The interaction between cellular constituents of gastrointestinal (GI) tract and commensal microflora is essential for the maintenance of mucosal barrier, promotion of the development of the GI system and modulation of enteric functions such as motility, secretion, mucosal immunity and visceral sensitivity. Alterations in the composition of the gut microflora have been associated to several GI disorders (e.g. inflammatory bowel disease, IBD, and irritable bowel syndrome, IBS) while changes in intestinal microbiota during infancy and adolescence, caused by infection or antibiotic therapy, appear to predispose to the onset of these diseases. Furthermore, dysfunctions of the enteric nervous system (ENS) such structural abnormalities and/or changes in the content of neurotransmitters, have been associated with the onset of IBD and 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 triggers beneficial signals to ensure tissue integrity under physiological and pathological conditions. Polymorphisms in genes encoding TLRs, including TLR2 or TLR4, have been associated with different phenotypes of disease extent and severity in patients with GI disorders. In this study we characterized structural and functional alterations of murine ENS induced by: i) anomalies in the composition of the microbiota, ii) changes in innate immunity response, mediated by TLR2 and/or TLR4 following recognition of gut commensal microflora, iii) alterations in the expression of the protein catechol-O-methyltransferase (COMT), involved in the turnover of several neurotransmitters present in both the ENS and the central nervous system, such as dopamine and other catecholamines. Functional and structural studies in male mice C57BL/6J WT and TLR2-/- (21 ± 5 days old) highlighted the presence of significant alterations of intestinal contractility and ENS architecture in ileal preparations of genetically modified mice. Once demonstrated that the deletion of the gene encoding for TLR2 determines ENS structural and functional abnormalities, it was examined whether TLR2-mediated functional anomalies were hematopoietic cell-independent. Therefore, bone marrow chimeric mice were generated and experimental transfers were as follows: WT donors into WT recipients (WT?WT), WT donors into TLR2-/- recipients (WT? TLR2-/-), TLR2-/- donors into TLR2-/- recipients (TLR2-/-? TLR2-/-), and TLR2-/- donors into WT recipients (TLR2-/-? WT). Contractility experiments conducted in bone marrow chimeric mice evidenced that the structure and function of ENS were similar in WT mice given either WT or TLR2?/? bone marrow, indicating that TLR2 signaling in nonhematopoietic cells is a main contributor to ENS health. To investigate the role of the TLR2-microbiota axis in the homeostasis of ENS and enteric smooth muscle we depleted gut microbiota by intragastric administration of a cocktail of broad spectrum antibiotics (50 mg/kg vancomycin, 100 mg/kg neomycin, 100 mg/kg metronidazol and 100 mg/kg ampicillin) twice a day for 14 days in adolescent mice (aged 21 ± 5, ABX). Mice resulted to be successfully depleted after antibiotic treatment and displayed significantly smaller spleens and enlarged ceca, macroscopically phenocopying germ-free mice. This condition, already highlighted in IBS subjects, appears to be due to a delayed emptying of feces from enlarged cecum, due to impaired motility. Functional studies in ABX mice revealed a significant decrease in gastrointestinal transit, accompanied by alterations in the rate of fecal pellet expulsion and stool water content, to suggest that continuous presence of microbial stimuli is required to control intestinal motility and potentially mucosal barrier permeability. Immunohistochemical analysis of ileal preparations from ABX mice showed abnormalities in the distribution and expression of the the pan-neuronal marker HuC/D, the glial structural protein GFAP (glial fibrillary acidic protein) and the cytoplasmatic and nuclear glial calcium-binding protein S100?. Overall these observations highlight the primary role of commensal microbiota in the preservation of the structural integrity of the enteric neuronal and glial network. Given the importance of proper composition of commensal microbiota in the maintenance of neuronal network and neurochemical coding of the ENS intestinal contractility was evaluated in isolated ileal segments from control and ABX mice. These analyses evidenced impaired neuromuscular function associated to antibiotic treatment to further underline that proper neuromuscular function relies on a correct composition of gut microbiota.
The primary role of TLR2 signaling in controlling gut motor function was further confirmed by testing the effect of TLR2 engagement by Pam3-CSK4 (a TLR2/TLR1 agonist) in ABX mice. Intraperitoneal supplementation with Pam3CSK4, during the second week of antibiotic treatment, partially corrected these anomalies in ENS structure and intestinal contraction, supporting the presence of a dialogue between commensal microbiota and TLR2, essential for the modulation of neuromuscular function. To highlight the key role of gut microbiota?TLR2-ENS axis in maintaining intestinal function and development of the ENS, male C57Bl/6 mice (2 weeks old) were daily treated subcutaneously with OxPAPC (a TLR2 and TLR4 inhibitor) for 7 days. In vivo inhibition of both TLR2 and TLR4 determined a significant alteration of receptor and non-receptor-mediated neuromuscular responses, in a manner similar to that found in TLR2-deficient mice, providing evidence that TLR2 and TLR4 signaling is essential in ensuring the structural and functional integrity of the SNE during adolescence. Then, we investigate changes in gene expression of GluN1 subunit of N-Methyl-D-Aspartate (NMDA) receptor of the neurotransmitter glutamate in the myenteric plexus of ileal preparations from control and ABX mice. Antibiotic-mediated depletion of commensal microflora determined increased mRNA levels of GluN1, suggesting that commensal microbiota is involved in modulating visceral sensitivity.
Finally, the role of brain-gut axis in ENS homeostasis was assessed in an animal model of psychiatric disease, characterized by the genetic reduction of catechol-o-methyltransferase (COMT), an enzyme responsible for the degradation of catecholamines. In female animals genetic-driven COMT defective activity determined increased levels of NO associated to altered ENS architecture, neurochemical coding and visceral sensitivity. We cannot exclude that such changes may be involved in the pathogenesis of IBS in female patients, underlining a potential link with psychiatric disorders
