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

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

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

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 ERT2knock 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): Lgr5high (stem cells), Lgr5low (daughter cells) and Lgr5negative (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]

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

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

Abstract 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