Intestinal serotonergic system is modulated by Toll-like receptor 9

Intestinal serotonergic system is a key modulator of intestinal homeostasis; however, its regulation is still unclear. Toll-like receptor 9 (TLR9), an innate immune receptor, detects different external agents in the intestine, preserving intestinal integrity. Since little is known about TLR9 role in the intestine, our aim was to address the potential regulation between TLR9 and intestinal serotonergic system. Caco-2/TC7 cell line and intestinal tract of Tlr9−/− mice were used in this study. Serotonin uptake studies were performed, and molecular expression of different serotonergic components was analyzed by western blot and real-time PCR. Our results show that TLR9 activation inhibits serotonin transporter activity and expression, involving p38/MAPK and ERK/MAPK intracellular pathways, and reciprocally, serotonin increases TLR9 expression. Supporting this interaction, serotonin transporter, serotonin receptors and serotonin producer enzymes were found altered in intestinal tract of Tlr9−/− mice. We conclude that TLR9 could contribute to intestinal homeostasis by modulation of intestinal serotonergic system. (AU)

Intestinal serotonergic system is modulated by Toll-like receptor 9.

Intestinal serotonergic system is a key modulator of intestinal homeostasis; however, its regulation is still unclear. Toll-like receptor 9 (TLR9), an innate immune receptor, detects different external agents in the intestine, preserving intestinal integrity. Since little is known about TLR9 role in the intestine, our aim was to address the potential regulation between TLR9 and intestinal serotonergic system. Caco-2/TC7 cell line and intestinal tract of Tlr9-/- mice were used in this study. Serotonin uptake studies were performed, and molecular expression of different serotonergic components was analyzed by western blot and real-time PCR. Our results show that TLR9 activation inhibits serotonin transporter activity and expression, involving p38/MAPK and ERK/MAPK intracellular pathways, and reciprocally, serotonin increases TLR9 expression. Supporting this interaction, serotonin transporter, serotonin receptors and serotonin producer enzymes were found altered in intestinal tract of Tlr9-/- mice. We conclude that TLR9 could contribute to intestinal homeostasis by modulation of intestinal serotonergic system.

Oxidized phospholipids affect small intestine neuromuscular transmission and serotonergic pathways in juvenile mice.

BACKGROUND: Oxidized phospholipid derivatives (OxPAPCs) act as bacterial lipopolysaccharide (LPS)-like damage-associated molecular patterns. OxPAPCs dose-dependently exert pro- or anti-inflammatory effects by interacting with several cellular receptors, mainly Toll-like receptors 2 and 4. It is currently unknown whether OxPAPCs may affect enteric nervous system (ENS) functional and structural integrity. METHODS: Juvenile (3 weeks old) male C57Bl/6 mice were treated intraperitoneally with OxPAPCs, twice daily for 3 days. Changes in small intestinal contractility were evaluated by isometric neuromuscular responses to receptor and non-receptor-mediated stimuli. Alterations in ENS integrity and serotonergic pathways were assessed by real-time PCR and confocal immunofluorescence microscopy in longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs). Tissue levels of serotonin (5-HT), tryptophan, and kynurenine were measured by HPLC coupled to UV/fluorescent detection. KEY RESULTS: OxPAPC treatment induced enteric gliosis, loss of myenteric plexus neurons, and excitatory hypercontractility, and reduced nitrergic neurotransmission with no changes in nNOS+ neurons. Interestingly, these changes were associated with a higher functional response to 5-HT, altered immunoreactivity of 5-HT receptors and serotonin transporter (SERT) together with a marked decrease in 5-HT levels, shifting tryptophan metabolism toward kynurenine production. CONCLUSIONS AND INFERENCES: OxPAPC treatment disrupted structural and functional integrity of the ENS, affecting serotoninergic tone and 5-HT tissue levels toward a higher kynurenine content during adolescence, suggesting that changes in intestinal lipid metabolism toward oxidation can affect serotoninergic pathways, potentially increasing the risk of developing functional gastrointestinal disorders during critical stages of development.

Anticancer Activity of Alkynylgold(I) with P(NMe2)3 Phosphane in Mouse Colon Tumors and Human Colon Carcinoma Caco-2 Cell Line.

