermentable fiber generates short-chain fatty acids (SCFA), for example, butyrate, in the colonic lumen which serves as a chemoprotective histone deacetylase inhibitor and/or as an acetylation substrate for histone acetylases. In addition, n-3 polyunsaturated fatty acids (n-3 PUFA) in fish oil can affect the chromatin landscape by acting as ligands for tumor suppressive nuclear receptors. In an effort to gain insight into the global dimension of post-translational modification of histones (including H3K4me3 and H3K9ac) and clarify the chemoprotective impact of dietary bioactive compounds on transcriptional control in a preclinical model of colon cancer, we generated high-resolution genome-wide RNA (RNA-Seq) and "chromatin-state" (H3K4me3-seq and H3K9ac-seq) maps for intestinal (epithelial colonocytes) crypts in rats treated with a colon carcinogen and fed diets containing bioactive (i) fish oil, (ii) fermentable fiber (a rich source of SCFA), (iii) a combination of fish oil plus pectin, or (iv) control, devoid of fish oil or pectin. In general, poor correlation was observed between differentially transcribed (DE) and enriched genes (DERs) at multiple epigenetic levels. The combinatorial diet (fish oil + pectin) uniquely affected transcriptional profiles in the intestinal epithelium, for example, upregulating lipid catabolism and beta-oxidation associated genes. These genes were linked to activated ligand-dependent nuclear receptors associated with n-3 PUFA and were also correlated with the mitochondrial L-carnitine shuttle and the inhibition of lipogenesis. These findings demonstrate that the chemoprotective fish oil + pectin combination diet uniquely induces global histone state modifications linked to the expression of chemoprotective genes.

Aryl Hydrocarbon Receptor (AhR) Ligands as Selective AhR Modulators: Genomic Studies.

The aryl hydrocarbon receptor (AhR) binds structurally diverse ligands that vary from the environmental toxicant 2,3,7,8-tetrachlorodibenzo-B-dioxin (TCDD) to AhR- active pharmaceuticals and health-promoting phytochemicals. There are remarkable differences in the toxicity of TCDD and related halogenated aromatics (HAs) vs. health promoting AhR ligands, and genomic analysis shows that even among the toxic HAs, there are differences in their regulation of genes and pathways. Thus, like ligands for other receptors, AhR ligands are selective AhR modulators (SAhRMs) which exhibit variable tissue-, organ- and species-specific genomic and functional activities.

The non-invasive exfoliated transcriptome (exfoliome) reflects the tissue-level transcriptome in a mouse model of NSAID enteropathy.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most frequently used classes of medications in the world, yet they induce an enteropathy that is associated with high morbidity and mortality. A major limitation to better understanding the pathophysiology and diagnosis of this enteropathy is the difficulty of obtaining information about the primary site of injury, namely the distal small intestine. We investigated the utility of using mRNA from exfoliated cells in stool as a means to surveil the distal small intestine in a murine model of NSAID enteropathy. Specifically, we performed RNA-Seq on exfoliated cells found in feces and compared these data to RNA-Seq from both the small intestinal mucosa and colonic mucosa of healthy control mice or those exhibiting NSAID-induced enteropathy. Global gene expression analysis, data intersection, pathway analysis, and computational approaches including linear discriminant analysis (LDA) and sparse canonical correlation analysis (CCA) were used to assess the inter-relatedness of tissue (invasive) and stool (noninvasive) datasets. These analyses revealed that the exfoliated cell transcriptome closely mirrored the transcriptome of the small intestinal mucosa. Thus, the exfoliome may serve as a non-invasive means of detecting and monitoring NSAID enteropathy (and possibly other gastrointestinal mucosal inflammatory diseases).

Comprehensive site-specific whole genome profiling of stromal and epithelial colonic gene signatures in human sigmoid colon and rectal tissue.

