Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli.

Adherent-invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

Altered Calreticulin expression in human colon cancer: maintenance of Calreticulin expression is associated with mucinous differentiation.

Calreticulin is an endoplasmic reticulum luminal calcium-binding chaperone involved in various cellular functions and is a ligand for the scavenger receptor CD91. Recent studies, based on proteomic approaches on whole tissue samples containing both neoplastic and non-neoplastic cells, have shown alterations of Calreticulin expression in colon carcinomas, albeit with divergent results. The aims of this study were: 1) to assess the expression of Calreticulin and its receptor CD91 in 58 human colon adenocarcinomas, compared with paired normal mucosa, using a semi-quantitative immunohistochemical analysis, and 2) to examine associations between the tumour phenotypic features, and Calreticulin and/or CD91 expressions. Calreticulin expression was down-regulated in 51.7% human colon adenocarcinomas. Accordingly, quantitative immunoblot analysis showed that Calreticulin expression was significantly lower in human colonic cancer cell lines than in preparations of isolated human normal colonic epithelial cells. CD91 was co-expressed with Calreticulin in both normal colonic epithelial cells and pericryptic myofibroblasts. Calreticulin and CD91, that characterize the 'amateur phagocyte' function of epithelial cells, were both down-regulated in 48% of adenocarcinomas. Finally, Calreticulin expression was significantly associated with the mucinous differentiation of the tumour. Collectively, these results show that Calreticulin is likely to play a pivotal role in the differentiation of human colonic adenocarcinomas.

The in vitro manipulation of carbohydrate metabolism: a new strategy for deciphering the cellular defence mechanisms against nitric oxide attack.

This study was aimed at examining the effects of manipulating the carbohydrate source of the culture medium on the cellular sensitivity of epithelial cells to an oxidative attack. Our rationale was that substituting galactose for glucose in culture media would remove the protection afforded by glucose utilization in two major metabolic pathways, i.e. anaerobic glycolysis and/or the pentose phosphate pathway (PPP), which builds up cellular reducing power. Indeed, we show that the polarized human colonic epithelial cell line HT29-Cl.16E was sensitive to the deleterious effects of the NO donor PAPANONOate [3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine] only in galactose-containing medium. In such medium NO attack led to cytotoxic and apoptotic cell death, associated with formation of derivatives of NO auto-oxidation (collectively termed NOx) and peroxynitrite, leading to intracellular GSH depletion and nitrotyrosine formation. The addition of 2-deoxyglucose, a non-glycolytic substrate, to galactose-fed cells protected HT29-Cl. 16E cells from NO attack and maintained control GSH levels through its metabolic utilization in the PPP, as shown by (14)CO(2) production from 2-deoxy[1-(14)C]glucose. Therefore, increasing the availability of reducing equivalents without interfering with energy metabolism is able to prevent NO-induced cell injury. Finally, this background provides the conceptual framework for establishing nutritional manipulation of cellular metabolic pathways that could provide new means for (i) deciphering the mechanisms of cell injury by reactive nitrogen species and reactive oxygen species at the whole-cell level and (ii) establishing the hierarchy of intracellular defence mechanisms against these attacks.

Interleukin 1 and interleukin 1beta converting enzyme (caspase 1) expression in the human colonic epithelial barrier. Caspase 1 downregulation in colon cancer.

BACKGROUND: Interleukin (IL) 1beta converting enzyme (now known as caspase 1) is able to process pro-IL-1beta into its active form and is involved in proapoptotic signalling. AIMS: To characterise IL-1 and caspase 1 expression in human colonic epithelial cells. METHODS: Intracellular IL-1 content (IL-1alpha and IL-1beta) was measured by ELISA in freshly isolated human normal colonocytes. Caspase 1 expression was determined both at the mRNA level using in situ hybridisation and reverse transcription polymerase chain reaction, and at the protein level by immunoblotting experiments using antibodies specific for the proform of caspase 1 and for its cleavage forms. RESULTS: Low amounts of IL-1beta were found in nearly all preparations (92%), and IL-1alpha was detected in only 45% of human colonocyte preparations. The normal colonic epithelium strongly expressed caspase 1, both at the mRNA level and at the protein level in its latent form. In contrast, caspase 1 was not expressed in colon cancer (primary colonic adenocarcinomas and cancer cell lines). CONCLUSIONS: The demonstration that the human colonic epithelial barrier is able to express caspase 1 and its substrate IL-1beta reinforces the concept that, under certain conditions, the epithelium could trigger an inflammatory reaction. In addition, the finding that caspase 1 was downregulated in colonic adenocarcinomas supports the concept that disrupted apoptosis pathways may be involved in tumour formation and/or may confer resistance to treatment.

