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The gut microbiota has been implicated in chronic pain disorders, including irritable bowel syndrome (IBS), yet specific pathophysiological mechanisms remain unclear. We showed that decreasing intake of fermentable carbohydrates improved abdominal pain in patients with IBS, and this was accompanied by changes in the gut microbiota and decreased urinary histamine concentrations. Here, we used germ-free mice colonized with fecal microbiota from patients with IBS to investigate the role of gut bacteria and the neuroactive mediator histamine in visceral hypersensitivity. Germ-free mice colonized with the fecal microbiota of patients with IBS who had high but not low urinary histamine developed visceral hyperalgesia and mast cell activation. When these mice were fed a diet with reduced fermentable carbohydrates, the animals showed a decrease in visceral hypersensitivity and mast cell accumulation in the colon. We observed that the fecal microbiota from patients with IBS with high but not low urinary histamine produced large amounts of histamine in vitro. We identifiedKlebsiella aerogenes, carrying a histidine decarboxylase gene variant, as a major producer of this histamine. This bacterial strain was highly abundant in the fecal microbiota of three independent cohorts of patients with IBS compared with healthy individuals. Pharmacological blockade of the histamine 4 receptor in vivo inhibited visceral hypersensitivity and decreased mast cell accumulation in the colon of germ-free mice colonized with the high histamine-producing IBS fecal microbiota. These results suggest that therapeutic strategies directed against bacterial histamine could help treat visceral hyperalgesia in a subset of patients with IBS with chronic abdominal pain.

Chronic constipation can have one or more of many etiologies, and a diagnosis based on symptoms is not sufficient as a basis for treatment, in particular surgery. To investigate the cause of chronic constipation in a patient with complete absence of spontaneous bowel movements. High-resolution colonic manometry was performed to assess motor functions of the colon, rectum, the sphincter of O’Beirne and the anal sphincters. Normal colonic motor patterns were observed, even at baseline, but a prominent high-pressure zone at the rectosigmoid junction, the sphincter of O’Beirne, was consistently present. In response to high-amplitude propagating pressure waves (HAPWs) that were not consciously perceived, the sphincter and the anal sphincters would not relax and paradoxically contract, identified as autonomous dyssynergia. Rectal bisacodyl evoked marked HAPW activity with complete relaxation of the sphincter of O’Beirne and the anal sphincters, indicating that all neural pathways to generate the coloanal reflex were intact but had low sensitivity to physiological stimuli. A retrograde propagating cyclic motor pattern initiated at the sphincter of O’Beirne, likely contributing to failure of content to move into the rectum. Chronic constipation without the presence of spontaneous bowel movements can be associated with normal colonic motor patterns but a highly exaggerated pressure at the rectosigmoid junction: the sphincter of O’Beirne, and failure of this sphincter and the anal sphincters to relax associated with propulsive motor patterns. The sphincter of O’Beirne can be an important part of the pathophysiology of chronic constipation.

Background Corticosteroids (CS) have been used extensively to induce remission in Crohn’s disease (CD); however, they are associated with severe side effects. We hypothesized that the administration of an exclusive enteral nutrition (EEN) formula to CS would lead to increased CD remission rates and to decreased CS-related adverse events. We proposed to undertake a pilot study comparing EEN and CS therapy to CS alone to assess decrease symptoms and inflammatory markers over 6 weeks. Aim The overall aim was to assess study feasibility based on recruitment rates and acceptability of treatment in arms involving EEN Methods The pilot study intended to recruit 100 adult patients with active CD who had been prescribed CS to induce remission as part of their care. The patients were randomized to one of three arms: (i) standard-dose CS; (ii) standard-dose CS plus EEN (Modulen 1.5 kcal); or (iii) short-course CS plus EEN. Results A total of 2009 CD patients attending gastroenterology clinics were screened from October 2018 to November 2019. Prednisone was prescribed to only 6.8% (27/399) of patients with active CD attending outpatient clinics. Of the remaining 372 patients with active CD, 34.8% (139/399) started or escalated immunosuppressant or biologics, 49.6% (198/399) underwent further investigation and 8.8% (35/399) were offered an alternative treatment (e.g., antibiotics, surgery or investigational agents in clinical trials). Only three patients were enrolled in the study (recruitment rate 11%; 3/27), and the study was terminated for poor recruitment. Conclusion The apparent decline in use of CS for treatment of CD has implications for CS use as an entry criterion for clinical trials.