The strength of associations between various exposures (e.g., diet, tobacco, chemopreventive agents) and colorectal cancer risk may partially depend on the complex interaction between epithelium and stroma across anatomic subsites. Currently, baseline data describing genome-wide coding and long noncoding gene expression profiles in the healthy colon specific to tissue type and location are lacking. Therefore, colonic mucosal biopsies from 10 healthy participants who were enrolled in a clinical study to evaluate effects of lignan supplementation on gut resiliency were used to characterize the site-specific global gene expression signatures associated with stromal vs. epithelial cells in the sigmoid colon and rectum. Using RNA-seq, we demonstrate that tissue type and location patterns of gene expression and upstream regulatory pathways are distinct. For example, consistent with a key role of stroma in the crypt niche, mRNAs associated with immunoregulatory and inflammatory processes (i.e., CXCL14, ANTXR1), smooth muscle contraction (CALD1), proliferation and apoptosis (GLP2R, IGFBP3), and modulation of extracellular matrix (MMP2, COL3A1, MFAP4) were all highly expressed in the stroma. In comparison, HOX genes (HOXA3, HOXD9, HOXD10, HOXD11, and HOXD-AS2, a HOXD cluster antisense RNA 2), and WNT5B expression were also significantly higher in sigmoid colon compared with the rectum. These findings provide strong impetus for considering colorectal tissue subtypes and location in future observational studies and clinical trials designed to evaluate the effects of exposures on colonic health.

A New Model to Study the Role of Arachidonic Acid in Colon Cancer Pathophysiology.

A significant increase in cyclooxygenase 2 (COX2) gene expression has been shown to promote cylcooxygenase-dependent colon cancer development. Controversy associated with the role of COX2 inhibitors indicates that additional work is needed to elucidate the effects of arachidonic acid (AA)-derived (cyclooxygenase and lipoxygenase) eicosanoids in cancer initiation, progression, and metastasis. We have recently developed a novel Fads1 knockout mouse model that allows for the investigation of AA-dependent eicosanoid deficiency without the complication of essential fatty acid deficiency. Interestingly, the survival rate of Fads1-null mice is severely compromised after 2 months on a semi-purified AA-free diet, which precludes long-term chemoprevention studies. Therefore, in this study, dietary AA levels were titrated to determine the minimal level required for survival, while maintaining a distinct AA-deficient phenotype. Null mice supplemented with AA (0.1%, 0.4%, 0.6%, 2.0%, w/w) in the diet exhibited a dose-dependent increase (P < 0.05) in AA, PGE2, 6-keto PGF1α, TXB2, and EdU-positive proliferative cells in the colon. In subsequent experiments, null mice supplemented with 0.6% AA diet were injected with a colon-specific carcinogen (azoxymethane) in order to assess cancer susceptibility. Null mice exhibited significantly (P < 0.05) reduced levels/multiplicity of aberrant crypt foci (ACF) as compared with wild-type sibling littermate control mice. These data indicate that (i) basal/minimal dietary AA supplementation (0.6%) expands the utility of the Fads1-null mouse model for long-term cancer prevention studies and (ii) that AA content in the colonic epithelium modulates colon cancer risk. Cancer Prev Res; 9(9); 750-7. ©2016 AACR.

Distinct Adipose Depots from Mice Differentially Respond to a High-Fat, High-Salt Diet.

BACKGROUND: Dietary factors such as high-sodium or high-fat (HF) diets have been shown to induce a proinflammatory phenotype. However, there is limited information with respect to how microenvironments of distinct intra-abdominal adipose depots respond to the combination of a high-salt, HF diet. OBJECTIVE: We tested the hypothesis that HF feeding would cause changes in distinct adipose depots, which would be further amplified by the addition of high salt to the diet. METHODS: Twenty-seven male C57BL6 mice were fed an HF diet (60% of kcal from fat), an HF + high-salt diet (4% wt:wt), a control diet [low-fat (LF);10% of kcal from fat], or an LF + high-salt diet for 12 wk. The main sources of fat in the diets were corn oil and lard. Adipokines in serum and released from adipose tissue organ cultures were measured by immunoassays. QIAGEN's Ingenuity Pathway Analysis was used to perform functional analysis of the RNA-sequencing data from distinct adipose depots. RESULTS: Diet-induced obesity resulted in a classical inflammatory phenotype characterized by increased concentrations of circulating inflammatory mediators (38-56%) and reduced adiponectin concentrations (27%). However, high-salt feeding did not exacerbate the HF diet-induced changes in adipokines and cytokines. Leptin and interleukin-6 were differentially released from adipose depots and HF feeding impaired adiponectin and resistin secretion across all 3 depots (34-48% and 45-83%, respectively). The addition of high salt to the HF diet did not further modulate secretion in cultured adipose tissue experiments. Although gene expression data from RNA sequencing indicated a >4.3-fold upregulation of integrin αX (Itgax) with HF feeding in all 3 depots, markers of cellular function were differentially expressed in response to diet across depots. CONCLUSION: Collectively, these findings highlight the role of distinct adipose depots in mice in the development of obesity and emphasize the importance of selecting specific depots to study the effects of therapeutic interventions on adipose tissue function.

