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).