Elena Layunta

Neomycin and bacitracin reduce the intestinal permeability in mice and increase the expression of some tight-junction proteins

BACKGROUND Tight-junction (TJ) proteins regulate paracellular permeability. Gut permeability can be modulated by commensal microbiota. Manipulation of the gut microbiota with antibiotics like bacitracin and neomycin turned out to be useful for the treatment of diarrhoea induced by Clostridium difficile or chemotherapy drugs. AIM To evaluate the effects of the microbiota depletion evoked by the oral administration of neomycin and bacitracin on the intestinal permeability and expression of TJ proteins in mice. METHODS Mice received neomycin and bacitracin orally for 7 days. Intestinal permeability was measured by the fluorescein-isothiocyanate-dextran (FITC-dextran) method. The gene expression of TJ proteins in the intestine was determined by real time-PCR. RESULTS FITC-dextran levels in serum were reduced by half in antibiotic-treated mice, indicating a reduction of intestinal permeability. Antibiotics increased the expression of zonula occludens 1 (ZO-1), junctional adhesion molecule A (JAM-A, and occludin in the ileum and ZO-1, claudin-3, and claudin-4 in the colon. CONCLUSION The combination of neomycin and bacitracin reduce intestinal permeability and increase the gene expression of ZO-1, junctional adhesion molecule A (JAM-A), and occludin in the ileum and ZO-1, claudin-3, and claudin-4 in the colon.

Intestinal Serotonin Transporter Inhibition by Toll-Like Receptor 2 Activation. A Feedback Modulation.

TLR2 is a microbiota recognition receptor that has been described to contribute to intestinal homeostasis and to ameliorate inflammatory intestinal injury. In this context, serotonin (5-HT) has shown to be an essential intestinal physiological neuromodulator that is also involved in intestinal inflammatory diseases. Since the interaction between TLR2 activation and the intestinal serotoninergic system remains non-investigated, our main aim was to analyze the effect of TLR2 on intestinal serotonin transporter (SERT) activity and expression and the intracellular pathways involved. Caco-2/TC7 cells were used to analyze SERT and TLR2 molecular expression and SERT activity by measuring 5-HT uptake. The results showed that apical TLR2 activation inhibits SERT activity in Caco-2/TC7 cells mainly by reducing SERT protein level either in the plasma membrane, after short-term TLR2 activation or in both the plasma membrane and cell lysate, after long-term activation. cAMP/PKA pathway appears to mediate short-term inhibitory effect of TLR2 on SERT; however, p38 MAPK pathway has been shown to be involved in both short- and long-term TLR2 effect. Reciprocally, 5-HT long-term treatment yielded TLR2 down regulation in Caco-2/TC7 cells. Finally, results from in vivo showed an augmented intestinal SERT expression in mice Tlr2-/-, thus confirming our inhibitory effect of TLR2 on intestinal SERT in vitro. The present work infers that TLR2 may act in intestinal pathophysiology, not only by its inherent innate immune role, but also by regulating the intestinal serotoninergic system.

IL-10 counteracts proinflammatory mediator evoked oxidative stress in Caco-2 cells.

Oxidative stress is thought to play a key role in the development of intestinal damage in intestinal inflammatory diseases. Several molecules are involved in the intestinal inflammation, either as pro- or anti-inflammatory factors; however, their effects on intestinal oxidative stress seem to be controversial. This work analyzes the contribution of pro- and anti-inflammatory molecules to the balance of oxidative damage in intestinal epithelial cells, as well as their effects on cellular antioxidant enzyme activity. With this purpose, the lipid and protein oxidation, together with the activity of catalase, superoxide dismutase, and glutathione peroxidase, were determined in the Caco-2 cells treated with serotonin, adenosine, melatonin, and TNFα, as proinflammatory factors, and IL-10, as an anti-inflammatory cytokine. The results have shown that all the proinflammatory factors assayed increased oxidative damage. In addition, these factors also inhibited the activity of antioxidant enzymes in the cells, except melatonin. In contrast, IL-10 did not alter these parameters but was able to reduce the prooxidant effects yielded by serotonin, adenosine, melatonin, or TNFα, in part by restoring the antioxidant enzymes activities. In summary, proinflammatory factors may induce oxidative damage in intestinal epithelial cells, whereas IL-10 seems to be able to restore the altered redox equilibrium in Caco-2 cells.

Toll-like receptor 9 modifies intestinal serotonergic system.

legend N2D N1D 2LPEG N2D vs. 2LPEG N1D vs. 2LPEG EFFICACY Primary analysis set, n1⁄4 275 Primary analysis set, n1⁄4 275 Primary analysis set, n1⁄4 272 Primary endpoint: Patients with successful overall bowel cleansing efficacy (HCS) [n] 253 (92.0%) 245 (89.1%) 238 (87.5%) -4.00%* [0.055] -6.91%* [0.328] Supportive secondary endpoint: Patients with successful overall bowel cleansing efficacy (BBPS) [n] 249 (90.5%) 243 (88.4%) 232 (85.3%) n.a. n.a. Primary endpoint: Excellent plus Good cleansing rate in colon ascendens (primary analysis set) [n] 87 (31.6%) 93 (33.8%) 41 (15.1%) 8.11%* [50.001] 10.32%* [50.001] Key secondary endpoint: Adenoma detection rate, colon ascendens 11.6% 11.6% 8.1% -4.80%; 12.00%** [0.106] -4.80%; 12.00%** [0.106] Key secondary endpoint: Adenoma detection rate, overall colon 26.6% 27.6% 26.8% -8.47%; 8.02%** [0.569] -7.65%; 9.11%** [0.455] Key secondary endpoint: Polyp detection rate, colon ascendens 23.3% 18.6% 16.2% -1.41%; 15.47%** [0.024] -6.12%; 10.82%** [0.268] Key secondary endpoint: Polyp detection rate, overall colon 44.0% 45.1% 44.5% -8.85%; 8.00%** [0.579] –7.78%; 9.09%** [0.478] Compliance rates (min 75% of both doses taken) [n] 235 (85.5%) 233 (84.7%) 245 (90.1%) n.a. n.a. SAFETY Safety set, n1⁄4 262 Safety set, n1⁄4 269 Safety set, n1⁄4 263 All treatment-emergent adverse events [n] 77 89 53 n.a. n.a. Patients with any related treatment-emergent adverse event [n] 30 (11.5%) 40 (14.9%) 20 (7.6%) n.a. n.a. *1⁄4 97.5% 1-sided CI; **1⁄4 95% 2-sided CI; n.a.1⁄4 not applicable. United European Gastroenterology Journal 4(5S) A219

Toll-like receptors 2 and 4 modulate intestinal IL-10 differently in ileum and colon:

Background Inflammatory bowel diseases are consequence of an intestinal homeostasis breakdown in which innate immune dysregulation is implicated. Toll-like receptor (TLR)2 and TLR4 are immune recognition receptors expressed in the intestinal epithelium, the first physical-physiological barrier for microorganisms, to inform the host of the presence of Gram-positive and Gram-negative organisms. Interleukin (IL)-10 is an essential anti-inflammatory cytokine that contributes to maintenance of intestinal homeostasis. Aim Our main aim was to investigate intestinal IL-10 synthesis and release, and whether TLR2 and TLR4 are determinants of IL-10 expression in the intestinal tract. Methods We used Caco-2 cell line as an enterocyte-like cell model, and also ileum and colon from mice deficient in TLR2, TLR4 or TLR2/4 to test the involvement of TLR signaling. Results Intestinal epithelial cells are able to synthesize and release IL-10 and their expression is increased after TLR2 or TLR4 activation. IL-10 regulation seems to be tissue specific, with IL-10 expression in the ileum regulated by a compensation between TLR2 and TLR4 expression, whereas in the colon, TLR2 and TLR4 affect IL-10 expression independently. Conclusions Intestinal epithelial cells could release IL-10 in response to TLR activation, playing an intestinal tissue-dependent and critical intestinal immune role.

Listeria monocytogenes Inhibits Serotonin Transporter in Human Intestinal Caco-2 Cells

Listeria monocytogenes is a Gram-positive bacterium that can cause a serious infection. Intestinal microorganisms have been demonstrated to contribute to intestinal physiology not only through immunological responses but also by modulating the intestinal serotonergic system. Serotonin (5-HT) is a neuromodulator that is synthesized in the intestinal epithelium and regulates the whole intestinal physiology. The serotonin transporter (SERT), located in enterocytes, controls intestinal 5-HT availability and therefore serotonin’s effects. Infections caused by L. monocytogenes are well described as being due to the invasion of intestinal epithelial cells; however, the effect of L. monocytogenes on the intestinal epithelium remains unknown. The main aim of this work, therefore, was to study the effect of L. monocytogenes on SERT. Caco2/TC7 cell line was used as an enterocyte-like in vitro model, and SERT functional and molecular expression assays were performed. Our results demonstrate that living L. monocytogenes inhibits serotonin uptake by reducing SERT expression at the brush border membrane. However, neither inactivated L. monocytogenes nor soluble metabolites were able to affect SERT. The results also demonstrate that L. monocytogenes yields TLR2 and TLR10 transcriptional changes in intestinal epithelial cells and suggest that TLR10 is potentially involved in the inhibitory effect observed on SERT. Therefore, L. monocytogenes, through TLR10-mediated SERT inhibition, may induce increased intestinal serotonin availability and potentially contributing to intestinal physiological changes and the initiation of the inflammatory response.