Early functional effects of Clostridium difficile toxin A on human colonocytes.

BACKGROUND & AIMS: Previous in vitro studies have shown that Clostridium difficile toxin A is able to directly affect the intestinal epithelial barrier function. The aim of this study was to examine the early effects of toxin A on mucin exocytosis and determine whether this toxin can induce the production of the chemokine interleukin 8 (IL-8) from human colonic epithelial cells. METHODS: Two model systems were used: the HT29-CI.16E colonic goblet cell line and primary cultures of human normal colonocytes. RESULTS: Toxin A exerted a rapid and dose-related inhibition of stimulated mucin exocytosis without altering baseline (constitutive) mucin exocytosis from HT29-CI.16E cells. Toxin A was also able to induce the secretion of IL-8 from both HT29-CI.16E cells and primary cultures of human normal colonocytes, as early as 2-3 hours of incubation. CONCLUSIONS: The results show that while toxin A is able to down-regulate stimulated mucin exocytosis, it is able to up-regulate the secretion of an important chemoattractant chemokine, IL-8. These modifications illustrate the ability of colonocytes to recruit inflammatory and immune cells that will eventually bring about major mucosal damage.

Role of calcium in carbachol- and neurotensin-induced mucin exocytosis in a human colonic goblet cell line and cross-talk with the cyclic AMP pathway.

The mechanisms of Ca(2+)-induced mucin secretion were examined in monolayers of the differentiated epithelial colon cell line C1.16E by combined measurements of free intracellular Ca2+ ([Ca2+]i) using a fluorescence indicator and mucous secretion using a specific and sensitive electrophoretic assay. Carbachol, a cholinergic agonist, induced an initial concentration-dependent [Ca2+]i peak increasing from 129 +/- 3 nM (basal [Ca2+]i) to 608 +/- 101 nM at 1 x 10(-4) M carbachol with an ED50 of 7 microM, and this was followed by a lower-level plateau. These biphasic effects were reversed by the muscarinic-receptor antagonist atropine. In the absence of extracellular Ca2+, the initial [Ca2+]i peak was maintained while the sustained plateau was abolished. The regulatory peptide neurotensin caused a monophasic transient rise in [Ca2+]i followed by a very rapid return to baseline. The neurotensin-induced rise in [Ca2+]i was concentration-dependent with an ED50 of 4 nM, and was maximal at 1 x 10(-6) M (598 +/- 127 nM). The [Ca2+]i response to neurotensin was not significantly affected by extracellular Ca2+ depletion. Carbachol-induced mucin exocytosis was concentration-dependent with an ED50 of 15 microM, and was inhibited by 35% upon removal of extracellular Ca2+. Neurotensin caused a concentration-dependent rise in mucous secretion with an ED50 of 36 nM, not significantly affected upon removal of extracellular Ca2+. Together our results suggest that while the mucin secretory response to carbachol depends on both the release of Ca2+ from intracellular stores and a Ca2+ influx from external medium, the secretory response to neurotensin is based solely on intracellular Ca2+ mobilization. Finally, evaluation of the cross-talk between the cyclic AMP pathway stimulated by vasoactive intestinal peptide (VIP) and the Ca2+ pathway stimulated by neurotensin or carbachol led to the conclusion that the potentiated secretory response elicited by the combined action of carbachol and VIP requires extracellular Ca2+.