BACKGROUND AND PURPOSEGut microbiota is essential for the development of the gastrointestinal system, including the enteric nervous system (ENS). Perturbations of gut microbiota in early life have the potential to alter neurodevelopment leading to functional bowel disorders later in life. We examined the hypothesis that gut dysbiosis impairs the structural and functional integrity of the ENS, leading to gut dysmotility in juvenile mice. EXPERIMENTAL APPROACHTo induce gut dysbiosis, broad-spectrum antibiotics were administered by gavage to juvenile (3weeks old) male C57Bl/6 mice for 14 days. Bile acid composition in the intestinal lumen was analysed by liquid chromatography-mass spectrometry. Changes in intestinal motility were evaluated by stool frequency, transit of a fluorescent-labelled marker and isometric muscle responses of ileal full-thickness preparations to receptor and non-receptor-mediated stimuli. Alterations in ENS integrity were assessed by immunohistochemistry and Western blot analysis. KEY RESULTSAntibiotic treatment altered gastrointestinal transit, luminal bile acid metabolism and bowel architecture. Gut dysbiosis resulted in distorted glial network, loss of myenteric plexus neurons, altered cholinergic, tachykininergic and nitrergic neurotransmission associated with reduced number of nNOS neurons and different ileal distribution of the toll-like receptor TLR2. Functional defects were partly reversed by activation of TLR2 signalling. CONCLUSIONS AND IMPLICATIONSGut dysbiosis caused complex morpho-functional neuromuscular rearrangements, characterized by structural defects of the ENS and increased tachykininergic neurotransmission. Altogether, our findings support the beneficial role of enteric microbiota for ENS homeostasis instrumental in ensuring proper gut neuromuscular function during critical stages of development. Abbreviations

Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity.

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 ± 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′-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.

The present study was designed to examine the role of enteric glial cells (EGCs) in colonic neuromuscular dysfunctions in a mouse model of high‐fat diet (HFD)‐induced obesity. C57BL/6J mice were fed with HFD or standard diet (SD) for 1, 2, or 8 weeks. Colonic interleukin (IL)‐1β, IL‐6, and malondialdehyde (MDA) levels were measured. Expression of occludin in colonic tissues was examined by western blot. Substance P (SP), S100β, GFAP, and phosphorylated mitogen‐activated protein kinase 1 (pERK) were assessed in whole mount specimens of colonic plexus by immunohistochemistry. Colonic tachykininergic contractions, elicited by electrical stimulation or exogenous SP, were recorded in the presence or absence of fluorocitrate (FC). To mimic exposure to HFD, cultured EGCs were incubated with palmitate (PA) and/or lipopolysaccharide (LPS). SP and IL‐1β levels were assayed in the culture medium by ELISA. HFD mice displayed an increase in colonic IL‐1β and MDA, and a reduction of occludin at week 2. These changes occurred to a greater extent at week 8. In vitro electrically evoked tachykininergic contractions were enhanced in HFD mice after 2 or 8 weeks, and they were blunted by FC. Colonic IL‐6 levels as well as substance P and S100β density in myenteric ganglia of HFD mice were increased at week 8, but not at week 1 or 2. In cultured EGCs, co‐incubation with palmitate plus LPS led to a significant increase in both SP and IL‐1β release. HFD‐induced obesity is characterized by a hyperactivation of EGCs and is involved in the development of enteric motor disorders through an increase in tachykininergic activity and release of pro‐inflammatory mediators.

Enteric glial cells (EGCs) influence nitric oxide (NO)− and adenosine diphosphate (ADP)− mediated signaling in the enteric nervous system (ENS). Since Toll-like receptor 4 (TLR4) participates to EGC homoeostasis, this study aimed to evaluate the possible involvement of EGCs in the alterations of the inhibitory neurotransmission in TLR4−/− mice. Ileal segments from male TLR4−/− and wild-type (WT) C57BL/6J mice were incubated with the gliotoxin fluoroacetate (FA). Alterations in ENS morphology and neurochemical coding were investigated by immunohistochemistry whereas neuromuscular responses were determined by recording non-adrenergic non-cholinergic (NANC) relaxations in isometrically suspended isolated ileal preparations. TLR4−/− ileal segments showed increased iNOS immunoreactivity associated with enhanced NANC relaxation, mediated by iNOS-derived NO and sensitive to P2Y1 inhibition. Treatment with FA diminished iNOS immunoreactivity and partially abolished NO− and ADP− mediated relaxation in the TLR4−/− mouse ileum, with no changes of P2Y1 and connexin-43 immunofluorescence distribution in the ENS. After FA treatment, S100β and GFAP immunoreactivity in TLR4−/− myenteric plexus was reduced to levels comparable to those observed in WT. Our findings show the involvement of EGCs in the alterations of ENS architecture and in the increased purinergic and nitrergic-mediated relaxation, determining gut dysmotility in TLR4−/− mice.

