Toll-Like Receptor 4 Modulates Small Intestine Neuromuscular Function through Nitrergic and Purinergic Pathways

Objective: Toll-like receptors (TLRs) play a pivotal role in the homeostatic microflora-host crosstalk. TLR4-mediated modulation of both motility and enteric neuronal survival has been reported mainly for colon with limited information on the role of TLR4 in tuning structural and functional integrity of enteric nervous system (ENS) and in controlling small bowel motility. Methods: Male TLR4 knockout (TLR4-/-, 9 \ub1 1 weeks old) and sex- and age-matched wild-type (WT) C57BL/6J mice were used for the experiments. Alterations in ENS morphology and neurochemical code were assessed by immunohistochemistry whereas neuromuscular function was evaluated by isometric mechanical activity of ileal preparations following receptor and non-receptor-mediated stimuli and by gastrointestinal transit. Results: The absence of TLR4 induced gliosis and reduced the total number of neurons, mainly nNOS+ neurons, in ileal myenteric plexus. Furthermore, a lower cholinergic excitatory response with an increased inhibitory neurotransmission was found together with a delayed gastrointestinal transit. These changes were dependent on increased ileal non-adrenergic non-cholinergic (NANC) relaxations mediated by a complex neuronal-glia signaling constituted by P2X7 and P2Y1 receptors, and NO produced by nNOS and iNOS. Conclusion: We provide novel evidence that TLR4 signaling is involved in the fine-tuning of P2 receptors controlling ileal contractility, ENS cell distribution, and inhibitory NANC neurotransmission via the combined action of NO and adenosine-5\u2032-triphosphate (ATP). For the first time, this study implicates TLR4 at regulating the crosstalk between glia and neurons in small intestine and helps to define its role in gastrointestinal motor abnormalities during dysbiosis

Influence of Tilia tomentosa Moench Extract on Mouse Small Intestine Neuromuscular Contractility

Functional gastrointestinal disorders (FGIDs) are characterized by abdominal pain, bloating and bowel disturbances. FGID therapy is primarily symptomatic, including treatment with herbal remedies. Flower extract of Tilia tomentosa Moench (TtM) is occasionally used as an anti-spasmodic in popular medicine. Since its effect on intestinal response is unknown, we evaluated the influence of TtM extract on small intestine contractility. Ileal preparations from C57BL/6J mice were mounted in organ baths to assess changes in muscle tension, following addition of TtM extract (0.5-36 mu g/mL) or a vehicle (ethanol). Changes in contractile response to receptor- and non-receptor-mediated stimuli were assessed in ileal preparations pretreated with 12 mu g/mL TtM. Alterations in the enteric nervous system neuroglial network were analyzed by confocal immunofluorescence. Increasing addition of TtM induced a marked relaxation in ileal specimens compared to the vehicle. Pretreatment with TtM affected cholinergic and tachykininergic neuromuscular contractions as well as K+-induced smooth muscle depolarization. Following incubation with TtM, a significant reduction in non-adrenergic non-cholinergic-mediated relaxation sensitive to N omega-Nitro-L-arginine methyl ester hydrochloride (pan-nitric oxide synthase inhibitor) was found. In vitro incubation of intestinal specimens with TtM did not affect the myenteric plexus neuroglial network. Our findings show that TtM-induced intestinal relaxation is mediated by nitric oxide pathways, providing a pharmacological basis for the use of TtM in FGIDs

Spray-drying Microencapsulation of an Extract from Tilia tomentosa Moench Flowers: Physicochemical Characterization and in Vitro Intestinal Activity

Silver linden (Tilia tomentosa Moench, TtM) flowers possess several health-promoting properties, especially at the neurological level, such as intestinal relaxation activity associated with specific flavonols, particularly quercetin and kaempferol derivatives. However, such molecules are susceptible to degradation upon different triggers like heat, light and extreme pH values. To overcome the scarce stability of TtM flowers bioactive molecules and make them suitable for developing functional food and supplements, we applied microencapsulation. Spray-drying microencapsulation of TtM flowers extract was performed using three starch-derived wall materials: maltodextrin 12 DE (MD12) and 19 DE (MD19), and OSA-modified starch (OSA-S). The stability of total phenols, flavanols, and antioxidant capacity was monitored for 70 days under accelerated stress conditions (40 °C/70% RH) by HPLC and spectrophotometric methods, and the intestinal contractile activity was tested in a murine model. In comparison to MD12 and MD19, OSA-S stood out for the higher encapsulation efficiency of quercetin and kaempferol glycosides (+ 36-47% compared to MD12 and + 18-24% compared to MD19) and stability thereof (half-life on average + 30% compared to MD12 and + 51% compared to MD19). The intestinal contractile activity of OAS-S powders resulted comparable to the original extract, indicating that flavonols were biologically active and accessible. Our results underly the potential advantages of OSA-S encapsulated formulation as a functional ingredient for the development of nutraceutical products

