New Concepts of the Interplay Between the Gut Microbiota and the Enteric Nervous System in the Control of Motility.

Propulsive gastrointestinal (GI) motility is critical for digestive physiology and host defense. GI motility is finely regulated by the intramural reflex pathways of the enteric nervous system (ENS). The ENS is in turn regulated by luminal factors: diet and the gut microbiota. The gut microbiota is a vast ecosystem of commensal bacteria, fungi, viruses, and other microbes. The gut microbiota not only regulates the motor programs of the ENS but also is critical for the normal structure and function of the ENS. In this chapter, we highlight recent research that has shed light on the microbial mechanisms of interaction with the ENS involved in the control of motility. Toll-like receptor signaling mechanisms have been shown to maintain the structural integrity of the ENS and the neurochemical phenotypes of enteric neurons, in part through the production of trophic factors including glia-derived neurotrophic factor. Microbiota-derived short-chain fatty acids and/or single-stranded RNA regulates the synthesis of serotonin in enterochromaffin cells, which are involved in the initiation of enteric reflexes, among other functions. Further evidence suggests a crucial role for microbial modulation of serotonin in maintaining the integrity of the ENS through enteric neurogenesis. Understanding the microbial pathways of enteric neural control sheds new light on digestive health and provides novel treatment strategies for GI motility disorders.

Colitis-associated microbiota drives changes in behaviour in male mice in the absence of inflammation.

Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal tract. IBD are associated with a high prevalence of cognitive, behavioural and emotional comorbidities, including anxiety and depression. The link between IBD and the development of behavioural comorbidities is poorly understood. As the intestinal microbiota profoundly influences host behaviour, we sought to determine whether the altered gut microbiota associated with intestinal inflammation contributes to the development of behavioural abnormalities. Using the dextran sulphate sodium (DSS) model of colitis, we characterized intestinal inflammation, behaviour (elevated plus maze and tail suspension test) and the composition of the microbiota in male mice. Cecal contents from colitic mice were transferred into germ-free (GF) or antibiotic (Abx)-treated mice, and behaviour was characterized in recipient mice. Gene expression was measured using qPCR. DSS colitis was characterized by a significant reduction in body weight and an increase in colonic inflammatory markers. These changes were accompanied by increased anxiety-like behaviour, an altered gut microbiota composition, and increased central Tnf expression. Transfer of the cecal matter from colitic mice induced similar behavioural changes in both GF and Abx-treated recipient mice, with no signs of colonic or neuroinflammation. Upon characterization of the microbiota in donor and recipient mice, specific taxa were found to be associated with behavioural changes, notably members of the Lachnospiraceae family. Behavioural abnormalities associated with intestinal inflammation are transmissible via transfer of cecal matter, suggesting that alterations in the composition of the gut microbiota play a key role in driving behavioural changes in colitis.

Intestinal microbiota shapes gut physiology and regulates enteric neurons and glia.

BACKGROUND: The intestinal microbiota plays an important role in regulating gastrointestinal (GI) physiology in part through interactions with the enteric nervous system (ENS). Alterations in the gut microbiome frequently occur together with disturbances in enteric neural control in pathophysiological conditions. However, the mechanisms by which the microbiota regulates GI function and the structure of the ENS are incompletely understood. Using a mouse model of antibiotic (Abx)-induced bacterial depletion, we sought to determine the molecular mechanisms of microbial regulation of intestinal function and the integrity of the ENS. Spontaneous reconstitution of the Abx-depleted microbiota was used to assess the plasticity of structure and function of the GI tract and ENS. Microbiota-dependent molecular mechanisms of ENS neuronal survival and neurogenesis were also assessed. RESULTS: Adult male and female Abx-treated mice exhibited alterations in GI structure and function, including a longer small intestine, slower transit time, increased carbachol-stimulated ion secretion, and increased intestinal permeability. These alterations were accompanied by the loss of enteric neurons in the ileum and proximal colon in both submucosal and myenteric plexuses. A reduction in the number of enteric glia was only observed in the ileal myenteric plexus. Recovery of the microbiota restored intestinal function and stimulated enteric neurogenesis leading to increases in the number of enteric glia and neurons. Lipopolysaccharide (LPS) supplementation enhanced neuronal survival alongside bacterial depletion, but had no effect on neuronal recovery once the Abx-induced neuronal loss was established. In contrast, short-chain fatty acids (SCFA) were able to restore neuronal numbers after Abx-induced neuronal loss, demonstrating that SCFA stimulate enteric neurogenesis in vivo. CONCLUSIONS: Our results demonstrate a role for the gut microbiota in regulating the structure and function of the GI tract in a sex-independent manner. Moreover, the microbiota is essential for the maintenance of ENS integrity, by regulating enteric neuronal survival and promoting neurogenesis. Molecular determinants of the microbiota, LPS and SCFA, regulate enteric neuronal survival, while SCFA also stimulates neurogenesis. Our data reveal new insights into the role of the gut microbiota that could lead to therapeutic developments for the treatment of enteric neuropathies. Video abstract.

