Co-culture Of HT-29 Cells Research Articles

P047 Superoxide production and enhanced NOX2 induction through JAK/STAT signalling mediate IL-6-induced inflammatory responses of colon epithelial cells

IL-6 plays an important role in the pathogenesis of inflammatory bowel disease (IBD), which is supported by close correlation between IL-6 production and severity of the disease in IBD patients. IL-6 enhances expression of adhesion molecules that are required for the recruitment process of neutrophils and monocytes to lesion sites. The regulation of those molecule expressions by NF-κB, a redox-sensitive transcription factor, implies that IL-6-induced adhesion molecule expression may also be associated with reactive oxygen species (ROS) producing activity of IL-6. Present study focused on whether NADPH oxidase is involved in IL-6-induced ROS production and changes in the protein expressions. The IL-6-induced adhesion of monocytes (U937 cell line) to colon epithelial cells (HT-29 cell line) was examined by co-culture of HT-29 cells with U937 cells that were already labelled with BCECF-AM (10 μg/ml). After 3 h treatment with IL-6, BCECF fluorescence from adhered cells was measured. To identify signalling pathway, siRNA transfection, RT-PCR and Western blot analyses were performed. ROS was measured by lucigenin chemiluminescence assay. IL-6 significantly increased U937 monocytic cell adhesion to HT-29 colonic epithelial cells, which was accompanied by upregulation of adhesion molecules (ICAM-1 and VCAM-1) and repression of junction proteins (E-cadherin and claudin). Concurrently, IL-6 significantly increased superoxide production in a time-dependent manner, which matched significant induction of NOX2 and its regulatory subunits. The IL-6-induced ROS production and protein expression changes were attenuated by pretreatment with NADPH oxidase inhibitors (VAS-2840, and DPI) and STAT3 inhibitor (stattic), but not by inhibitors against other enzymes, such as cytosolic COX-2 (celecoxib), mitochondria (antimycin A), xanthine oxidase (allopurinol), and iNOS (NAME). Similarly, tofacitinib, a JAK inhibitor, and sttattic blocked IL-6-induced superoxide production and NOX2 induction. Taken together, our results suggest that IL-6-activated JAK/STAT signalling is linked to superoxide production and NOX2 induction, resulting in IL-6-induced inflammatory responses of colon epithelial cells.

Gender Dimorphism in the Gut: Mucosal Protection by Estrogen Stimulation of IgA Transcytosis

Laboratory studies demonstrate gender dimorphism following trauma/hemorrhagic shock (T/HS). These differences have been attributed to estrogen (E2) levels. Maintenance of gut barrier function by E2 following T/HS has been recently described. However, the mechanisms are not clear. The principle humoral defense mechanism of the gut is provided by secretory immunoglobulin IgA. It is transported across intestinal epithelial cells (IEC) by a specific transmembrane protein receptor (polyimmunoglobulin receptor, pIgR). Transport of IgA (transcytosis) may be influenced by a number of factors. We postulated that there may be differences in IgA transcytosis and IEC pIgR expression in response to sex hormones. We studied this in vitro. Confluent HT-29 IEC monolayers were established in a two-chamber cell culture system. E2 or dihydrotestosterone (DHT) was added for 72 hours; then dimeric IgA (dIgA) was added to the basal chamber (4°C, to obtain maximal pIgR binding of dIgA). Apical media were sampled at intervals and recovery of secretory immunoglobulin IgA quantitated by enzyme-linked immunosorbent assay. PIgR expression in HT-29 cells was quantitated as mean fluorescence intensity using flow cytometry. Monolayer integrity was confirmed by serial measurement of transepithelial electrical resistance. IgA transcytosis increased fourfold in 12-hour versus 3-hour culture periods in the control experiments. A similar finding was noted in the DHT experiments on IgA transcytosis. There were dramatic increases in IgA transcytosis across HT-29 cells exposed to E2.This was apparent at both 3- and 12-hour experimental time points and exhibited a dose-response effect. HT-29 cells cocultured with E2 increased pIgR expression in a time- and dose-dependent fashion. The greatest pIgR expression was noted following coculture of HT-29 cells with E2 for 6 days at the 1.0 μmol/L E2 concentration. The integrity of HT-29 monolayers in both the E2 and DHT treatment groups at T = 0 and 72 hours was assessed and showed no significant differences versus control cells. IgA transcytosis was augmented by E2 in a dose-response fashion. This effect was due to augmented intracellular trafficking of IgA and later partly due to increased pIgR expression. The dose-related effects of E2 on IgA transport confirm the findings in animal studies that improved outcomes in females can be related to the estrus cycle.