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Toll-like receptor 4, enteric nervous system and gut neuromuscular function in models of functional and inflammatory bowel disorders

The interaction between the constituents of the intestinal wall and commensal microflora is essential for the maintenance of mucosal barrier, the promotion of digestive system development and the modulation of gastrointestinal (GI) activities, such as motility, secretion, mucosal immunity and visceral sensitivity. Alterations in the gut microflora composition have been associated to several GI disorders (e.g. inflammatory bowel disease, IBD, and irritable bowel syndrome, IBS) while changes in intestinal microbiome during childhood and adolescence, caused by infections or antibiotics, predispose at the onset of these diseases. Furthermore, dysfunctions of the enteric nervous system (ENS) such as structural abnormalities and/or changes in the content of neurotransmitters have been associated with the onset of IBD and/or IBS. In this context, a sophisticated system of proteins, so-called Toll-like receptors (TLRs), plays a key role in mediating the inflammatory response against pathogens and activates beneficial signals to ensure tissue integrity in physiological and pathological conditions. Polymorphisms in the genes encoding TLRs, including TLR2 and TLR4, have been associated with different disease phenotypes in patients with GI disorders. Therefore, in this study, we evaluated the structural and functional alterations of murine ENS in the absence of the signal mediated by TLR4, a receptor of innate immunity, in mouse models of: i) ex vivo inhibition of enteric glial cells activities; ii) obesity induced by a high fat diet (HFD); iii) experimental colitis by in vivo administration of sodium dextran sulfate (DSS). Firstly, the role of the TLR4 receptor in maintaining intestinal function was investigated by performing in vitro contractility experiments, using ileal preparations from C57BL/6J WT mice exposed to a cocktail of antibiotics to induce microbiota depletion to be compared to mice deficient for TLR4 signaling. The effects provoked by the antibiotic-induced microbiota depletion are similar to those evidenced by the absence of TLR4 signaling on GI function, in particular, a significant reduction of the cholinergic excitatory contractile response accompanied by an altered ratio of neurons positive for the neuronal nitric oxide synthase (nNOS), associated to alterations in the distribution and expression of the glial S100β protein. These observations suggest that the lack of the intestinal microbiota, and, especially, the lack of TLR4 signaling may influence the integrity of the enteric neuronal and glial network. Given the importance of a correct commensal microbiota signal in maintaining the neuroglial network and the ENS neurochemical code, intestinal function was evaluated by in vitro contractility experiments using the isolated organ bath technique on ileal segments from WT and TLR4-/- mice. Thanks to functional and immunofluorescence studies with confocal microscopy, it was possible to demonstrate that the absence of the TLR4 signaling not only determines a significant decrease in the excitatory response but induces a marked increase in inhibitory neurotransmission mediated by both nitric oxide (produced both by nNOS than from the inducible NOS (iNOS)) and ATP, which mediated its actions through the P2Y1 purinergic receptor. In order to characterize the origin of the altered inhibitory tone in intestinal preparations of TLR4-/- mice, ex vivo experiments with the gliotoxin fluoroacetate were performed revealing the primary role of P2Y1 purinergic receptors, expressed in the enteric glia, in support of inhibitory transmission. Therefore, the evaluation of TLR4-/- mice highlighted the primary role of this receptor in modulating the interaction of both neuronal and glial signals of the ENS to ensure correct intestinal neuromuscular activity. In order to investigate the role of TLR4 in mild inflammatory conditions, the effects of obesity induced by high-fat diet (HFD; 60% kcal of lipids) on the functional and morphological integrity of the ENS were evaluated in WT and TLR4-/- mice. The HFD determines reduced cholinergic excitatory activity and increased inhibitory tone with consequent slower intestinal transit, associated with reactive gliosis. The absence of TLR4 protects mice from weight gain and the relative functional and structural neuromuscular anomalies induced by HFD, to highlight the primary role of this receptor in modulating the harmful effects of obesity in the GI tract, such as constipation. In TLR4-/- mice the serotonergic neurotransmission, mediated by 5-HT3 receptors, is increased and insensitive to ketanserin, an antagonist of 5-HT2A receptors, and was not subjected to changes following HFD to underline the influence of the signal mediated by the TLR4 in the modulation of the neuromuscular serotonergic response. These alterations were associated to significantly increased tissue levels of serotonin and its metabolites, tryptophan and kynurenine, induced by the absence of the TLR4 signaling and further enhanced by the HFD. Finally, we evaluated the importance of the TLR-ENS axis in maintaining ENS integrity in inflammatory conditions, obtained by inducing experimental colitis in WT and TLR4-/- mice. This model of experimental colitis, recognized for its simplicity, reproducibility, and versatility, offers the opportunity to mimic the inflammatory processes involved in the development of ulcerative colitis in humans and to study the involvement of immunity. DSS-induced colitis led to marked structural alterations in the neurochemical code of the ENS, which resulted in an increased excitatory neuromuscular response more marked in WT mice than in those deficient for TLR4. In TLR4-/- mice treated with DSS, the increased response to serotonin is markedly reduced and is mediated by 5-HT3 receptors but not by 5-HT2A receptors. These changes were associated to a marked reduction of serotonin and kynurenine tissue levels. These results further highlight the role of the TLR4 receptor in the modulation of neuroimmuno-mediated processes, involving both the serotonergic system and the enteric microbiota. In conclusion, our findings underline the key role of TLR4 signaling in ensuring gut homeostasis by finely tuning enteric glioplasticity, inhibitory neuromuscular response, mediated by iNOS-produced NO, and gut motility during pathological conditions, such as in case of low-grade systemic inflammation (e.g. obesity) or high-grade acute inflammation (i.e. IBD)