New alkynylgold(I) with P(NMe2)3 (HMPT) phosphane complexes, [Au(C≡C-R)(HMPT)] (R= 4-Ph, 4-MePh, 4-OMe, 4-Br, 4-Cl, 2-py, and 3-py) have been synthesized and characterized, including X-ray studies of complexes with R= 4-OMe and 4-Br; additionally, their physicochemical properties and anticancer activity have been tested. Due to the great water solubility of the HMPT phosphane, all the complexes exhibit an optimal balance of hydrophilicity/lipophilicity. Also, all of these complexes are quite stable in physiological conditions and interact well enough with the transport protein BSA. All complexes exhibit a higher anticancer activity against Caco-2 cells than cisplatin, and some of them do not present cytotoxic activity against enterocyte-like differentiated cells. The selective complexes are proapoptotic drugs by the exposure of phosphatidylserine, results that are also confirmed in primary cultures from mouse colon tumors. Complexes with a halogen unit also arrest the cell cycle in G2/M phase. It is thought that maybe these apoptosis processes are promoted by the observed oxidative damage in the membrane lipids, as a consequence of the inhibition of the thioredoxin reductase enzyme. Based on our results, we conclude that five of our complexes are good candidates to be used in chemotherapy.

Unique Properties and Behavior of Nonmercerized Type-II Cellulose Nanocrystals as Carbon Nanotube Biocompatible Dispersants.

Nanocellulose is increasingly being investigated as a paradigm of a sustainable nanomaterial because of its extraordinary physical and chemical properties, together with its renewable nature and worldwide abundance. The rich structural diversity of cellulose materials is represented by different crystalline allomorphs, from which types I and II stand out. While type I is naturally and ubiquitously present, type II is man-made and requires harsh and caustic synthesis conditions such as the so-called mercerization process. Here, we provide an optimal scenario to obtain either type-I or II nanocrystalline cellulose (NCC) by a mercerization-free method consisting only of the acid hydrolysis commonly used to produce nanocellulose from microcellulose. The possibility of having nonmercerized type-II NCC acquires a great relevance since this nanostructure shows particularly appealing properties. Moreover, an entangled and wrapped system arises when used as a dispersing agent for single-walled carbon nanotubes (SWCNTs), significantly different from that of type I. The biological testing of each NCC type and their respective SWCNT-NCC dispersions in human intestinal (Caco-2) cells reveals a general innocuous behavior in both cancer and normal stages of differentiation; however, the type-II-based SWCNT-NCC dispersions display cytotoxicity for cancer cells while enhancing mitochondrial metabolism of normal cells.

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.

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 NOD1's 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.

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.

TLR2, TLR3, and TLR4 activation specifically alters the oxidative status of intestinal epithelial cells.

Intestinal inflammatory diseases are the result of multiple processes, including mucosal oxidative stress and perturbed homeostasis between commensal bacteria and mucosal immunity. Toll-like receptors (TLRs) recognize molecular-associated microorganisms' patterns and trigger innate immunity responses contributing to intestinal homeostasis and inflammatory responses. However, TLRs effects on redox balance in intestinal mucosa remain unknown. Therefore, the present study analyzes the effect of TLR2, TLR3, and TLR4 on both oxidative damage of lipids and proteins, and the activity of antioxidant enzymes in enterocyte-like Caco-2 cells. The results show that the activation of these TLRs increased lipid and protein oxidation levels; however, the effect on the antioxidant enzymes activity is different depending on the TLR activated. These results suggest that the activation of TLR2, TLR3, and TLR4 might affect intestinal inflammation by not only their inherent innate immunity responses, but also their pro-oxidative effects on intestinal epithelial cells.

IL-10 modulates serotonin transporter activity and molecular expression in intestinal epithelial cells.