Toll‐like receptors‐2, ‐3 and ‐4 expression patterns on human colon and their regulation by mucosal‐associated bacteria

The colonic epithelium provides an interface between the host and micro-organisms colonising the gastrointestinal tract. Molecular recognition of bacteria is facilitated through Toll-like receptors (TLR). The colonic epithelium expresses relatively high levels of mRNA for TLR3 and less for TLR2 and -4. Little is known of the expression patterns and mode of induction of expression for these pattern recognition receptors in human colon. The aim of this study was to investigate their localization in the gut and induction of expression in epithelial cell lines by mucosal bacteria. TLR2 and -4 were expressed only in crypt epithelial cells, expression was lost as the cells matured and moved towards the gut lumen. In contrast, TLR3 was only produced in mature epithelial cells. HT29 and CACO-2 had different levels of expression for TLR1-4. Co-culture of HT29 cells with different mucosal isolates showed that they were highly responsive to bacterial challenge, with up-regulation of mRNA for TLR1-4. In contrast, CACO-2 cells were refractive to bacterial challenge, showing little difference in mRNA levels. TLR3 was induced in HT29 only by Gram-positive commensals with up-regulation of both mRNA and protein and an enhancement of the antiviral immune response. This pattern of expression allows induction of responsiveness to bacteria only by the crypt epithelium so that tolerance to commensal organisms can be maintained. In contrast, mature columnar epithelium is able to respond to viral pathogens, which are not part of the normal gut commensal microbiota.

Helicobacter pylori leads to decreased junctional expression of β-catenin in epithelial cells

Background: H.py/oriis implicated in the pathogenesis of gastric cancer. The cadberin-cotenin complex (of which ,8-catenin is a key signalling component) promotes stable adhesion of epithelial monolayers and is altered in several epithelial cancers, including gastric cancer+ Furthermore, germline mutations in the E-cadhenn gene have been described in familial gastric cancer. Aims: To assess the effect of H.pylori on cadberin-catenin expression in epithelial cell lines. Methods: Immunoblofting for a,/],~-catenin and E-cadherin was performed on lysed cell extracts of HeLa, AGS, RK13 and HT29 cells. H.pylonstrains 60190 (vac,4 slim1, caoA+) and Tx30a (vacA s2/m2, cagA-) were co-cultured with HT29 cells for 24 hours at 37C. Cells were also incubated with VacA (purified from 60190 by broth culture, precipitation, anion exchange and gel filtration chromatography). Using monoclonal antibodies and confocal microscopy, the distribution of E-cadherin and/3-eatanin was studied in two consecutive optical slices, taken 5/~m apart. Intensity of staining was graded as strong/weak/absent in 200 cell-cell junctions (systematic randomised selection) from treated and control cells. Experiments were performed on at least three occasions. Western blots were performed on cell extracts of HT29 cells for ~,/3,-/-catenin and E-cadberin comparing untreated cells with treated cells (60198,Tx30a co-culture and purified 60190 VacA). Results: Of the cell lines studied, only HT29 cells possessed the entire cadherin-catenin complex; and these molecules were Iocalised to junctional membrane domains. Co-culture of HT29 cells with H.pyloristr~n 60190 lead to a consistent decrease in junctional expression o f / ~ e n i n (mean of four experiments: strong staining in 84% of cell-cell junctions in untreated cells vs. 54% in H.pyloriexposed cells, p =0.05, paired t test). No redistribution of/Y-catsnin to nuclear regions was noted. Intensity and Iocalisation of E-cadberin remained unchanged. No gross changes ware seen with co-culture with strain Tx30a and with addition of purified VacA. Western blotting of untreated and treated HT29 cells revealed no maior alterations in levels of cadberin-catenin expression, Conclusion: Co-culture of a toxioenic caoA+ strain of H.pylori (but not a nontoxigenic cagAstrain)with epithelial cells leads to perturbation of/],.cetenin but has no effect on E-cadherin. We speculate that modification of junctional/]-eatanin may be important in the pathogenicity of H.py/ofi.

IL-8 secretion and neutrophil activation by HT-29 colonic epithelial cells

This study examines the ability of HT-29 human colonic epithelial cells to stimulate neutrophil migration and adhesion. Interleukin-8 (IL-8), a potent neutrophil chemoattractant, was detected in conditioned media from both unstimulated (1.1 ng/ml) and IL-1 beta-stimulated (16.1 ng/ml) HT-29 cultures. Conditioned medium from IL-1 beta-exposed HT-29 cells stimulated neutrophil migration (395% of control, P < 0.01), and this effect was completely inhibited by anti-IL-8 antibody. HT-29 medium also induced shedding of neutrophil L-selectin and increased expression of neutrophil CD11/CD18 adhesion receptors. Coculture of HT-29 cells with human endothelial cell monolayers resulted in increased neutrophil transendothelial migration (169% of control, P < 0.01), which was blocked by both anti-IL-8 and anti-CD18 antibody. Northern hybridization analysis demonstrated increased levels of mRNA for IL-8 and intercellular adhesion molecule-1 (ICAM-1) in cytokine-treated HT-29 cells. Cytokine stimulation of HT-29 monolayers was also associated with increased neutrophil adhesion to these cells. Neutrophil-HT-29 cell adhesion was blocked by monoclonal antibodies to neutrophil CD18 or to ICAM-1 on the HT-29 cells (86% and 56% inhibition, respectively, P < 0.01 for both). These data suggest that IL-8 secretion by activated colonic epithelial cells may contribute to neutrophil extravasation and tissue infiltration in intestinal inflammation.