ABSTRACT: Glycosylation-related gene expression in the mucus-secreting gastrointestinal cell line HT29-MTX-E12 in response to infection by Helicobacter pylori
Project description:The HT29 derivative cell line HT29-MTX-E12 (E12) produces an adherent mucus layer predominantly of the gastric MUC5AC mucin when grown on transwells. This mucus layer supports Helicobacter pylori survival in culture. E12 cells were infected with H. pylori and the transcriptome of infected and uninfected E12 were compared. Also included for comparison was the HT29 parent cell line grown on transwells. The study focused on glycosylation related genes as these regulate the structures that have a major role to play in pathogen-host cell interaction.
Project description:The HT29 derivative cell line HT29-MTX-E12 (E12) produces an adherent mucus layer predominantly of the gastric MUC5AC mucin when grown on transwells. E12 cells were infected with C. jejuni and the transcriptome of infected and uninfected E12 were compared. The study focused on glycosylation related genes as these regulate the structures that have a major role to play in pathogen-host cell interaction. This experiment is related to another experiment where the cell line (E12) is replaced by the parental cell line (HT29) and importantly HT29 cells do not produce an adherent mucus layer.
Project description:HT29 cells were infected with C. jejuni 11168 and the transcriptome of infected and uninfected HT29 were compared by micoarray analysis (Affymetrix). The study focused on glycosylation related genes as these regulate the structures that have a major role to play in pathogen-host cell interaction. This experiment is related to another experiment where the parental cell line (HT29) is replaced by the derivative cell line (E12). E12 cells secrete an adherent mucus layer whereas HT29 cells do not.
Project description:The multifunctional intestinal mucus layer plays a crucial role in human health. Our understanding of the human colonic mucus layer in terms of structure, function and has been largely dependent on expensive and advanced ex vivo or in vitro models, which often require high expertise. The mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used in more simple in vitro models, but produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured in Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2 and had a thicker mucus layer compared to conventional culturing methods. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased mucus production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell inserts for 15 days were subject to transcriptome analysis to investigate underlying molecular pathways at gene expression level. We also further characterized the model by measuring transepithelial resistance and pH and lactate production of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms in which mucus production is enhanced under SWMS and propose potential applications of this model in future studies.
Project description:Glycosylation-related gene expression in the mucous-secreting gastrointestinal cell line HT29-MTX-E12 in response to infection by Campylobacter jejuni
Project description:The aim of this study is to identify alterations induced in gastric mucosa of mice exposed to Pteridium aquilinum and/or infected with Helicobacter pylori, in order to identify genes that are induced by bracken fern exerts exacerbating effects on gastric lesions associated to the infection. Six groups of C57Bl/6 mice were be used: 1) control, 2) infected Helicobacter pylori, 3) treated with Bracken fern extract orogastrically, 4) treated with Bracken fern extract in drinking water, 5) infected Helicobacter pylori + treated with Bracken fern extract orogastrically, 6) infected Helicobacter pylori + treated with Bracken fern extract in drinking water. The infection procedure was performed using an orogastric inoculation of H.pylori (strain SS1) twice in the first week. The RNA isolation was done in triplicate (3 mice per each condition). Further evaluation of morphological alterations on gastric mucosa, proliferative index and induction of DNA strand breaks will be performed in the mice stomach exposed to Pteridium aquilinum infected or not with Helicobacter pylori. Alterations of glycosylation in gastric tissues will also evaluated.
Project description:Following the initial demonstration of Helicobacter pyloriâ??s pathogenic potential, evidence has been accumulated that H. pylori is the leading cause of gastric ulcers, carcinoma and lymphoma3. Cholesterol is a physiological constituent of membranes critical for their biophysical properties, but is stigmatised as mediating detrimental effects in obesity and cardiovascular disease. Since H. pylori is auxotrophic for cholesterol, we explored the assimilation of cholesterol by H. pylori upon infection. Here we show that H. pylori follows a cholesterol gradient and extracts the lipid from plasma membranes of epithelial cells for subsequent glycosylation. Cholesterol promotes phagocytosis of H. pylori by antigen-presenting cells such as macrophages and dendritic cells and enhances antigen-specific T cell responses. Consistently, cholesterol-rich diet during bacterial challenge leads to a reduction of the H. pylori burden in the stomach. Intrinsic a-glycosylation of cholesterol abrogates phagocytosis of H. pylori and subsequent T cell activation. Hence, we propose a novel mechanism regulating host-pathogen interaction which describes glycosylation of a lipid tipping the scales towards immune evasion or response. color-swap dye-reversal hybridizations
Project description:Following the initial demonstration of Helicobacter pylori’s pathogenic potential, evidence has been accumulated that H. pylori is the leading cause of gastric ulcers, carcinoma and lymphoma3. Cholesterol is a physiological constituent of membranes critical for their biophysical properties, but is stigmatised as mediating detrimental effects in obesity and cardiovascular disease. Since H. pylori is auxotrophic for cholesterol, we explored the assimilation of cholesterol by H. pylori upon infection. Here we show that H. pylori follows a cholesterol gradient and extracts the lipid from plasma membranes of epithelial cells for subsequent glycosylation. Cholesterol promotes phagocytosis of H. pylori by antigen-presenting cells such as macrophages and dendritic cells and enhances antigen-specific T cell responses. Consistently, cholesterol-rich diet during bacterial challenge leads to a reduction of the H. pylori burden in the stomach. Intrinsic a-glycosylation of cholesterol abrogates phagocytosis of H. pylori and subsequent T cell activation. Hence, we propose a novel mechanism regulating host-pathogen interaction which describes glycosylation of a lipid tipping the scales towards immune evasion or response. Keywords: disease state analysis
Project description:Helicobacter pylori (H. pylori) causes assorted gastrointestinal disorders. H. pylori-specific T cells develop during infection, which is a prerequisite for the induction of gastritis and malignancy. How the innate immune system senses H. pylori to prime T cells remains unclear. We report that cholesteryl glucosides in H. pylori activate innate immunity through C-type lectin receptors (CLRs). Cholesteryl acyl α-glucoside (αCAG) was identified as a ligand for Mincle (Clec4e). Upon H. pylori infection, T cell responses and gastritis were ameliorated in Mincle-deficient mice, although bacterial numbers were comparable.Thus the cholesteryl lipid–CLR axis exacerbates H. pylori-induced gastric inflammation with limited contribution to protective immunity.
Project description:Eosinophils are highly abundant throughout the gastrointestinal tract. However, their function in bacterially-induced gastrointestinal inflammatory disorders is poorly understood. In this study, we have examined the function and regulation of gastric eosinophils in the steady state and during infection with Helicobacter pylori. We find that eosinophils are recruited to sites of infection, make direct contact with live bacteria, and activate a signature transcriptional program.