Serotonin is a neuromodulator mainly synthesized by intestinal enterochromaffin cells that regulate overall intestinal physiology. The serotonin transporter (SERT) determines the final serotonin availability and has been described as altered in inflammatory bowel diseases. IL-10 is an anti-inflammatory cytokine that is involved in intestinal inflammatory processes and also contributes to intestinal mucosa homeostasis. The regulation of SERT by pro-inflammatory factors is well known; however, the effect of IL-10 on the intestinal serotoninergic system mediated by SERT remains unknown. Therefore, the aim of the present study is to determine whether IL-10 affects SERT activity and expression in enterocyte-like Caco-2 cells. Treatment with IL-10 was assessed and SERT activity was determined by 5-HT uptake. SERT mRNA and protein expression was analyzed using quantitative RT-PCR and western blotting. The results showed that IL-10 induced a dual effect on SERT after 6h of treatment. On one hand, IL-10, at a low concentration, inhibited SERT activity, and this effect might be explained by a non-competitive inhibition of SERT. On the other hand, IL-10, at a high concentration, increased SERT activity and molecular expression in the membrane of the cells. This effect was mediated by the IL-10 receptor and triggered by the PI3K intracellular pathway. Our results demonstrate that IL-10 modulates SERT activity and expression, depending on its extracellular conditions. This study may contribute to understand serotoninergic responses in intestinal pathophysiology.

Glucose transporters GLUT4 and GLUT8 are upregulated after facial nerve axotomy in adult mice.

Peripheral nerve axotomy in adult mice elicits a complex response that includes increased glucose uptake in regenerating nerve cells. This work analyses the expression of the neuronal glucose transporters GLUT3, GLUT4 and GLUT8 in the facial nucleus of adult mice during the first days after facial nerve axotomy. Our results show that whereas GLUT3 levels do not vary, GLUT4 and GLUT8 immunoreactivity increases in the cell body of the injured motoneurons after the lesion. A sharp increase in GLUT4 immunoreactivity was detected 3 days after the nerve injury and levels remained high on Day 8, but to a lesser extent. GLUT8 also increased the levels but later than GLUT4, as they only rose on Day 8 post-lesion. These results indicate that glucose transport is activated in regenerating motoneurons and that GLUT4 plays a main role in this function. These results also suggest that metabolic defects involving impairment of glucose transporters may be principal components of the neurotoxic mechanisms leading to motoneuron death.

Developmental regulation of glucose transporters GLUT3, GLUT4 and GLUT8 in the mouse cerebellar cortex.

Glucose uptake into the mammalian nervous system is mediated by the family of facilitative glucose transporter proteins (GLUT). In this work we investigate how the expression of the main neuronal glucose transporters (GLUT3, GLUT4 and GLUT8) is modified during cerebellar cortex maturation. Our results reveal that the levels of the three transporters increase during the postnatal development of the cerebellum. GLUT3 localizes in the growing molecular layer and in the internal granule cell layer. However, the external granule cell layer, Purkinje cell cytoplasm and cytoplasm of the other cerebellar cells lack GLUT3 expression. GLUT4 and GLUT8 have partially overlapping patterns, which are detected in the cytoplasm and dendrites of Purkinje cells, and also in the internal granule cell layer where GLUT8 displays a more diffuse pattern. The differential localization of the transporters suggests that they play different roles in the cerebellum, although GLUT4 and GLUT8 could also perform some compensatory or redundant functions. In addition, the increase in the levels and the area expressing the three transporters suggests that these roles become more important as development advances. Interestingly, the external granule cells, which have been shown to express the monocarboxylate transporter MCT2, express none of the three main neuronal GLUTs. However, when these cells migrate inwardly to differentiate in the internal granule cells, they begin to produce GLUT3, GLUT4 and GLUT8, suggesting that the maturation of the cerebellar granule cells involves a switch in their metabolism in such a way that they start using glucose as they mature.

Lipopolysaccharide induces alteration of serotonin transporter in human intestinal epithelial cells.