Behavioural adaptations after antibiotic treatment in male mice are reversed by activation of the aryl hydrocarbon receptor.

The intestinal microbiota plays an important role in regulating brain functions and behaviour. Microbiota-dependent changes in host physiology have been suggested to be key contributors to psychiatric conditions. However, specific host pathways modulated by the microbiota involved in behavioural control are lacking. Here, we assessed the role of the aryl hydrocarbon receptor (Ahr) in modulating microbiota-related alterations in behaviour in male and female mice after antibiotic (Abx) treatment. Mice of both sexes were treated with Abx to induce bacterial depletion. Mice were then tested in a battery of behavioural tests, including the elevated plus maze and open field tests (anxiety-like behaviour), 3 chamber test (social preference), and the tail suspension and forced swim tests (despair behaviour). Behavioural measurements in the tail suspension test were also performed after microbiota reconstitution and after administration of an Ahr agonist, ß-naphthoflavone. Gene expression analyses were performed in the brain, liver, and colon by qPCR. Abx-induced bacterial depletion did not alter anxiety-like behaviour, locomotion, or social preference in either sex. A sex-dependent effect was observed in despair behaviour. Male mice had a reduction in despair behaviour after Abx treatment in both the tail suspension and forced swim tests. A similar alteration in despair behaviour was observed in Ahr knockout mice. Despair behaviour was normalized by either microbiota recolonization or Ahr activation in Abx-treated mice. Ahr activation by ß-naphthoflavone was confirmed by increased expression of the Ahr-target genes Cyp1a1, Cyp1b1, and Ahrr. Our results demonstrate a role for Ahr in mediating the behaviours that are regulated by the crosstalk between the intestinal microbiota and the host. Ahr represents a novel potential modulator of behavioural conditions influenced by the intestinal microbiota.

Brain TNF drives post-inflammation depression-like behavior and persistent pain in experimental arthritis.

Patients with rheumatoid arthritis experience chronic pain, depression and fatigue, even when inflammation of the joints is well controlled. To study the relationship between arthritis, depression, and sustained pain when articular inflammation is no longer observed, we tested the hypothesis that brain TNF drives post-inflammation depression-like behavior and persistent pain in experimental arthritis. The murine model of antigen-induced arthritis (AIA) was used to evaluate the effects of knee inflammation on sustained pain and depression-like behavior. We measured joint pain using an automated dynamic plantar algesiometer and depression-like behavior with the tail suspension test. Cytokines were measured by Luminex assay and ELISA. TNF in the brain was blocked by intracerebroventricular injection of anti-TNF antibodies. Histological damage and elevated levels of cytokines were observed in the knee 24 h after antigen treatment, but not at 13 days. Reduced pain thresholds were seen 24 h and 13 days after treatment. Depression-like behavior was observed on day 13. Treatment with the antidepressant imipramine reduced both depression-like behavior and persistent pain. However, blocking joint pain with the analgesic dipyrone did not alter depression-like behavior. Elevated levels of TNF, CCL2, and CXCL-1 were observed in the hippocampus 24 h after treatment, with TNF remaining elevated at day 13. Intracerebroventricular infusion of an anti-TNF antibody blocked depression-like behavior and reduced persistent pain. We have demonstrated that depression-like behavior and pain is sustained in AIA mice after the resolution of inflammation. These changes are associated with elevated levels of TNF in the hippocampus and are dependent upon brain TNF. The findings reveal an important mechanistic link between the expression of chronic pain and depression in experimental arthritis. Furthermore, they suggest treating depression in rheumatoid arthritis may positively impact other debilitating features of this condition.

