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

Introduction: The recognition of intestinal microbiota is in part carried out by toll-like receptors (TLR), which are responsible for initiating the innate immune response. Alterations in the intestinal microbiota and its recognition may contribute to the development of intestinal inflammatory pathologies. Otherwise, hydrogen sulphide (H2S) is an endogenous gaseous signalling molecule and it potentially plays a relevant role in the intestinal motility. In mammals, two pyridoxalphosphate-dependent enzymes are responsible for H2S synthesis: cystathionine b-synthase (CBS) and cystathionine -lyase (CSE)..

Estudio comparativo de diversos tipos de esencias de espliego españolas

Resumen de la tesis Doctoral realizada por M. MARTIN MESONERO Y dirigida por el Prof. Dr. J. CABO TORRES.Estimamos que en el estudio de las esencias naturales las más modernas técnicas cromatográficas en capa fina y fase gaseosa constituyen un magnifico complemento de las técnicas analíticas clásicas en este campo. Ello se aprecia con meridiana claridad en el presente estudio sobre esencias de espliego españolas, pues las conclusiones obtenidas mediante las técnicas aludidas no sólo se han complementado sino que ofrecen en conjunto gran coherencia. En efecto, se obtienen resultados comunes que ofrecen precisamente los puntos de partida en que se cimentan otras conclusiones especificas aportadas aisladamente por alguna de las técnicas empleadas. Por ejemplo está el caso presentado por la muestra de espliego procedente de Zaragoza, en que el dato analítico clásico de determinación de ésteres acusaba una diferencia tímidamente señalada en algún otro índice físico o químico; la cromatografía en capa fina y la cromatografía gaseosa subrayaron la aparente anomalía -más la primera que la segunda- corroborándose después que en efecto, dicha muestra contenía cierta cantidad de acetato de linalilo. La C. C. F., es particularmente útil en el aspecto cualitativo de los componentes de la esencia. Nos atrevemos a decir que en este aspecto es más aconsejable su empleo que la C. G., ya que a su rapidez une lo sencillo de su instrumental y el ser mucho más económica que ésta; en este aspecto cualitativo queremos puntualizar que la imagen final -el cromatograma- posee en la C. C. F., una fuerza plástica, pues en vez de picos más o menos agudos se estudian manchas de tamaño variable y de tonalidades diferentes para cada reactivo de revelado e incluso frente a uno mismo. La cromatografía gaseosa además de ratificarse mutuamente con la de capa fina en lo cualitativo, se muestra de gran utilidad especialmente en las consideraciones de tipo cuantitativo de los componentes detectados. El presente estudio sobre esencias españolas de espliego, referidas a las procedentes de las regiones de mayor producción, nos ha permitido afirmar que los tres componentes principales con diferencia notable sobre los demás son: linalol, eucaliptol y alcanfor, y entre los restantes se han identificado también el terpineol, acetato de terpenilo, canfeno, pineno, y 8 -pineno; en la únIca muestra procedente de Zaragoza existe además en cantidad apreciable acetato de linalilo. Al tomar en consideración el interesante aspecto de la posible influencie ecológica, pal"e1ce que, desde el punto de 'Vista cualitati'vo dicha influencia es nula como lo demuestra la homogeneidad de las imágenes cromatográficas. Sin embargo, hay que admitir una excepción positiva cual es la esencia de Zaragoza, con su acetato de linalilo que según dijimos no existe en las demás. Naturalmente, es preciso corroborar tal punto estudiando esencias de otras "campañas". Sin embargo, en lo cuantitativo dicha influencia ecológica se hace algo más patente: así, los tres componentes mayoritarios se sitúan en orden decreciente linalol>eucaliptol> alcanfor, 'excepto en las muestras de Soria en el que el orden es en este sentido: eucaliptol >linalol > alcanfor; por otra parte es variable el orden de los que llamamos componentes minoritarios

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

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

This is the final version of the article. Available from Public Library of Science via the DOI in this record.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.This work was funded by grants from the Spanish Ministry of Science and Innovation and the European Regional Development Fund (ERDF/FEDER) (BFU2010-18971), Zaragoza University (UZ2014-BIO-03), European Social Found (ESF) and the Aragon Regional Government (B61) and the Foundation for the Study of Inflammatory Bowel Diseases in Aragón (ARAINF 012/2008). ARAID Foundation supported J.P. (SAF2014-54763-C2-1-R) and E. Layunta is a PhD student fellow from Aragon Regional Government (B022/13). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Toll-like receptor 9 modifies intestinal serotonergic system.