This review examines preclinical and clinical studies relevant to our understanding of how the bidirectional gut-brain axis influences the natural history of inflammatory bowel disease. Preclinical studies provide proof of concept that preexisting behavioral illness, such as depression, results in increased susceptibility to inflammatory stimuli and that commonly used classes of antidepressants protect against this vulnerability. However, clinical studies suggesting behavioral illness as a risk factor for IBD and a protective role for antidepressants have relied primarily on symptom-reporting rather than objective measurements of inflammation. In terms of gut-to-brain signaling, there is emerging evidence from preclinical and clinical observation that intestinal inflammation alters brain functions, including the induction of mood disorders, alteration of circadian rhythm both centrally and peripherally, and changes in appetitive behaviors. Furthermore, preclinical studies suggest that effective treatment of intestinal inflammation improves associated behavioral impairment. Taken together, the findings of this review encourage a holistic approach to the management of patients with IBD, accommodating lifestyle issues that include the avoidance of sleep deprivation, optimized nutrition, and the monitoring and appropriate management of behavioral disorders. The review also acknowledges the need for better-designed clinical studies evaluating the impact of behavioral disorders and their treatments on the natural history of IBD, utilizing hard end points to assess changes in the inflammatory process as opposed to reliance on symptom-based assessments. The findings of the review also encourage a better understanding of changes in brain function and circadian rhythm induced by intestinal inflammation., The review adopts a translational approach examining proof-of-concept from preclinical studies and clinical evidence supporting the role of the bidirectional gut-brain axis in IBD and describes implications for optimal management.

Background & Aims Serotonin (5-hydroxytryptamine [5-HT]) is synthesized mainly within enterochromaffin (EC) cells in the gut, and tryptophan hydroxylase 1 (Tph1) is the rate-limiting enzyme for 5-HT synthesis in EC cells. Accumulating evidence suggests the importance of gut microbiota in intestinal inflammation. Considering the close proximity of EC cells and the microbes, we investigated the influence of gut-derived 5-HT on the microbiota and the susceptibility to colitis. Methods Gut microbiota of Tph1-/- and Tph1+/- mice were investigated by deep sequencing. Direct influence of 5-HT on bacteria was assessed by using in vitro system of isolated commensals. The indirect influence of 5-HT on microbiota was assessed by measuring antimicrobial peptides, specifically β-defensins, in the colon of mice and HT-29 colonic epithelial cells. The impact of gut microbiota on the development of dextran sulfate sodium–induced colitis was assessed by transferring gut microbiota from Tph1-/- mice to Tph1+/- littermates and vice versa, as well as in germ-free mice. Results A significant difference in microbial composition between Tph1-/- and Tph1+/- littermates was observed. 5-HT directly stimulated and inhibited the growth of commensal bacteria in vitro, exhibiting a concentration-dependent and species-specific effect. 5-HT also inhibited β-defensin production by HT-29 cells. Microbial transfer from Tph1-/- to Tph1+/- littermates and vice versa altered colitis severity, with microbiota from Tph1-/- mice mediating the protective effects. Furthermore, germ-free mice colonized with microbiota from Tph1-/- mice exhibited less severe dextran sulfate sodium–induced colitis. Conclusions These findings demonstrate a novel role of gut-derived 5-HT in shaping gut microbiota composition in relation to susceptibility to colitis, identifying 5-HT–microbiota axis as a potential new therapeutic target in intestinal inflammatory disorders., Graphical abstract