NOD1 downregulates intestinal serotonin transporter and interacts with other pattern recognition receptors

Serotonin (5‐HT) is an essential gastrointestinal modulator whose effects regulate the intestinal physiology. 5‐HT effects depend on extracellular 5‐HT bioavailability, which is controlled by the serotonin transporter (SERT) expressed in both the apical and basolateral membranes of enterocytes. SERT is a critical target for regulating 5‐HT levels and consequently, modulating the intestinal physiology. The deregulation of innate immune receptors has been extensively studied in inflammatory bowel diseases (IBD), where an exacerbated defense response to commensal microbiota is observed. Interestingly, many innate immune receptors seem to affect the serotonergic system, demonstrating a new way in which microbiota could modulate the intestinal physiology. Therefore, our aim was to analyze the effects of NOD1 activation on SERT function, as well as NOD1s interaction with other immune receptors such as TLR2 and TLR4. Our results showed that NOD1 activation inhibits SERT activity and expression in Caco‐2/TC7 cells through the extracellular signal‐regulated kinase (ERK) signaling pathway. A negative feedback between 5‐HT and NOD1 expression was also described. The results showed that TLR2 and TLR4 activation seems to regulate NOD1 expression in Caco‐2/TC7 cells. To assess the extend of cross‐talk between NOD1 and TLRs, NOD1 expression was measured in the intestinal tract (ileum and colon) of wild type mice and mice with individual knockouts of TLR2, and TLR4 as well as double knockout TLR2/TLR4 mice. Hence, we demonstrate that NOD1 acts on the serotonergic system decreasing SERT activity and molecular expression. Additionally, NOD1 expression seems to be modulated by 5‐HT and other immune receptors as TLR2 and TLR4. This study could clarify the relation between both the intestinal serotonergic system and innate immune system, and their implications in intestinal inflammation.

NOD2 Modulates Serotonin Transporter and Interacts with TLR2 and TLR4 in Intestinal Epithelial Cells

Background/Aims: Serotonin (5-HT) is a chief modulator of intestinal activity. The effects of 5-HT depend on its extracellular availability, which is mainly controlled by serotonin transporter (SERT), expressed in enterocytes. On the other hand, innate immunity, mediated by Toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs), is known to control intestinal microbiota and maintain intestinal homeostasis. The dysregulation of the intestinal serotonergic system and innate immunity has been observed in inflammatory bowel diseases (IBD), the incidence of which has severely increased all over the world. The aim of the present study, therefore, was to analyze the effect of NOD2 on intestinal SERT activity and expression, as well as to study the crosstalk of NOD2 with TLR2 and TLR4. Methods: Intestinal epithelial cell line Caco-2/TC7 was used to analyze SERT activity and SERT, NOD2, TLR2 and TLR4 molecular expression by real-time PCR and western blotting. Moreover, intestinal tract (ileum and colon) from mice deficient in TLR2, TLR4 or TLR2/4 receptors was used to test the interdependence of NOD2 with these TLR receptors. Results: NOD2 activation inhibits SERT activity in Caco-2/TC7 cells, mainly due to the decrement of SERT molecular expression, with RIP2/RICK being the intracellular pathway involved in this effect. This inhibitory effect on SERT would yield an increment of extracellular 5-HT availability. In this sense, 5-HT strongly inhibits NOD2 expression. In addition, NOD2 showed greater interdependence with TLR2 than with TLR4. Indeed, NOD2 expression significantly increased in both cells treated with TLR2 agonists and the intestinal tract of Tlr2-/- mice. Conclusions: It may be inferred from our data that NOD2 could play a role in intestinal pathophysiology not only through its inherent innate immune role but also due to its interaction with other receptors as TLR2 and the modulation of the intestinal serotonergic system decreasing SERT activity and expression.

Toll-like receptor 2 modulates the inhibitory motor response induced by hydrogen sulphide in mouse colon

No: OP015 Table 1: lesions where cold forceps avulsion and snare tip soft coagulation (CFA and STSC) was used in the resection of PANL or NNL; p values represent comparison to LSL. Two stage procedures were excluded. SD – standard deviation, IQR – interquartile range, SC1 – surveillance colonoscopy 1, ICV – ileocaecal valve, PANL – previously attempted non lifting lesion, NNL – naı̈ve non lifting lesion. PANL n1⁄4 33 p NNL n1⁄4 50 p LSL n1⁄4 650