The microbiota-derived metabolite indole decreases mucosal inflammation and injury in a murine model of NSAID enteropathy.

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most frequently used classes of medications in the world. Unfortunately, NSAIDs induce an enteropathy associated with high morbidity and mortality. Although the pathophysiology of this condition involves the interaction of the gut epithelium, microbiota, and NSAIDs, the precise mechanisms by which microbiota influence NSAID enteropathy are unclear. One possible mechanism is that the microbiota may attenuate the severity of disease by specific metabolite-mediated regulation of host inflammation and injury. The microbiota-derived tryptophan-metabolite indole is abundant in the healthy mammalian gut and positively influences intestinal health. We thus examined the effects of indole administration on NSAID enteropathy. Mice (n = 5 per group) were treated once daily for 7 days with an NSAID (indomethacin; 5 mg/kg), indole (20 mg/kg), indomethacin plus indole, or vehicle only (control). Outcomes compared among groups included: microscopic pathology; fecal calprotectin concentration; proportion of neutrophils in the spleen and mesenteric lymph nodes; fecal microbiota composition and diversity; small intestinal mucosal transcriptome; and, fecal tryptophan metabolites. Co-administration of indole with indomethacin: significantly reduced mucosal pathology scores, fecal calprotectin concentrations, and neutrophilic infiltration of the spleen and mesenteric lymph nodes induced by indomethacin; modulated NSAID-induced perturbation of the microbiota, fecal metabolites, and inferred metagenome; and, abrogated a pro-inflammatory gene expression profile in the small intestinal mucosa induced by indomethacin. The microbiota-derived metabolite indole attenuated multiple deleterious effects of NSAID enteropathy, including modulating inflammation mediated by innate immune responses and altering indomethacin-induced shift of the microbiota.

Data describing the effects of dietary bioactive agents on colonic stem cell microRNA and mRNA expression.

With the identification of Lgr5 as a definitive marker for intestinal stem cells, we used the highly novel, recently described, Lgr5-EGFP-IRES-cre ER (T2) knock in mouse model. Mice were injected with azoxymethane (AOM, a colon carcinogen) or saline (control) and fed a chemo-protective diet containing n-3 fatty acids and fermentable fiber (n-3 PUFA+pectin) or a control diet (n-6 PUFA + cellulose). Single cells were isolated from colonic mucosa crypts and three discrete populations of cells were collected via fluorescence activated cell sorting (FACS): Lgr5(high) (stem cells), Lgr5(low) (daughter cells) and Lgr5(negative) (differentiated cells). microRNA profiling and RNA sequencing were performed from the same sample and analyzed. These data refer to 'Comparative effects of diet and carcinogen on microRNA expression in the stem cell niche of the mouse colonic crypt' (Shah et al., 2016) [5].

Comparative effects of diet and carcinogen on microRNA expression in the stem cell niche of the mouse colonic crypt.