Introduction: Toll-like receptor 9 (TLR9) is expressed in intestinal epithelial cells, which recognize microbiota developing different responses 1. Several studies have shown that TLR9 seems to be involved in Inflammatory Bowel Diseases (IBD) due to an inappropriate defensive response against microorganisms 2. Moreover, intestinal serotonergic system is also altered in IBD, where extracellular serotonin (5–HT) levels are increased 3. 5-HT bioavailability is mainly regulated by the serotonin transporter (SERT), expressed in enterocytes 4. Aims & Methods: The aim of the present study was to analyse whether TLR9 activation affects SERT expression and activity, and expression of other elements from the serotonergic system (TPH1, TPH2 and 5-HT receptors). Human enterocyte-like Caco-2 cells, and ileum and colon from TLR9-/- mice and Dextran Sulphate Sodium (DSS) mouse colitis model were used as experimental models. mRNA expression was determined by RT-qPCR, and protein expression by western blot..

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

Toll-like receptor 9 activation affects intestinal serotonin transporter activity and expression in Caco-2 cells

Background: Toll-like Receptor 9 (TLR9) is expressed mainly in the endosomal membrane of intestinal cells and mediates intestinal host-microbiota interaction. Serotonin (5-HT) is an intestinal neuromodulator involved in the intestinal immunity and homeostasis. In addition, a high level of 5-HT has been described in intestinal inflammation. 5-HT intestinal availability is mainly regulated by the serotonin transporter (SERT) expressed in enterocytes. Aim: The interaction of TLR9 with serotoninergic system remains known. Therefore, the aim of the present study was to assess the effects of TLR9 activation on SERT activity and expression. Methods: Caco-2 cells and colon from wild type (WT) and TLR9 C57BL/10 mice were used in this study. SERT activity (5-HT uptake) in Caco-2 cells and SERT expression (RT-qPCR and western blotting) in both Caco-2 cells and colon from WT and TLR9 mice, were analyzed. TLR9 mRNA and protein levels were also measured in Caco-2 cells. Results: TLR9 activation in Caco-2 cells reduced SERT activity in a MyD88 independent-way. SERT mRNA and protein level in both cell lysate and brush border membrane, were also diminished. SERT protein expression in colon of TLR9 mice resulted augmented compared with WT mice. Interestingly, activation of TLR9 in Caco-2 cells diminished TLR9 mRNA and protein in the cell lysate; however, TLR9 protein in brush border resulted increased. Conclusions: The results of this work highlight the role of TLR9 as a mediator intestinal homeostasis and/or intestinal inflammation by regulating intestinal serotoninergic system

Protective effect of bovine lactoferrin against Cronobacter sakazakii in human intestinal Caco-2/TC7 cells

Milk is a source of bioactive proteins with defensive properties of great value for protecting the newborn. The activity of bovine milk lactoferrin (LF) was investigated as an antibacterial agent in the internalisation of the emergent pathogen Cronobacter sakazakii into Caco-2/TC7 cells, a model of human intestinal epithelium. The effect of LF on oxidative stress and expression of Toll-like receptors (TLR) was also investigated. LF exerted a clear antibacterial activity against C. sakazakii, as well as an inhibitory effect on C. sakazakii adhesion and internalisation into Caco-2/TC7 cells. Incubation with C. sakazakii induced an increase in oxidative stress on the lipid fraction of Caco-2/TC7 cells, which was reversed by LF. Additionally, LF altered the expression of TLR, with a clear decrease in the expression levels of TLR4 at 24 h of incubation. These results suggest that LF has very interesting properties as a potential ingredient for functional foods

Intrinsic and Selective Activity of Functionalized Carbon Nanotube/Nanocellulose Platforms Against Colon Cancer Cells

[Abstract] Given their large surface area and versatile chemical reactivity, single-walled carbon nanotubes (SWCNTs) are regarded as the basis of new pharmacological complexes. In this study, SWCNTs are chemically functionalized with fluorescein, folic acid, and capecitabine, a drug that is commonly used against colorectal cancer. These functionalized SWCNTs are dispersed in water by taking advantage of their synergistic interaction with type-II nanocrystalline cellulose (II-NCC), and the resulting colloidal system is tested in vitro on both normal (differentiated) and cancerous (proliferative) human colon cells (Caco-2). The functionalized SWCNT/II-NCC hybrids show a higher activity than the reference (capecitabine) against the Caco-2 cancer cell line. However, this effect appears to be intrinsically associated with the SWCNT/II-NCC complex, particularly boosted by fluorescein, as the presence of capecitabine is not required. In addition, confocal microscopy fluorescence imaging using cell cultures highlights the enormous potential of this nanohybrid platform for colon cancer theranostics.This research was funded by the regional government of Aragón, DGA (Grupos Reconocidos DGA-T03_17R, DGA-T03_20R and DGA-A20_20R), together with associated EU Regional Development Funds, and also the Spanish MINEICO through a “Juan de la Cierva Incorporación” contract, and their associated research funds (ref. IJCI-2016–27789). A.C. thanks the Xunta de Galicia for an “Atracción de Talento” research grant (no. ED431H 2020/17)Gobierno de Aragón; DGA-T03_17RGobierno de Aragón; DGA-T03_20RGobierno de Aragón; DGA-A20_20RXunta de Galicia; ED431H 2020/1