Enteropathogenic Escherichia coli (EPEC) inactivate innate immune responses prior to compromising epithelial barrier function

Enteropathogenic Escherichia coli (EPEC) infection of the human small intestine induces severe watery diarrhoea linked to a rather weak inflammatory response despite EPEC's in vivo capacity to disrupt epithelial barrier function. Here, we demonstrate that EPEC flagellin triggers the secretion of the pro-inflammatory cytokine, interleukin (IL)-8, from small (Caco-2) and large (T84) intestinal epithelia model systems. Interestingly, IL-8 secretion required basolateral infection of T84 cells implying that flagellin must penetrate the epithelial barrier. In contrast, apical infection of Caco-2 cells induced IL-8 secretion but less potently than basolateral infections. Importantly, infection of Caco-2, but not T84 cells rapidly inhibited IL-8 secretion by a mechanism dependent on the delivery of effectors through a translocation system encoded on the locus of enterocyte effacement (LEE). Moreover, EPEC prevents the phosphorylation-associated activation of multiple kinase pathways regulating IL-8 gene transcription by a mechanism apparently independent of LEE-encoded effectors and four non-LEE-encoded effectors. Crucially, our studies reveal that EPEC inhibits the capacity of the cells to secrete IL-8 in response to bacterial antigens and inflammatory cytokines prior to disrupting barrier function by a distinct mechanism. Thus, these findings also lend themselves to a plausible mechanism to explain the absence of a strong inflammatory response in EPEC-infected humans

Anti-Diarrheal Mechanism of the Traditional Remedy Uzara via Reduction of Active Chloride Secretion

BACKGROUND AND PURPOSE: The root extract of the African Uzara plant is used in traditional medicine as anti-diarrheal drug. It is known to act via inhibition of intestinal motility, but malabsorptive or antisecretory mechanisms are unknown yet. EXPERIMENTAL APPROACH: HT-29/B6 cells and human colonic biopsies were studied in Ussing experiments in vitro. Uzara was tested on basal as well as on forskolin- or cholera toxin-induced Cl(-) secretion by measuring short-circuit current (I(SC)) and tracer fluxes of (22)Na(+) and (36)Cl(-). Para- and transcellular resistances were determined by two-path impedance spectroscopy. Enzymatic activity of the Na(+)/K(+)-ATPase and intracellular cAMP levels (ELISA) were measured. KEY RESULTS: In HT-29/B6 cells, Uzara inhibited forskolin- as well as cholera toxin-induced I(SC) within 60 minutes indicating reduced active chloride secretion. Similar results were obtained in human colonic biopsies pre-stimulated with forskolin. In HT-29/B6, the effect of Uzara on the forskolin-induced I(SC) was time- and dose-dependent. Analyses of the cellular mechanisms of this Uzara effect revealed inhibition of the Na(+)/K(+)-ATPase, a decrease in forskolin-induced cAMP production and a decrease in paracellular resistance. Tracer flux experiments indicate that the dominant effect is the inhibition of the Na(+)/K(+)-ATPase. CONCLUSION AND IMPLICATIONS: Uzara exerts anti-diarrheal effects via inhibition of active chloride secretion. This inhibition is mainly due to an inhibition of the Na(+)/K(+)-ATPase and to a lesser extent to a decrease in intracellular cAMP responses and paracellular resistance. The results imply that Uzara is suitable for treating acute secretory diarrhea

Saccharomyces boulardii Improves Intestinal Cell Restitution through Activation of the α2β1 Integrin Collagen Receptor

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the cessation of inflammation and the migration of enterocytes to repair the damaged epithelium. Lyophilized Saccharomyces boulardii (Sb, Biocodex) is a nonpathogenic yeast widely used as a therapeutic agent for the treatment and prevention of diarrhea and other gastrointestinal disorders. In this study, we determined whether Sb could accelerate enterocyte migration. Cell migration was determined in Sb force-fed C57BL6J mice and in an in vitro wound model. The impact on α2β1 integrin activity was assessed using adhesion assays and the analysis of α2β1 mediated signaling pathways both in vitro and in vivo. We demonstrated that Sb secretes compounds that enhance the migration of enterocytes independently of cell proliferation. This enhanced migration was associated with the ability of Sb to favor cell-extracellular matrix interaction. Indeed, the yeast activates α2β1 integrin collagen receptors. This leads to an increase in tyrosine phosphorylation of cytoplasmic molecules, including focal adhesion kinase and paxillin, involved in the integrin signaling pathway. These changes are associated with the reorganization of focal adhesion structures. In conclusion Sb secretes motogenic factors that enhance cell restitution through the dynamic regulation of α2β1 integrin activity. This could be of major importance in the development of novel therapies targeting diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases

