Premysl Bercik

The Intestinal Microbiota Affect Central Levels of Brain-Derived Neurotropic Factor and Behavior in Mice

BACKGROUND & AIMS Alterations in the microbial composition of the gastrointestinal tract (dysbiosis) are believed to contribute to inflammatory and functional bowel disorders and psychiatric comorbidities. We examined whether the intestinal microbiota affects behavior and brain biochemistry in mice. METHODS Specific pathogen-free (SPF) BALB/c mice, with or without subdiaphragmatic vagotomy or chemical sympathectomy, or germ-free BALB/c mice received a mixture of nonabsorbable antimicrobials (neomycin, bacitracin, and pimaricin) in their drinking water for 7 days. Germ-free BALB/c and NIH Swiss mice were colonized with microbiota from SPF NIH Swiss or BALB/c mice. Behavior was evaluated using step-down and light preference tests. Gastrointestinal microbiota were assessed using denaturing gradient gel electrophoresis and sequencing. Gut samples were analyzed by histologic, myeloperoxidase, and cytokine analyses; levels of serotonin, noradrenaline, dopamine, and brain-derived neurotropic factor (BDNF) were assessed by enzyme-linked immunosorbent assay. RESULTS Administration of oral antimicrobials to SPF mice transiently altered the composition of the microbiota and increased exploratory behavior and hippocampal expression of BDNF. These changes were independent of inflammatory activity, changes in levels of gastrointestinal neurotransmitters, and vagal or sympathetic integrity. Intraperitoneal administration of antimicrobials to SPF mice or oral administration to germ-free mice did not affect behavior. Colonization of germ-free BALB/c mice with microbiota from NIH Swiss mice increased exploratory behavior and hippocampal levels of BDNF, whereas colonization of germ-free NIH Swiss mice with BALB/c microbiota reduced exploratory behavior. CONCLUSIONS The intestinal microbiota influences brain chemistry and behavior independently of the autonomic nervous system, gastrointestinal-specific neurotransmitters, or inflammation. Intestinal dysbiosis might contribute to psychiatric disorders in patients with bowel disorders.

The interplay between the intestinal microbiota and the brain

The intestinal microbiota consists of a vast bacterial community that resides primarily in the lower gut and lives in a symbiotic relationship with the host. A bidirectional neurohumoral communication system, known as the gut–brain axis, integrates the host gut and brain activities. Here, we describe the recent advances in our understanding of how the intestinal microbiota communicates with the brain via this axis to influence brain development and behaviour. We also review how this extended communication system might influence a broad spectrum of diseases, including irritable bowel syndrome, psychiatric disorders and demyelinating conditions such as multiple sclerosis.

Innate and Adaptive Immunity Cooperate Flexibly to Maintain Host-Microbiota Mutualism

Maintaining Mutual Ignorance Our gut is colonized by trillions of bacteria that do not activate the immune system because of careful compartmentalization. Such compartmentalization means that our immune system is “ignorant” of these microbes and thus it has been proposed that loss of compartmentalization might result in an immune response to the colonizing bacteria. Microorganisms are sensed by cells that express pattern recognition receptors, such as Toll-like receptors, which recognize patterns specific to those microbes. Slack et al. (p. 617) show that Toll-like receptor–dependent signaling is required to maintain compartmentalization of bacteria to the gut of mice. In the absence of Toll-dependent signaling, intestinal bacteria disseminated throughout the body and the mice mounted a high-titer antibody response against them. This antibody response was of great functional importance because, despite the loss of systemic ignorance to intestinal microbes, the mice were tolerant of the bacteria. Thus, in the absence of innate immunity, the adaptive immune system can compensate so that host and bacterial mutualism can be maintained. Mouse immune systems interact to ensure tolerance to nonpathogenic bacteria in the gut. Commensal bacteria in the lower intestine of mammals are 10 times as numerous as the body’s cells. We investigated the relative importance of different immune mechanisms in limiting the spread of the intestinal microbiota. Here, we reveal a flexible continuum between innate and adaptive immune function in containing commensal microbes. Mice deficient in critical innate immune functions such as Toll-like receptor signaling or oxidative burst production spontaneously produce high-titer serum antibodies against their commensal microbiota. These antibody responses are functionally essential to maintain host-commensal mutualism in vivo in the face of innate immune deficiency. Spontaneous hyper-activation of adaptive immunity against the intestinal microbiota, secondary to innate immune deficiency, may clarify the underlying mechanisms of inflammatory diseases where immune dysfunction is implicated.