There is mounting evidence that noncoding microRNAs (miRNA) are modulated by select chemoprotective dietary agents. For example, recently we demonstrated that the unique combination of dietary fish oil (containing n-3 fatty acids) plus pectin (fermented to butyrate in the colon) (FPA) up-regulates a subset of putative tumor suppressor miRNAs in intestinal mucosa, and down-regulates their predicted target genes following carcinogen exposure as compared to control (corn oil plus cellulose (CCA)) diet. To further elucidate the biological effects of diet and carcinogen modulated miR's in the colon, we verified that miR-26b and miR-203 directly target PDE4B and TCF4, respectively. Since perturbations in adult stem cell dynamics are generally believed to represent an early step in colon tumorigenesis and to better understand how the colonic stem cell population responds to environmental factors such as diet and carcinogen, we additionally determined the effects of the chemoprotective FPA diet on miRNAs and mRNAs in colonic stem cells obtained from Lgr5-EGFP-IRES-creER(T2) knock-in mice. Following global miRNA profiling, 26 miRNAs (P<0.05) were differentially expressed in Lgr5(high) stem cells as compared to Lgr5(negative) differentiated cells. FPA treatment up-regulated miR-19b, miR-26b and miR-203 expression as compared to CCA specifically in Lgr5(high) cells. In contrast, in Lgr5(negative) cells, only miR-19b and its indirect target PTK2B were modulated by the FPA diet. These data indicate for the first time that select dietary cues can impact stem cell regulatory networks, in part, by modulating the steady-state levels of miRNAs. To our knowledge, this is the first study to utilize Lgr5(+) reporter mice to determine the impact of diet and carcinogen on miRNA expression in colonic stem cells and their progeny.

Non-invasive analysis of intestinal development in preterm and term infants using RNA-Sequencing.

The state and development of the intestinal epithelium is vital for infant health, and increased understanding in this area has been limited by an inability to directly assess epithelial cell biology in the healthy newborn intestine. To that end, we have developed a novel, noninvasive, molecular approach that utilizes next generation RNA sequencing on stool samples containing intact epithelial cells for the purpose of quantifying intestinal gene expression. We then applied this technique to compare host gene expression in healthy term and extremely preterm infants. Bioinformatic analyses demonstrate repeatable detection of human mRNA expression, and network analysis shows immune cell function and inflammation pathways to be up-regulated in preterm infants. This study provides incontrovertible evidence that whole-genome sequencing of stool-derived RNA can be used to examine the neonatal host epithelial transcriptome in infants, which opens up opportunities for sequential monitoring of gut gene expression in response to dietary or therapeutic interventions.

Differential effects of 2- and 3-series E-prostaglandins on in vitro expansion of Lgr5+ colonic stem cells.

Arachidonic acid (20:4(Δ5,8,11,14), AA)-derived prostaglandin E2 (PGE2) promotes colon cancer development. In contrast, chemoprotective n-3 polyunsaturated fatty acids supplant AA, thereby decreasing PGE2 biosynthesis in colonocytes, with eicosapentaenoic acid (20:5(Δ5,8,11,14,17), EPA) in particular being metabolized to a novel 3-series E-prostaglandin (PGE3), a putative anti-tumorigenic-cyclooxygenase metabolite. Because transformation of adult stem cells is an extremely important route toward initiating intestinal cancer, we utilized the leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescent protein-internal ribosome entry site (IRES)-creER(T2) knock-in mouse model to isolate and culture colonic organoids, in order to document ex vivo responses to exogenous PGE2 and PGE3. Colonic crypts were isolated from transgenic mice and cultured in a Matrigel-based three-dimensional platform. Organoids were treated with exogenous PGE2, PGE3 or dimethyl sulfoxide (vehicle control) for 5 days and the number of viable organoids was recorded daily. Subsequently, samples were processed for immunohistochemistry, flow cytometry and real-time PCR analyses. PGE2 promoted optimal organoid growth and induced significantly higher levels of cell proliferation (P < 0.05) compared with PGE3 and control. In contrast, the Lgr5-green fluorescent protein-positive stem cell number was uniquely elevated by >2-fold in PGE2-treated cultures compared with PGE3 and control. This coincided with the upregulation of stem-cell-related Sox9, Axin2 and Cd44 messenger RNAs. Our results demonstrate that relative to AA-derived PGE2, a known promoter of colon tumorigenesis, EPA-derived PGE3 has diminished ability to support colonic stem cell expansion in mouse colonic organoids.

Alteration of colonic stem cell gene signatures during the regenerative response to injury.