Intestinal fungi are causally implicated in microbiome assembly and immune development in mice.

The gut microbiome consists of a multi-kingdom microbial community. Whilst the role of bacteria as causal contributors governing host physiological development is well established, the role of fungi remains to be determined. Here, we use germ-free mice colonized with defined species of bacteria, fungi, or both to differentiate the causal role of fungi on microbiome assembly, immune development, susceptibility to colitis, and airway inflammation. Fungal colonization promotes major shifts in bacterial microbiome ecology, and has an independent effect on innate and adaptive immune development in young mice. While exclusive fungal colonization is insufficient to elicit overt dextran sulfate sodium-induced colitis, bacterial and fungal co-colonization increase colonic inflammation. Ovalbumin-induced airway inflammation reveals that bacterial, but not fungal colonization is necessary to decrease airway inflammation, yet fungi selectively promotes macrophage infiltration in the airway. Together, our findings demonstrate a causal role for fungi in microbial ecology and host immune functionality, and therefore prompt the inclusion of fungi in therapeutic approaches aimed at modulating early life microbiomes.

Colitis-Induced Microbial Perturbation Promotes Postinflammatory Visceral Hypersensitivity.

BACKGROUND & AIMS: Despite achieving endoscopic remission, more than 20% of inflammatory bowel disease patients experience chronic abdominal pain. These patients have increased rectal transient receptor potential vanilloid-1 receptor (TRPV1) expression, a key transducer of inflammatory pain. Because inflammatory bowel disease patients in remission exhibit dysbiosis and microbial manipulation alters TRPV1 function, our goal was to examine whether microbial perturbation modulated transient receptor potential function in a mouse model. METHODS: Mice were given dextran sodium sulfate (DSS) to induce colitis and were allowed to recover. The microbiome was perturbed by using antibiotics as well as fecal microbial transplant (FMT). Visceral and somatic sensitivity were assessed by recording visceromotor responses to colorectal distention and using hot plate/automated Von Frey tests, respectively. Calcium imaging of isolated dorsal root ganglia neurons was used as an in vitro correlate of nociception. The microbiome composition was evaluated via 16S rRNA gene variable region V4 amplicon sequencing, whereas fecal short-chain fatty acids (SCFAs) were assessed by using targeted mass spectrometry. RESULTS: Postinflammatory DSS mice developed visceral and somatic hyperalgesia. Antibiotic administration during DSS recovery induced visceral, but not somatic, hyperalgesia independent of inflammation. FMT of postinflammatory DSS stool into antibiotic-treated mice increased visceral hypersensitivity, whereas FMT of control stool reversed antibiotics' sensitizing effects. Postinflammatory mice exhibited both increased SCFA-producing species and fecal acetate/butyrate content compared with controls. Capsaicin-evoked calcium responses were increased in naive dorsal root ganglion neurons incubated with both sodium butyrate/propionate alone and with colonic supernatants derived from postinflammatory mice. CONCLUSIONS: The microbiome plays a central role in postinflammatory visceral hypersensitivity. Microbial-derived SCFAs can sensitize nociceptive neurons and may contribute to the pathogenesis of postinflammatory visceral pain.

Behavioral adaptations in a relapsing mouse model of colitis.

