Novel chicken two-dimensional intestinal model comprising all key epithelial cell types and a mesenchymal sub-layer.

The intestinal epithelium plays a variety of roles including providing an effective physical barrier and innate immune protection against infection. Two-dimensional models of the intestinal epithelium, 2D enteroids, are a valuable resource to investigate intestinal cell biology and innate immune functions and are suitable for high throughput studies of paracellular transport and epithelial integrity. We have developed a chicken 2D enteroid model that recapitulates all major differentiated cell lineages, including enterocytes, Paneth cells, Goblet cells, enteroendocrine cells and leukocytes, and self-organises into an epithelial and mesenchymal sub-layer. Functional studies demonstrated the 2D enteroids formed a tight cell layer with minimal paracellular flux and a robust epithelial integrity, which was maintained or rescued following damage. The 2D enteroids were also able to demonstrate appropriate innate immune responses following exposure to bacterial endotoxins, from Salmonella enterica serotype Typhimurium and Bacillus subtilis. Frozen 2D enteroids cells when thawed were comparable to freshly isolated cells. The chicken 2D enteroids provide a useful ex vivo model to study intestinal cell biology and innate immune function, and have potential uses in screening of nutritional supplements, pharmaceuticals, and bioactive compounds.

Diabetic Phenotype in the Small Intestine of Zucker Diabetic Fatty Rats.

BACKGROUND/AIMS: In contrast to streptozotocin (STZ)-induced rodent models of diabetes, there are no thorough characterizations of the intestinal phenotype and the underlying changes in the global gene-expression of genetic models of diabetes, such as the Zucker diabetic fatty (ZDF) rat. The aim of the present study was to characterize the intestine in the ZDF rat. METHODS: The intestine of ZDF rats and lean controls was examined macroscopically and histologically, and ribonucleic acid sequencing (RNAseq) was performed in samples of jejunal mucosa. RESULTS: We observed an increased mass and length of the small and large intestines in ZDF rats. RNAseq showed an increased expression of Pdk2 and Pdk4, which are involved in the regulation of glucose and fatty acid metabolism, and increased expression of genes involved in gluconeogenesis and peroxisomal beta-oxidation in jejunal mucosa. CONCLUSION: Intestinal enlargement in ZDF rats is likely driven by increased food intake, since (i) it also occurs in obese and normoglycemic Zucker fatty rats, and (ii) insulin treatment of STZ-induced diabetic rats reduced the food intake and mass of the small intestine. Results from RNAseq indicate that small intestinal epithelial cells in ZDF rats have developed insulin resistance, and support that a normal physiological effect of insulin in the enterocytes is the regulation of glucose metabolism.

Distinct inflammatory and cytopathic characteristics of Escherichia coli isolates from inflammatory bowel disease patients.

Escherichia coli (E. coli) may be implicated in the pathogenesis of inflammatory bowel disease (IBD), as implied from a higher prevalence of mucosa-associated E. coli in the gut of IBD-affected individuals. However, it is unclear whether different non-diarrheagenic E. coli spp. segregate from each other in their ability to promote intestinal inflammation. Herein we compared the inflammation-inducing properties of non-diarrheagenic LF82, 691-04A, E. coli Nissle 1917 (ECN) and eleven new intestinal isolates from different locations in five IBD patients and one healthy control. Viable E. coli were cultured with human monocyte-derived dendritic cells (moDCs) and monolayers of intestinal epithelial cells (IECs), followed by analysis of secreted cytokines, intracellular levels of reactive oxygen species and cellular death. The IBD-associated E. coli LF82 induced the same dose-dependent inflammatory cytokine profile as ECN and ten of the new E. coli isolates displayed as high level IL-12p70, IL-1ß, IL-23 and TNF-α from moDCs irrespective of their site of isolation (ileum/colon/faeces), disease origin (diseased/non-diseased) or known virulence factors. Contrarily, 691-04A and one new IBD E. coli isolate induced a different cellular phenotype with enhanced killing of moDCs and IECs, coupled to elevated IL-18. The cytopathic nature of 691-04A and one other IBD E. coli isolate suggests that colonization with specific non-diarrheagenic E. coli could promote intestinal barrier leakage and profound intestinal inflammation, while LF82, ECN and the remaining non-diarrheagenic E. coli isolates hold notorious pro-inflammatory characteristics that can progress inflammation in case of intestinal barrier leakage.

Human milk oligosaccharides differentially support gut barrier integrity and enhance Th1 and Th17 cell effector responses in vitro.

