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)..

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 in ammatory 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 de cient 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 signi cantly 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 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..

Oxidative stress balance between pro- and anti-inflammatory factors in human intestinal epithelial cells

Background: Oxidative stress plays a key role in the development of intestinal inflammatory diseases. Several pro-inflammatory mediators may generate oxidation products that exacerbate the inflammatory damage. Gastrointestinal molecules, like serotonin (5- HT), adenosine and melatonin, which are involved in intestinal physiology, have also been described as intestinal pro-inflammatory factors; whereas IL-10, a known anti-inflammatory cytokine, has also been implicated in intestinal pathophysiology. Aim: The aim of this study was to analyze the contribution of pro-inflammatory and anti-inflammatory molecules to oxidative stress balance, as well as to assess their effect on cellular antioxidant enzymes activity, in intestinal epithelial cells. Methods: Caco-2 cells were treated with the different molecules, and the oxidative stress was determined by measuring lipid peroxidation (MDA+4HDA) and protein carbonyl levels. The activity of the anti-oxidant enzymes (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) was also analyzed in treated cells. Results: Pro-inflammatory factors (5-HT, adenosine, melatonin and TNFa) increased oxidative damage in both lipids and proteins. Theses molecules, except melatonin, also inhibited the activity of antioxidant enzymes. With regard to IL-10, this cytokine was not shown to alter cellular oxidative damage, but was able to reduce the oxidative damage g by pro-inflamatory factors, and to restore their effects on anti-oxidant enxymes activities. Unexpectedly, IL-10, together with melatonin, was found to increase the antioxidant activity above the control. Conclusions: The anti-oxidant effect of IL-10 emphasizes the role of this cytokine as a potential therapy for the treatment of intestinal inflammation induced by pro-inflammatory molecules

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

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

Expression of microbiota recognition receptors and intestinal serotoninergic system in two mouse models of colitis

Background: The intestinal microbiota patterns recognition receptors TLRs and NODs (PRRs), and the intestinal serotoninergic system, may contribute to intestinal responses to microbiota and alter intestinal homeostasis/inflammation e.g. in inflammatory bowel disease. Aim: We examined two mouse colitis models (Dextran Sulfate Sodium (DSS) or Lymphocyte Transfer (LT) mouse colitis) and compared the expression of PRRs, implicated in innate immunity, and some elements of the intestinal serotoninergic system. Methods: In ileum and colon from DSS or LT mouse colitis animals, TLRs, NODs, serotonin transporter (SERT) and serotonin receptors (5-HTRs) mRNA expression was measured by RT-qPCR. SERT protein expression was analyzed by western blotting. Results: In DSS ileum, TLR9, 5HTR1A, 5-HTR4 and 5-HTR7 mRNA levels were over-expressed, and SERT expression reduced; in DSS colon, NOD2, TLR2, TLR9, and 5-HTR7 mRNAs were increased; however, 5HTR1A, 5-HTR2B, 5-HTR3, and 5HTR4 mRNA levels were diminished, as well as SERT expression. On the other hand, in LT ileum, TLR9, 5-HTR1A, 5- HT2A, and 5-HT2B mRNAs were reduced and, although SERT mRNA was not altered, SERT protein level was reduced; in colon of LT mouse model, TLR2, TLR9, 5-HTR1A, and 5-HTR7 mRNA levels and SERT expression were increased; however, TLR4, NOD1, 5- HTR2B, 5-HT3, 5-HT4 mRNA levels were reduced

Detailed stratified GWAS analysis for severe COVID-19 in four European populations

Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of a well-characterized cohort of 3255 COVID-19 patients with respiratory failure and 12 488 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen (HLA) region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a ~ 0.9-Mb inversion polymorphism that creates two highly differentiated haplotypes and characterized the potential effects of the inversion in detail. Our data, together with the 5th release of summary statistics from the COVID-19 Host Genetics Initiative including non-Caucasian individuals, also identified a new locus at 19q13.33, including NAPSA, a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung