Dataset Information

Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform.

ABSTRACT: In vitro models of the human intestinal epithelium derived from primary stem cells are much needed for the study of intestinal immunology in health and disease. Here, we describe an intestinal monolayer cultured on a porous membrane with accessible basal and apical surfaces for assay of intestinal cytokine production in response to stimuli. The system was composed of a differentiated, confluent epithelial monolayer derived from human primary stem cells obtained from small or large intestine. Interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) were the most abundant inflammatory cytokines produced by the intestinal epithelium. The epithelium from all five tested regions of the intestine preferentially secreted into the apical reservoir of the monolayer, with a 26-fold greater concentration of IL-8 present in the apical reservoir of the colonic monolayer relative to that in the basal reservoir. Upon application of tumor-necrosis factor ? (TNF-?) to the basal surface of the colonic monolayer, the IL-8 concentration significantly increased in the basal, but not the apical, reservoir. A dose-dependent elevation of IL-8 in the basal reservoir was observed for TNF-?-stimulation of the monolayer but not for an organoid-based platform. To demonstrate the utility of the monolayer system, 88 types of dietary metabolites or compounds were screened for their ability to modulate IL-8 production in the basal reservoir of the intestinal monolayer in the absence or presence of TNF-?. No dietary metabolite or compound caused an increase in IL-8 in the basal reservoir in the absence of TNF-?. After addition of TNF-? to the monolayer, two compounds (butyrate and gallic acid) suppressed IL-8 production, suggesting their potential anti-inflammatory effects, whereas the dietary factor forskolin significantly increased IL-8 production. These results demonstrate that the described human-intestinal-monolayer platform has the potential for assays and screening of metabolites and compounds that alter the inflammatory response of the intestine.

SUBMITTER: Wang Y

PROVIDER: S-EPMC6309958 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform.

Wang Yuli Y Kim Raehyun R Hwang Shee-Hwan J SJ Dutton Johanna J Sims Christopher E CE Allbritton Nancy L NL

Analytical chemistry 20180921 19

In vitro models of the human intestinal epithelium derived from primary stem cells are much needed for the study of intestinal immunology in health and disease. Here, we describe an intestinal monolayer cultured on a porous membrane with accessible basal and apical surfaces for assay of intestinal cytokine production in response to stimuli. The system was composed of a differentiated, confluent epithelial monolayer derived from human primary stem cells obtained from small or large intestine. Int ...[more]

PMID: 30199234

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Project description:Enteroendocrine (EE) cells within the intestinal epithelium produce a range of hormones that have key roles in modulating satiety and feeding behavior in humans. The regulation of hormone release from EE cells as a potential therapeutic strategy to treat metabolic disorders is highly sought after by the pharmaceutical industry. However, functional studies are limited by the scarcity of EE cells (or surrogates) in both in vivo and in vitro systems. Enterochromaffin (EC) cells are a subtype of EE cells that produce serotonin (5HT). Here, we explored simple strategies to enrich EC cells in in vitro monolayer systems derived from human primary intestinal stem cells. During differentiation of the monolayers, the EC cell lineage was significantly altered by both the culture method [air-liquid interface (ALI) vs submerged] and the presence of vasoactive intestinal peptide (VIP). Compared with traditional submerged cultures without VIP, VIP-assisted ALI culture significantly boosted the number of EC cells and their 5HT secretion by up to 430 and 390%, respectively. The method also increased the numbers of other subtypes of EE cells such as L cells. Additionally, this method generated monolayers with enhanced barrier integrity, so that directional (basal or apical) 5HT secretion was measurable. For all donor tissues, the enriched EC cells improved the signal-to-background ratio and reliability of 5HT release assays. The enhancement in the 5HT secretion behavior was consistent over time from a single donor, but significant variation in the amount of secreted 5HT was present among tissues derived from five different donors. To demonstrate the utility of the EC-enriched monolayer system, 13 types of pungent food ingredients were screened for their ability to stimulate 5HT secretion. Curcumin found in the spice turmeric derived from the Curcuma longa plant was found to be the most potent secretagogue. This EC-enriched cell monolayer platform can provide a valuable analytical tool for the high-throughput screening of nutrients and gut microbial components that alter the secretion of 5HT.

Project description:Background: Interleukin-22 (IL-22) impacts the integrity of intestinal epithelia and has been associated with the development of colitis-associated cancer and inflammatory bowel diseases (IBD). Previous data suggest that IL-22 protects the mucosal barrier and promotes wound healing and barrier defect. We hypothesized, that IL-22 modulates cell polarity of intestinal epithelial cells (IECs) acting on tight junction assembly. The aim of the study was to investigate IL-22-dependent mechanisms in the reprogramming of intestinal epithelia. Methods: IECs were exposed to IL-22 at various concentrations. IECs in Matrigel® were grown to 3-dimensional cysts in the presence or absence of IL-22 and morphology and expression of polarity proteins were analyzed by confocal microscopy. Epithelial cell barrier (TER and sandwich assay) and TJ assembly analysis (calcium-switch assay) were performed. TJ and cell polarity protein expression were assessed by western blotting and confocal microscopy. Cell migration and invasion assays were performed. Induction of epithelial-mesenchymal transition (EMT) was assessed by RT-qPCR analysis and western blotting. Signaling pathway analyses were performed by phosphoblotting and functional assays after blocking STAT3 and ERK signaling pathways. Using the toxoplasma-model of terminal ileitis, IL-22-knock-out mice were compared to wild-type littermates, analyzed for barrier function using one-path-impedance-analysis and macromolecular flux (H3-mannitol, Ussing-chambers). Results: IECs exhibited a barrier defect after IL-22 exposure. TJ protein distribution and expression were severely impaired. Delayed recovery in the calcium-switch assay was observed suggesting a defect in TJ assembly. Analyzing the 3D-cyst model, IL-22 induced multi-lumen and aberrant cysts, and altered the localization of cell polarity proteins. Cell migration and invasion was caused by IL-22 as well as induction of EMT. Interestingly, only inhibition of the MAPK pathway, rescued the TJal barrier defect, while blocking STAT3 was relevant for cell survival. In addition, ileal mucosa of IL-22 deficient mice was protected from the barrier defect seen in Toxoplasma gondii-induced ileitis in wild type mice shown by significantly higher Re values and correspondingly lower macromolecule fluxes. Conclusion: IL-22 impairs intestinal epithelial cell barrier by inducing EMT, causing defects in epithelial cell polarity and increasing cell motility and cell invasion. IL-22 modulates TJ protein expression and mediates tight junctional (TJal) barrier defects via ERK pathway.

Dataset Information

Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform.

Publications

Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform.

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