Human milk oligosaccharides (HMOs) can modulate the intestinal barrier and regulate immune cells to favor the maturation of the infant intestinal tract and immune system, but the precise functions of individual HMOs are unclear. To determine the structure-dependent effects of individual HMOs (representing different structural classes) on the intestinal epithelium as well as innate and adaptive immune cells, we assessed fucosylated (2'FL and 3FL), sialylated (3'SL and 6'SL) and neutral non-fucosylated (LNT and LNT2) HMOs for their ability to support intestinal barrier integrity, to stimulate the secretion of chemokines from intestinal epithelial cells, and to modulate cytokine release from LPS-activated dendritic cells (DCs), M1 macrophages (MØs), and co-cultures with naïve CD4+ T cells. The fucosylated and neutral non-fucosylated HMOs increased barrier integrity and protected the barrier following an inflammatory insult but exerted minimal immunomodulatory activity. The sialylated HMOs enhanced the secretion of CXCL10, CCL20 and CXCL8 from intestinal epithelial cells, promoted the secretion of several cytokines (including IL-10, IL-12p70 and IL-23) from LPS-activated DCs and M1 MØs, and increased the secretion of IFN-γ and IL-17A from CD4+ T cells primed by LPS-activated DCs and MØs while reducing the secretion of IL-13. Thus, 3'SL and 6'SL supported Th1 and Th17 responses while reducing Th2 responses. Collectively, our data show that HMOs exert structure-dependent effects on the intestinal epithelium and possess immunomodulatory properties that confer benefits to infants and possibly also later in life.

Clinical Studies on the Supplementation of Manufactured Human Milk Oligosaccharides: A Systematic Review.

Human milk oligosaccharides (HMOs) are a major component of human milk. They are associated with multiple health benefits and are manufactured on a large scale for their addition to different food products. In this systematic review, we evaluate the health outcomes of published clinical trials involving the supplementation of manufactured HMOs. We screened the PubMed database and Cochrane Library, identifying 26 relevant clinical trials and five publications describing follow-up studies. The clinical trials varied in study populations, including healthy term infants, infants with medical indications, children, and adults. They tested eight different HMO structures individually or as blends in varying doses. All trials included safety and tolerance assessments, and some also assessed growth, stool characteristics, infections, gut microbiome composition, microbial metabolites, and biomarkers. The studies consistently found that HMO supplementation was safe and well tolerated. Infant studies reported a shift in outcomes towards those observed in breastfed infants, including stool characteristics, gut microbiome composition, and intestinal immune markers. Beneficial gut health and immune system effects have also been observed in other populations following HMO supplementation. Further clinical trials are needed to substantiate the effects of HMO supplementation on human health and to understand their structure and dose dependency.

Ex vivo intestinal adhesion of Escherichia coli LF82 in Crohn's disease.

Adherent-invasive Escherichia coli (AIEC) are reported to inhabit the gut mucosa in Crohn's disease (CD), however, little is known about the importance of host factors for the interplay between AIEC and the human gut. To examine if differences in bacterial adhesion patterns are disease associated, the AIEC-prototype strain LF82 was evaluated for its ability to adhere to ileal and colonic biopsies from CD and healthy controls (HC). Moreover, the efficacy of the non-pathogenic E. coli Nissle 1917 (ECN) in averting LF82 adhesion to ileal mucosa was assessed. Similar numbers of LF82 adhered to biopsies from CD and HC. A significantly greater LF82 attachment to ileal versus colonic mucosa was found in HC (P < 0.01), however, not in CD. ECN did not reduce the adhesion of LF82 to ileal specimens in CD or HC. These results show that enhanced bacterial adhesion ability is unlikely to play any significant role in CD, thus implying that other host protective factors may be impaired in CD. Further, exclusion of LF82 attachment by ECN co-incubation does not appear to represent a relevant treatment regimen.

Elucidating the Biological Roles of Insulin and Its Receptor in Murine Intestinal Growth and Function.