Specific probiotic therapy attenuates antibiotic induced visceral hypersensitivity in mice

Background and aim: Abdominal pain and discomfort are common symptoms in functional disorders and are attributed to visceral hypersensitivity. These symptoms fluctuate over time but the basis for this is unknown. Here we examine the impact of changes in gut flora and gut inflammatory cell activity on visceral sensitivity. Methods: Visceral sensitivity to colorectal distension (CRD) was assessed at intervals in healthy mice for up to 12 weeks, and in mice before and after administration of dexamethasone or non-absorbable antibiotics with or without supplementation with Lactobacillus paracasei (NCC2461). Tissue was obtained for measurement of myeloperoxidase activity (MPO), histology, microbiota analysis, and substance P (SP) immunolabelling. Results: Visceral hypersensitivity developed over time in healthy mice maintained without sterile precautions. This was accompanied by a small increase in MPO activity. Dexamethasone treatment normalised MPO and CRD responses. Antibiotic treatment perturbed gut flora, increased MPO and SP immunoreactivity in the colon, and produced visceral hypersensitivity. Administration of Lactobacillus paracasei in spent culture medium normalised visceral sensitivity and SP immunolabelling, but not intestinal microbiota counts. Conclusion: Perturbations in gut flora and in inflammatory cell activity alter sensory neurotransmitter content in the colon, and result in altered visceral perception. Changes in gut flora may be a basis for the variability of abdominal symptoms observed in functional gastrointestinal disorders and may be prevented by specific probiotic administration.

Chronic Gastrointestinal Inflammation Induces Anxiety-Like Behavior and Alters Central Nervous System Biochemistry in Mice

BACKGROUND & AIMS Clinical and preclinical studies have associated gastrointestinal inflammation and infection with altered behavior. We investigated whether chronic gut inflammation alters behavior and brain biochemistry and examined underlying mechanisms. METHODS AKR mice were infected with the noninvasive parasite Trichuris muris and given etanercept, budesonide, or specific probiotics. Subdiaphragmatic vagotomy was performed in a subgroup of mice before infection. Gastrointestinal inflammation was assessed by histology and quantification of myeloperoxidase activity. Serum proteins were measured by proteomic analysis, circulating cytokines were measured by fluorescence activated cell sorting array, and serum tryptophan and kynurenine were measured by liquid chromatography. Behavior was assessed using light/dark preference and step-down tests. In situ hybridization was used to assess brain-derived neurotrophic factor (BDNF) expression in the brain. RESULTS T muris caused mild to moderate colonic inflammation and anxiety-like behavior that was associated with decreased hippocampal BDNF messenger RNA (mRNA). Circulating tumor necrosis factor-α and interferon-γ, as well as the kynurenine and kynurenine/tryptophan ratio, were increased. Proteomic analysis showed altered levels of several proteins related to inflammation and neural function. Administration of etanercept, and to a lesser degree of budesonide, normalized behavior, reduced cytokine and kynurenine levels, but did not influence BDNF expression. The probiotic Bifidobacterium longum normalized behavior and BDNF mRNA but did not affect cytokine or kynurenine levels. Anxiety-like behavior was present in infected mice after vagotomy. CONCLUSIONS Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry, which can be normalized by inflammation-dependent and -independent mechanisms, neither of which requires the integrity of the vagus nerve.

Proton Pump Inhibitors Exacerbate NSAID-Induced Small Intestinal Injury by Inducing Dysbiosis

BACKGROUND & AIMS Proton pump inhibitors (PPIs) and nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used classes of drugs, with the former frequently coprescribed to reduce gastroduodenal injury caused by the latter. However, suppression of gastric acid secretion by PPIs is unlikely to provide any protection against the damage caused by NSAIDs in the more distal small intestine. METHODS Rats were treated with antisecretory doses of omeprazole or lanzoprazole for 9 days, with concomitant treatment with anti-inflammatory doses of naproxen or celecoxib on the final 4 days. Small intestinal damage was blindly scored, and changes in hematocrit were measured. Changes in small intestinal microflora were evaluated by denaturing gradient gel electrophoresis and reverse-transcription polymerase chain reaction. RESULTS Both PPIs significantly exacerbated naproxen- and celecoxib-induced intestinal ulceration and bleeding in the rat. Omeprazole treatment did not result in mucosal injury or inflammation; however, there were marked shifts in numbers and types of enteric bacteria, including a significant reduction (∼80%) of jejunal Actinobacteria and Bifidobacteria spp. Restoration of small intestinal Actinobacteria numbers through administration of selected (Bifidobacteria enriched) commensal bacteria during treatment with omeprazole and naproxen prevented intestinal ulceration/bleeding. Colonization of germ-free mice with jejunal bacteria from PPI-treated rats increased the severity of NSAID-induced intestinal injury, as compared with mice colonized with bacteria from vehicle-treated rats. CONCLUSIONS PPIs exacerbate NSAID-induced intestinal damage at least in part because of significant shifts in enteric microbial populations. Prevention or reversal of this dysbiosis may be a viable option for reducing the incidence and severity of NSAID enteropathy.