Inflammatory bowel disease (IBD) is characterized by relapsing periods of gut inflammation, and is comorbid with depression, anxiety, and cognitive deficits. Animal models of IBD that explore the behavioral consequences almost exclusively use acute models of gut inflammation, which fails to recapitulate the cyclic, chronic nature of IBD. This study sought to identify behavioral differences in digging, memory, and stress-coping strategies in mice exposed to one (acute) or three (chronic) cycles of gut inflammation, using the dextran sodium sulfate (DSS) model of colitis. Similar levels of gut pathology were observed between acute and chronically exposed mice, although mice in the chronic treatment had significantly shorter colons, suggesting more severe disease. Behavioral measures revealed an unexpected pattern in which chronic treatment evoked fewer deficits than acute treatment. Specifically, acutely-treated mice showed alterations in measures of object burying, novel object recognition, object location memory, and stress-coping (forced swim task). Chronically-treated animals, however, showed similar alterations in object burying, but not the other measures. These data suggest an adaptive or tolerizing effect of repeated cycles of peripheral gut inflammation on mnemonic function and stress-coping, whereas some other behaviors continue to be affected by gut inflammation. We speculate that the normalization of some functions may involve the reversion to the baseline state of the hypothalamic-pituitary-adrenal axis and/or levels of neuroinflammation, which are both activated by the first exposure to the colitic agent.

Antibiotic treatment affects the expression levels of copper transporters and the isotopic composition of copper in the colon of mice.

Copper is a critical enzyme cofactor in the body but also a potent cellular toxin when intracellularly unbound. Thus, there is a delicate balance of intracellular copper, maintained by a series of complex interactions between the metal and specific copper transport and binding proteins. The gastrointestinal (GI) tract is the primary site of copper entry into the body and there has been considerable progress in understanding the intricacies of copper metabolism in this region. The GI tract is also host to diverse bacterial populations, and their role in copper metabolism is not well understood. In this study, we compared the isotopic fractionation of copper in the GI tract of mice with intestinal microbiota significantly depleted by antibiotic treatment to that in mice not receiving such treatment. We demonstrated variability in copper isotopic composition along the length of the gut. A significant difference, ∼1.0‰, in copper isotope abundances was measured in the proximal colon of antibiotic-treated mice. The changes in copper isotopic composition in the colon are accompanied by changes in copper transporters. Both CTR1, a copper importer, and ATP7A, a copper transporter across membranes, were significantly down-regulated in the colon of antibiotic-treated mice. This study demonstrated that isotope abundance measurements of metals can be used as an indicator of changes in metabolic processes in vivo. These measurements revealed a host-microbial interaction in the GI tract involved in the regulation of copper transport.

Copaiba Oil Decreases Oxidative Stress and Inflammation But not Colon Damage in Rats with TNBS-Induced Colitis.

BACKGROUND: TNBS-induced colitis is an experimental immunopathology in rats that shares many features with human inflammatory bowel diseases. Copaiba oleoresin is extracted from plants of the genus Copaifera and is shown to reduce inflammation. OBJECTIVE: The aim of this study was to investigate the action of copaiba oil (C. reticulata Ducke) on inflammation and oxidative status in the distal colon of colitic rats. METHODS: Acute and subchronic colitis were induced in Wistar rats by an intracolonic enema with 2,4,6-trinitrobenzenesulfonic acid (TNBS). The colonic morphology was assessed by histological analysis and the oxidative stress parameters were measured in the intestinal homogenate. The liver damage markers were measured in the plasma. Control and colitic rats were orally treated either with one single dose (acute colitis) of copaiba oil (1.15 g Kg-1) or once a day during seven days (subchronic colitis). RESULTS: The intestinal morphology was severely modified by acute and subchronic colitis, as indicated by the intramural infiltration of polymorphonuclear cells and the increased thickness of all colon layers. The levels of TBARS, protein carbonyl groups and reactive oxygen species (ROS) were increased in the intestine of colitic rats. Copaiba oil did not attenuate the inflammatory damage in acute and subchronic colitis, but it decreased the activity of myeloperoxidase, leukocyte infiltration and oxidative stress in the colon. The level of plasma bilirubin and the activity of alkaline phosphatase were both increased in treated healthy and colitic rats. CONCLUSION: Copaiba oil decreased oxidative stress and inflammation but did not prevent intestinal damage in the colon of colitic rats. The alterations of plasma markers of hepatic damage caused by the oil seem to be associated to its harmful action on the liver.