The role of the intestinal insulin receptor (IR) is not well understood. We therefore explored the effect of insulin (300 nmol/kg per day for 12 days) on the intestine in sex-matched C57Bl/6J mice. The intestinal and metabolic profiles were also characterized in male and female intestinal-epithelial IR knockout (IE-irKO) mice compared with all genetic controls on a chow diet or Western diet (WD) for 4 to 12 weeks. Insulin treatment did not affect intestinal size, intestinal resistance, or metabolic genes, but it reduced proximal-colon crypt depth and acutely increased colonic serine/threonine-specific protein kinase B (AKT) activation. Feeding with a WD increased body weight and fasting insulin level and decreased oral glucose tolerance in C57Bl/6J and IE-irKO mice. However, although the overall responses of the IE-irKO mice were not different from those of Villin-Cre (Vil-Cre):IRfl/+ and IRfl/fl controls, profound differences were found for female control Vil-Cre mice, which demonstrated reduced food intake, body weight, jejunal glucose transport, oral glucose tolerance, and fasting insulin and cholesterol levels. Vil-Cre mice also had smaller intestines compared with those of IE-irKO and IRfl/fl mice and greater insulin-mediated activation of jejunal IR and AKT. In summary, gain- and loss-of-function studies, with and without caloric overload, indicate that insulin did not exert remarkable effects on intestinal metabolic or morphologic phenotype except for a small effect on the colon. However, the transgenic control Vil-Cre mice displayed a distinct phenotype compared with other control and knockout animals, emphasizing the importance of thoroughly characterizing genetically modified mouse models.

Quantitative Proteomics of Intestinal Mucosa From Male Mice Lacking Intestinal Epithelial Insulin Receptors.

The goal of the present study was to determine whether loss of the insulin receptor alters the molecular landscape of the intestinal mucosa, using intestinal-epithelial insulin receptor knockout (IE-irKO) mice and both genetic (IRfl/fl and Villin-cre) controls. Quantitative proteomic analysis by liquid chromatography mass spectrometry was applied to jejunal and colonic mucosa from mice fed a normal chow diet and mice fed a Western diet (WD). Jejunal mucosa from IE-irKO mice demonstrated alterations in all intestinal cell lineages: Paneth, goblet, absorptive, and enteroendocrine cells. Only goblet and absorptive cells were affected in the colon. Also, a marked effect of WD consumption was found on the gut proteome. A substantial reduction was detected in Paneth cell proteins with antimicrobial activity, including lysozyme C-1, angiogenin-4, cryptdin-related sequence 1C-3 and -2, α-defensin 17, and intelectin-1a. The key protein expressed by goblet cells, mucin-2, was also reduced in the IE-irKO mice. Proteins involved in lipid metabolism, including aldose reductase-related protein 1, 15-hydroxyprostaglandin dehydrogenase, apolipoprotein A-II, and pyruvate dehydrogenase kinase isozyme 4, were increased in the mucosa of WD-fed IE-irKO mice compared with controls. In contrast, expression of the nutrient-responsive gut hormones, glucose-dependent insulinotropic polypeptide and neurotensin, was reduced in the jejunal mucosa of IE-irKO mice, and the expression of proteins of the P-type adenosine triphosphatases and the solute carrier-transporter family was reduced in the colon of WD-fed IE-irKO mice. In conclusion, IE-irKO mice display a distinct molecular phenotype, suggesting a biological role of insulin and its receptor in determining differentiated cell specificity in the intestinal epithelium.

Are NOD2 polymorphisms linked to a specific disease endophenotype of Crohn's disease?

The complex and yet unknown etiology of Crohn's disease (CD) might consist of various disease endophenotypes, each of which represent their own pathogenesis. This review focuses on the disease endophenotype linked to polymorphisms in the nucleotide-binding oligomerization domain containing 2 (NOD2) protein and on the importance of established adherent-invasive E. coli (AIEC) in ileal mucosa. To date, there are several reports pointing to the implications of NOD2 polymorphisms in epithelial and immunological responses against microbes, but the pathological significance of NOD2 mutations in CD is not yet clarified. The enhanced number of pathogenic E. coli in the ileal mucosa of CD as compared to healthy controls may result from a genetically based failure in one of the intestinal bacteria sensing systems, like NOD2, making the ileal epithelium more prone to colonization with microbes harboring specific properties such as AIEC. Increasing the focus on defining subgroups of patients with similar disease initiations, mechanisms of action, and manifestations in CD may be pivotal for the development and implementation of future individualized treatment strategies of benefit for the single patient at an early stage.

Quantification of specific E. coli in gut mucosa from Crohn's disease patients.

We here present a method based on qRT-PCR to quantify E. coli LF82 in intestinal human samples. Two different primer-probe sets were designed to detect LF82, and a third to target total E. coli. The assay showed high robustness and specificity for detection of LF82 in the presence of intestinal tissue.