Intestinal serotoninergic activity and serotonin transporter (SERT) function have been shown to be altered in intestinal inflammatory diseases. Serotonin (5-HT) plays a critical role in the regulation of gastrointestinal physiology. Activity of 5-HT depends on its extracellular availability, partly modulated by SERT that transports 5-HT into the cell. Lipopolysaccharide (LPS) is a component of Gram-negative bacteria outer membrane, which acts as a potent activator of the inflammatory system in the intestine. The aim of this work was to determine, in the enterocyte-like cell line Caco-2, whether LPS treatment affects serotoninergic activity by acting on SERT. The results demonstrate that LPS treatment diminishes SERT activity in a dose- and period-dependent way. The kinetic study shows that V(max) was significantly reduced after treatment with LPS. The LPS effect on 5-HT uptake was, in part, mediated by protein kinase C (PKC) activation. The molecular expression of SERT revealed that LPS treatment did not affect the mRNA level or the SERT protein content in cell homogenate. The level of SERT protein, however, was reduced on brush border membrane. The LPS effect might be due to an alteration of the intracellular traffic of SERT which may, in part, be mediated by PKC activity.

Expression of GLUT8 in mouse intestine: identification of alternative spliced variants.

GLUT8 is a facilitative glucose transporter composed of 10 exons coding for a 477 amino acids protein. It is mainly expressed in the testis, but it has also been studied in a number of tissues such as brain, adipose tissue, and liver. In this work, we have characterized the expression of GLUT8 in the small and large intestine under normal physiological conditions. Protein assay revealed low GLUT8 protein levels in the intestine compared to the testis, with higher levels in the colon than in the small intestine. Immunohistochemistry studies showed an intracellular localization of GLUT8 in enterocytes and colonocytes with a supranuclear distribution next to the apical membrane. GLUT8 immunoreactivity was also detected in the crypt cells. Interestingly, we have identified three additional transcriptional variants in mouse intestine (mGLUT-SP1, mGLUT8-SP2, and mGLUT8-SP3) produced by the deletion of one, two, and four exons, respectively, whereas only the entire mRNA was detected in the testis. Expression of these alternative variants did not have an effect on glucose consumption in 3T3-L1 cells. Although the specific function of GLUT8 in intestine remains unclear, the alternative splicing of GLUT8 could reflect a mechanism for the regulation of the gene expression in a tissue-specific manner by targeting GLUT8 mRNA for nonsense-mediated decay.

Effect of long-term fluoxetine treatment on the human serotonin transporter in Caco-2 cells.

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) broadly used in the treatment of human mood disorders and gastrointestinal diseases involving the serotoninergic system. The effectiveness of this therapy depends on repeated long-term treatment. Most of the long-term studies in vivo of SSRI effects on serotoninergic activity have focused on their effects on autoreceptors or postsynaptic receptors. The chronic effect of SSRIs on the activity of the serotonin transporter (SERT) has been less studied and the results have been contradictory. The aim of this study was to determine the specific effect of long-term fluoxetine treatment on human serotonin transporter (hSERT) in vitro, by using the human enterocyte-like cell line Caco-2. Results show that fluoxetine diminished the 5-HT uptake in a concentration-dependent way and that this effect was reversible. Fluoxetine affected mainly the hSERT transport rate by reducing the availability of the transporter in the membrane with no significant alteration of either the total hSERT protein content or the hSERT mRNA level. These results suggest that the effect of fluoxetine on the expression of hSERT is post-translational and has shown itself to be independent of PKC and PKA activity. This study may be useful to clarify the effect of the long-term fluoxetine therapy in both gastrointestinal and central nervous system disorders.

Differential expression of glucose transporter GLUT8 during mouse spermatogenesis.

GLUT8 is a facilitative glucose transporter expressed at high levels in the testis. In this study, we analyzed the GLUT8 expression in mouse testis during spermatogenesis by RT-PCR, Western blot and immunohistochemistry methods. Our results show that GLUT8 expression is limited to spermatids and spermatozoa in the testis. Expression begins when round spermatids are formed at postnatal day 24. The expression persists throughout spermiogenesis, and it is also detected in spermatozoa, but it is absent in more immature germ cells, Sertoli cells and interstitial tissue. GLUT8 immunoreactivity is always restricted to the acrosomic system in a manner that matches the acrosome system formation. The GLUT8 expression is mainly associated with the acrosomic membrane in the acrosome, although significant immunoreactivity is also found inside the acrosomic lumen. The specific GLUT8 location suggests that this transporter plays a pivotal role in the fuel supply of spermatozoa, and in the traffic of sugars during the capacitation and fertilization processes.