Constitutive androstane receptor regulates the intestinal mucosal response to injury.

BACKGROUND AND PURPOSE: The pathogenesis of the inflammatory bowel diseases (IBD), comprising Crohn's disease (CD) and ulcerative colitis (UC), involves aberrant interactions between a genetically susceptible individual, their microbiota and environmental factors. Alterations in xenobiotic receptor expression and function are associated with increased risk for IBD. Here, we have assessed the role of the constitutive androstane receptor (CAR), a xenobiotic receptor closely related to the pregnane X receptor, in the regulation of intestinal mucosal homeostasis. EXPERIMENTAL APPROACH: CAR expression was assessed in intestinal mucosal biopsies obtained from CD and UC patients, and in C57/Bl6 mice exposed to dextran sulphate sodium (DSS; 3.5% w/v in drinking water) to evoke intestinal inflammation and tissue damage. CAR-deficient mice were exposed to DSS and mucosal healing assessed. Modulation of wound healing by CAR was assessed in vitro. The therapeutic potential of CAR activation was evaluated, using 3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP), a selective rodent CAR agonist. KEY RESULTS: CAR expression was reduced in CD and UC samples, compared with expression in healthy controls. This was reproduced in our DSS studies, where CAR expression was reduced in colitic mice. CAR-deficient mice exhibited reduced healing following DSS exposure. In vitro, CAR activation accelerated intestinal epithelial wound healing by enhancing cell migration. Lastly, treating mice with TCPOBOP, following induction of colitis, enhanced mucosal healing. CONCLUSION AND IMPLICATIONS: Our results support the notion that xenobiotic sensing is altered during intestinal inflammation, and suggest that CAR activation may prove effective in enhancing mucosal healing in patients with IBD.

Food restriction beginning at lactation interferes with the cellular dynamics of the mucosa and colonic myenteric innervation in adult rats.

The effects of food restriction (FR) on the morphoquantitative aspects of the wall and myenteric neurons of the proximal colon in adult rats were analysed. FR was imposed by duplication of the experimental brood size in relation to the control brood during lactation. The FR group received a 50% reduction of food from weaning until 90 days of age. Samples of the colon underwent histological processing to morphometrically analyze the crypts, muscularis mucosae, tunica mucosa, and muscularis externa. We determined the number of goblet cells and serotoninergic enteroendocrine cells, and morphoquantitatively studied the myenteric neuronal population. FR caused hypertrophy in the tunica mucosa, increase in crypt depth and in the muscular layer of the mucosa, a decrease in the thickness of the tunica muscularis and in the number of goblet cells and an increase in serotoninergic cells. A higher neuronal density in the ganglia and a reduction of the cell profile area were observed in the FR group. FR imposed since lactation led to hypertrophy of the tunica mucosa, a reduction of neutral mucin production, atrophy of the tunica muscularis, and an increase in the survival neuronal in adult rats, attributable to an increase in the number of serotoninergic enteroendocrine cells in mucosa.

Effects of an Agaricus blazei aqueous extract pretreatment on paracetamol-induced brain and liver injury in rats.

The action of an Agaricus blazei aqueous extract pretreatment on paracetamol injury in rats was examined not only in terms of the classical indicators (e.g., levels of hepatic enzymes in the plasma) but also in terms of functional and metabolic parameters (e.g., gluconeogenesis). Considering solely the classical indicators for tissue damage, the results can be regarded as an indication that the A. blazei extract is able to provide a reasonable degree of protection against the paracetamol injury in both the hepatic and brain tissues. The A. blazei pretreatment largely prevented the increased levels of hepatic enzymes in the plasma (ASP, ALT, LDH, and ALP) and practically normalized the TBARS levels in both liver and brain tissues. With respect to the functional and metabolic parameters of the liver, however, the extract provided little or no protection. This includes morphological signs of inflammation and the especially important functional parameter gluconeogenesis, which was impaired by paracetamol. Considering these results and the long list of extracts and substances that are said to have hepatoprotective effects, it would be useful to incorporate evaluations of functional parameters into the experimental protocols of studies aiming to attribute or refute effective hepatoprotective actions to natural products.