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.

The role played by adenosine A2B receptors (A2BRs) in the regulation of enteric glial cell (EGC) functions remains unclear. This study was aimed at investigating the involvement of A2BRs in the control of EGC functions in a model of obesity. C57BL/6 mice were fed with standard diet (SD) or high fat diet (HFD) for eight weeks. Colonic tachykininergic contractions were recorded in the presence of BAY60-6583 (A2BRs agonist), MRS1754 (A2BRs antagonist), and the gliotoxin fluorocitrate. Immunofluorescence distribution of HuC/D, S100β, and A2BRs was assessed in whole mount preparations of colonic myenteric plexus. To mimic HFD, EGCs were incubated in vitro with palmitate (PA) and lipopolysaccharide (LPS), in the absence or in the presence of A2BR ligands. Toll-like receptor 4 (TLR4) expression was assessed by Western blot analysis. Interleukin-1β (IL-1β), substance P (SP), and glial cell derived neurotrophic factor (GDNF) release were determined by enzyme-linked immunosorbent assay (ELISA) assays. MRS1754 enhanced electrically evoked tachykininergic contractions of colonic preparations from HFD mice. BAY60-6583 decreased the evoked tachykininergic contractions, with higher efficacy in HFD mice. Such effects were blunted upon incubation with fluorocitrate. In in vitro experiments on EGCs, PA and LPS increased TLR4 expression as well as IL-1β, GDNF, and SP release. Incubation with BAY60-6583 reduced TLR4 expression as well as IL-1β, GDNF, and SP release. Such effects were blunted by MRS1754. The present results suggest that A2BRs, expressed on EGCs, participate in the modulation of enteric inflammation and altered tachykininergic responses associated with obesity, thus representing a potential therapeutic target.

Background Oxidized phospholipid derivatives (OxPAPCs) act as bacterial lipopolysaccharide (LPS)–like damage‐associated molecular patterns. OxPAPCs dose‐dependently exert pro‐ or anti‐inflammatory effects by interacting with several cellular receptors, mainly Toll‐like receptors 2 and 4. It is currently unknown whether OxPAPCs may affect enteric nervous system (ENS) functional and structural integrity. Methods Juvenile (3 weeks old) male C57Bl/6 mice were treated intraperitoneally with OxPAPCs, twice daily for 3 days. Changes in small intestinal contractility were evaluated by isometric neuromuscular responses to receptor and non–receptor‐mediated stimuli. Alterations in ENS integrity and serotonergic pathways were assessed by real‐time PCR and confocal immunofluorescence microscopy in longitudinal muscle‐myenteric plexus whole‐mount preparations (LMMPs). Tissue levels of serotonin (5‐HT), tryptophan, and kynurenine were measured by HPLC coupled to UV/fluorescent detection. Key Results OxPAPC treatment induced enteric gliosis, loss of myenteric plexus neurons, and excitatory hypercontractility, and reduced nitrergic neurotransmission with no changes in nNOS+ neurons. Interestingly, these changes were associated with a higher functional response to 5‐HT, altered immunoreactivity of 5‐HT receptors and serotonin transporter (SERT) together with a marked decrease in 5‐HT levels, shifting tryptophan metabolism toward kynurenine production. Conclusions and Inferences OxPAPC treatment disrupted structural and functional integrity of the ENS, affecting serotoninergic tone and 5‐HT tissue levels toward a higher kynurenine content during adolescence, suggesting that changes in intestinal lipid metabolism toward oxidation can affect serotoninergic pathways, potentially increasing the risk of developing functional gastrointestinal disorders during critical stages of development.