Microbiota and host determinants of behavioural phenotype in maternally separated mice

Early-life stress is a determinant of vulnerability to a variety of disorders that include dysfunction of the brain and gut. Here we exploit a model of early-life stress, maternal separation (MS) in mice, to investigate the role of the intestinal microbiota in the development of impaired gut function and altered behaviour later in life. Using germ-free and specific pathogen-free mice, we demonstrate that MS alters the hypothalamic-pituitary-adrenal axis and colonic cholinergic neural regulation in a microbiota-independent fashion. However, microbiota is required for the induction of anxiety-like behaviour and behavioural despair. Colonization of adult germ-free MS and control mice with the same microbiota produces distinct microbial profiles, which are associated with altered behaviour in MS, but not in control mice. These results indicate that MS-induced changes in host physiology lead to intestinal dysbiosis, which is a critical determinant of the abnormal behaviour that characterizes this model of early-life stress.

The microbiota–gut–brain axis in gastrointestinal disorders: stressed bugs, stressed brain or both?

The gut–brain axis is the bidirectional communication between the gut and the brain, which occurs through multiple pathways that include hormonal, neural and immune mediators. The signals along this axis can originate in the gut, the brain or both, with the objective of maintaining normal gut function and appropriate behaviour. In recent years, the study of gut microbiota has become one of the most important areas in biomedical research. Attention has focused on the role of gut microbiota in determining normal gut physiology and immunity and, more recently, on its role as modulator of host behaviour (‘microbiota–gut–brain axis’). We therefore review the literature on the role of gut microbiota in gut homeostasis and link it with mechanisms that could influence behaviour. We discuss the association of dysbiosis with disease, with particular focus on functional bowel disorders and their relationship to psychological stress. This is of particular interest because exposure to stressors has long been known to increase susceptibility to and severity of gastrointestinal diseases.

Validation of the Rome III Criteria for the Diagnosis of Irritable Bowel Syndrome in Secondary Care

BACKGROUND & AIMS There are few validation studies of existing diagnostic criteria for irritable bowel syndrome (IBS). We conducted a validation study of the Rome and Manning criteria in secondary care. METHODS We collected complete symptom, colonoscopy, and histology data from 1848 consecutive adult patients with gastrointestinal symptoms at 2 hospitals in Hamilton, Ontario; the subjects then underwent colonoscopy. Assessors were blinded to symptom status. Individuals with normal colonoscopy and histopathology results, and no evidence of celiac disease, were classified as having no organic gastrointestinal disease. The reference standard used to define the presence of true IBS was lower abdominal pain or discomfort in association with a change in bowel habit and no organic gastrointestinal disease. Sensitivity, specificity, and positive and negative likelihood ratios, with 95% confidence intervals, were calculated for each diagnostic criteria. RESULTS In identifying patients with IBS, sensitivities of the criteria ranged from 61.9% (Manning) to 95.8% (Rome I), and specificities from 70.6% (Rome I) to 81.8% (Manning). Positive likelihood ratios ranged from 3.19 (Rome II) to 3.39 (Manning), and negative likelihood ratios from 0.06 (Rome I) to 0.47 (Manning). The level of agreement between diagnostic criteria was greatest for Rome I and Rome II (κ = 0.95), and lowest for Manning and Rome III (κ = 0.59). CONCLUSIONS Existing diagnostic criteria perform modestly in distinguishing IBS from organic disease. There appears to be little difference in terms of accuracy. More accurate ways of diagnosing IBS, avoiding the need for investigation, are required.

The intestinal microbiome, probiotics and prebiotics in neurogastroenterology

The brain-gut axis allows bidirectional communication between the central nervous system (CNS) and the enteric nervous system (ENS), linking emotional and cognitive centers of the brain with peripheral intestinal functions. Recent experimental work suggests that the gut microbiota have an impact on the brain-gut axis. A group of experts convened by the International Scientific Association for Probiotics and Prebiotics (ISAPP) discussed the role of gut bacteria on brain functions and the implications for probiotic and prebiotic science. The experts reviewed and discussed current available data on the role of gut microbiota on epithelial cell function, gastrointestinal motility, visceral sensitivity, perception and behavior. Data, mostly gathered from animal studies, suggest interactions of gut microbiota not only with the enteric nervous system but also with the central nervous system via neural, neuroendocrine, neuroimmune and humoral links. Microbial colonization impacts mammalian brain development in early life and subsequent adult behavior. These findings provide novel insights for improved understanding of the potential role of gut microbial communities on psychological disorders, most particularly in the field of psychological comorbidities associated with functional bowel disorders like irritable bowel syndrome (IBS) and should present new opportunity for interventions with pro- and prebiotics.