Dopamine transporter genetic reduction induces morpho-functional changes in the enteric nervous system

10sinoneAntidopaminergic gastrointestinal prokinetics are indeed commonly used to treat gastrointestinal motility disorders, although the precise role of dopaminergic transmission in the gut is still unclear. Since dopamine transporter (DAT) is involved in several brain disorders by mod-ulating extracellular dopamine in the central nervous system, this study evaluated the impact of DAT genetic reduction on the morpho-functional integrity of mouse small intestine enteric nervous system (ENS). In DAT heterozygous (DAT+/−) and wild-type (DAT+/+) mice (14 ± 2 weeks) alterations in small intestinal contractility were evaluated by isometrical assessment of neuromuscular responses to receptor and non-receptor-mediated stimuli. Changes in ENS integrity were studied by real-time PCR and confocal immunofluorescence microscopy in longitudinal muscle-myenteric plexus whole-mount preparations (). DAT genetic reduction resulted in a significant increase in dopamine-mediated effects, primarily via D1 receptor activation, as well as in reduced cholinergic response, sustained by tachykininergic and glutamatergic neurotransmission via NMDA receptors. These functional anomalies were associated to architectural changes in the neurochemical coding and S100β immunoreactivity in small intestine myenteric plexus. Our study provides evidence that genetic-driven DAT defective activity determines anomalies in ENS architecture and neurochemical coding together with ileal dysmotility, highlighting the involvement of dopaminergic system in gut disorders, often associated to neurological conditions.noneCerantola S.; Caputi V.; Contarini G.; Mereu M.; Bertazzo A.; Bosi A.; Banfi D.; Mantini D.; Giaroni C.; Giron M.C.Cerantola, S.; Caputi, V.; Contarini, G.; Mereu, M.; Bertazzo, A.; Bosi, A.; Banfi, D.; Mantini, D.; Giaroni, C.; Giron, M. C

Impact of Microbial Metabolites on Microbiota–Gut–Brain Axis in Inflammatory Bowel Disease

The complex bidirectional communication system existing between the gastrointestinal tract and the brain initially termed the “gut–brain axis” and renamed the “microbiota–gut–brain axis”, considering the pivotal role of gut microbiota in sustaining local and systemic homeostasis, has a fundamental role in the pathogenesis of Inflammatory Bowel Disease (IBD). The integration of signals deriving from the host neuronal, immune, and endocrine systems with signals deriving from the microbiota may influence the development of the local inflammatory injury and impacts also more distal brain regions, underlying the psychophysiological vulnerability of IBD patients. Mood disorders and increased response to stress are frequently associated with IBD and may affect the disease recurrence and severity, thus requiring an appropriate therapeutic approach in addition to conventional anti-inflammatory treatments. This review highlights the more recent evidence suggesting that alterations of the microbiota–gut–brain bidirectional communication axis may concur to IBD pathogenesis and sustain the development of both local and CNS symptoms. The participation of the main microbial-derived metabolites, also defined as “postbiotics”, such as bile acids, short-chain fatty acids, and tryptophan metabolites in the development of IBD-associated gut and brain dysfunction will be discussed. The last section covers a critical evaluation of the main clinical evidence pointing to the microbiome-based therapeutic approaches for the treatment of IBD-related gastrointestinal and neuropsychiatric symptoms