Since aberrant wound healing and chronic inflammation can promote malignant transformation, we determined whether dietary bioactive fish oil (FO)-derived n-3 polyunsaturated fatty acids (n-3 PUFA) modulate stem cell kinetics in a colitis-wounding model. Lgr5-LacZ and Lgr5-EGFP-IRES-creER(T2) mice were fed diets enriched with n-3 PUFA vs n-6 PUFA (control) and exposed to dextran sodium sulfate (DSS) for 5days in order to induce crypt damage and colitis throughout the colon. Stem cell number, cell proliferation, apoptosis, expression of stem cell (Lgr5, Sox9, Bmi1, Hopx, mTert, Ascl2, and DCAMKL-1) and inflammation (STAT3) markers were quantified. DSS treatment resulted in the ablation of Lgr5(+) stem cells in the distal colon, concurrent with the loss of distal crypt structure and proliferating cells. Lgr5, Ascl2 and Hopx mRNA expression levels were decreased in damaged colonic mucosa. Lgr5(+) stem cells reappeared at day 5 of DSS recovery, with normal levels attained by day 6 of recovery. There was no effect of diet on the recovery of stem cells. FO fed animals exhibited higher levels of phospho-STAT3 at all time points, consistent with a higher wounding by DSS in FO feeding. n-3 PUFA-fed mice exhibited a reduction in stem cell associated factors, Ascl2, Axin2 and EphB3. These results indicate that rapidly cycling Lgr5(+) stem cells residing at position 1 in the colon epithelium are highly susceptible to DSS-induced damage and that dietary cues can impact stem cell regulatory networks.

A metagenomic study of diet-dependent interaction between gut microbiota and host in infants reveals differences in immune response.

BACKGROUND: Gut microbiota and the host exist in a mutualistic relationship, with the functional composition of the microbiota strongly affecting the health and well-being of the host. Thus, it is important to develop a synthetic approach to study the host transcriptome and the microbiome simultaneously. Early microbial colonization in infants is critically important for directing neonatal intestinal and immune development, and is especially attractive for studying the development of human-commensal interactions. Here we report the results from a simultaneous study of the gut microbiome and host epithelial transcriptome of three-month-old exclusively breast- and formula-fed infants. RESULTS: Variation in both host mRNA expression and the microbiome phylogenetic and functional profiles was observed between breast- and formula-fed infants. To examine the interdependent relationship between host epithelial cell gene expression and bacterial metagenomic-based profiles, the host transcriptome and functionally profiled microbiome data were subjected to novel multivariate statistical analyses. Gut microbiota metagenome virulence characteristics concurrently varied with immunity-related gene expression in epithelial cells between the formula-fed and the breast-fed infants. CONCLUSIONS: Our data provide insight into the integrated responses of the host transcriptome and microbiome to dietary substrates in the early neonatal period. We demonstrate that differences in diet can affect, via gut colonization, host expression of genes associated with the innate immune system. Furthermore, the methodology presented in this study can be adapted to assess other host-commensal and host-pathogen interactions using genomic and transcriptomic data, providing a synthetic genomics-based picture of host-commensal relationships.

Dietary fish oil and curcumin combine to modulate colonic cytokinetics and gene expression in dextran sodium sulphate-treated mice.

Both fish oil (FO) and curcumin have potential as anti-tumour and anti-inflammatory agents. To further explore their combined effects on dextran sodium sulphate (DSS)-induced colitis, C57BL/6 mice were randomised to four diets (2 × 2 design) differing in fatty acid content with or without curcumin supplementation (FO, FO+2 % curcumin, maize oil (control, MO) or MO+2 % curcumin). Mice were exposed to one or two cycles of DSS in the drinking-water to induce either acute or chronic intestinal inflammation, respectively. FO-fed mice exposed to the single-cycle DSS treatment exhibited the highest mortality (40 %, seventeen of forty-three) compared with MO with the lowest mortality (3 %, one of twenty-nine) (P = 0·0008). Addition of curcumin to MO increased (P = 0·003) mortality to 37 % compared with the control. Consistent with animal survival data, following the one- or two-cycle DSS treatment, both dietary FO and curcumin promoted mucosal injury/ulceration compared with MO. In contrast, compared with other diets, combined FO and curcumin feeding enhanced the resolution of chronic inflammation and suppressed (P < 0·05) a key inflammatory mediator, NF-κB, in the colon mucosa. Mucosal microarray analysis revealed that dietary FO, curcumin and FO plus curcumin combination differentially modulated the expression of genes induced by DSS treatment. These results suggest that dietary lipids and curcumin interact to regulate mucosal homeostasis and the resolution of chronic inflammation in the colon.