Prostaglandin- and theophylline-induced Cl secretion in rat distal colon is inhibited by microtubule inhibitors

The aim of the present study was to examine the possible role of microtubules in chloride secretion by distal rat colon stimulated by prostaglandin (PGE 2 ) and theophylline. Distal colonic tissue from male rats was mounted in Ussing chambers, and short-circuit current (I sc ) was measured to assess chloride secretion. Three microtubule inhibitors, colchicine, nocodazole, and taxol, all inhibited the stimulated I sc and reduced the 60-min integrated secretory response to PGE 2 and theophylline (▪I sc dt) by 39–52%, whereas the inactive colchicine analog lumicolchicine did not. Atropine and tetrodotoxin had no effect on stimulated chloride secretion. To confirm the source of I sc , unidirectional 22 Na + and 36 Cl − fluxes were measured in tissues exposed to lumicolchicine (control) or colchicine. Control tissues absorbed both chloride [5.0 (1.1–8.6) (median and 95% confidence interval) μeq/cm 2 /hr] and sodium [2.8 (0.9–7.2) μeq/cm 2 /hr], and this net absorption was reduced by 96% and 79%, respectively, by treatment with PGE 2 and theophylline due to an increase in serosal-to-mucosal chloride and sodium movement. Colchicine-treated tissues exhibited similar net basal chloride and sodium absorption that was reduced by 71% and 75%, respectively, by treatment with PGE 2 and theophylline. Thus the PGE 2 - and theophylline-induced increase in chloride secretion was significantly reduced by colchicine ( P <0.05 by Wilcoxon rank-sum test), whereas colchicine had no effect on PGE 2 - and theophylline-induced changes in sodium fluxes. Furthermore, the colchinine-related changes in stimulated chloride secretion were numerically similar to colchicine-related changes in stimulated I sc . These findings indicate that microtubules are required for normal PGE 2 - and theophylline-induced chloride secretion in distal rat colon and suggest that induced chloride secretion may involve vesicular insertion of ion transporters into the plasma membrane or other microtubule-dependent regulatory processes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44414/1/10620_2005_Article_BF01299864.pd

Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line.

Serosal application of carbachol to T84 cell monolayers mounted in an Ussing chamber caused an immediate increase in short circuit current (Isc) that peaked within 5 min and declined rapidly thereafter, although a small increase in Isc persisted for approximately 30 min. The increase in Isc was detectable with 1 microM carbachol; half-maximal with 10 microM carbachol; and maximal with 100 microM carbachol. Unidirectional Na+ and Cl- flux measurements indicated that the increase in Isc was due to net Cl- secretion. Carbachol did not alter cellular cAMP, but caused a transient increase in free cytosolic Ca2+ ([Ca2+]i) from 117 +/- 7 nM to 160 +/- 15 nM. The carbachol-induced increase in Isc was potentiated by either prostaglandin E1 (PGE1) or vasoactive intestinal polypeptide (VIP), agents that act by increasing cAMP. Measurements of cAMP and [Ca2+]i indicated that the potentiated response was not due to changes in these second messengers. Studies of the effects of these agents on ion transport pathways indicated that carbachol, PGE1, or VIP each increased basolateral K+ efflux by activating two different K+ transport pathways on the basolateral membrane. The pathway activated by carbachol was not sensitive to barium, while that activated by PGE1 or VIP was; furthermore, their action on K+ efflux are additive. Our study indicates that carbachol causes Cl- secretion, and that this action may result from its ability to increase [Ca2+]i and basolateral K+ efflux. Carbachol's effect on Cl- secretion is greatly augmented in the presence of VIP or PGE1, which open a cAMP-sensitive Cl- channel on the apical membrane, accounting for a potentiated response