Background Studies of germ-free (GF) mice demonstrate that gut microbiota can influence behaviour by modulating neurochemical pathways in the brain, and that bacterial colonization normalizes behavioural deficits in GF-mice. Since disrupted GABAergic and glutamatergic signaling are reported in mood disorders, this study investigated the effect of gut microbiota manipulations on EIB-relevant gene expression in the brain. Methods GF Swiss-Webster mice were colonized with E. coli JM83, complex microbiota (specific-pathogen-free; SPF), or no microbiota, and compared with controls (n = 6/group). 21 synaptic genes representing GABAergic, glutamatergic, BDNF, and astrocytic functions were measured in the hippocampus, amygdala, and prefrontal cortex using quantitative PCR. Gene co-expression analysis was used to identify gene modules related to colonization status, and compared by permutation analysis. Gene expression profiles were compared to existing post-mortem cohorts of depressed subjects (n = 28 cases vs 28 controls). Results Region-specific alterations in gene expression were observed in GF-mice compared to controls. 58% of all genes (14/24) altered in GF-mice were normalized following SPF-colonization. GF-mice displayed disorganization of gene co-expression networks in all three brain regions (hippocampus, p = 0.0003; amygdala, p = 0.0012; mPFC, p = 0.0069), which was restored by SPF colonization in hippocampus (p v.s. GF = 0.0003, p v.s. control = 0.60). The hippocampal gene expression profile in GF-mice was significantly correlated with that in human depression (ρ = 0.51, p = 0.027), and this correlation was not observed after colonization. Conclusion Together, we show that the absence of gut microbiota disrupts the expression of EIB-relevant genes in mice, and colonization restores EIB-relevant expression, in ways that are relevant to human depression.

Background Irritable bowel syndrome (IBS) is one of the most common conditions seen by gastroenterologists for which there is no effective cure. The pathophysiology of IBS is multifactorial and poorly understood, but the condition is characterized by chronic abdominal pain accompanied by altered bowel habits in the absence of an underlying structural abnormality. IBS is frequently associated with psychiatric co-morbidities such as anxiety and depression and has been considered a disorder of gut-brain communication. We previously developed a humanized mouse model of IBS with co-morbid anxiety (IBS+A) by colonizing germ-free mice with fecal microbiota of IBS+A patients. Aims To test the therapeutic potential of the probiotic yeast Saccharomyces boulardii CNCM I-745 (S. bou) in preventing the transfer of the IBS+A phenotype and investigated underlying mechanisms. Methods Germ-free Swiss-Webster mice were colonized with fecal microbiota from an IBS+A patient or from a healthy subject (controls) and after three weeks they were gavaged daily for two weeks with 3g/kg/day of the probiotic S. bou (Biocodex–France) or water. Behavior, intestinal motility and permeability were assessed at sacrifice. Potential mechanisms were assessed by microbiota 16S rRNA gene sequencing, gene expression by Nanostring Counter Gene Expression and indole quantification by absorption using Kovak’s reagent. Results IBS+A colonized mice developed 25% faster gastrointestinal transit (P Conclusions Our data indicate that Saccharomyces boulardii CNCM I-745 supplementation improves the intestinal and behavioral phonotype that is induced by IBS+A microbiota in mice. Putative mechanisms include regulation of indole production by bacteria and regulation of host Trpv1 gene expression. The results prompt investigation of S. bou in IBS patients with co-morbid anxiety. Funding Agencies Biocodex – Gentilly – France

Background Probiotics are living microorganisms that, when administered in adequate amounts, confer a health benefit. It has been speculated that probiotics might help prevent preterm birth, but in two previous systematic reviews possible major increases in this risk have been suggested. Our objective was to perform a systematic review and meta-analysis of the risk of preterm birth and other adverse pregnancy outcomes in pregnant women taking probiotics, prebiotics or synbiotics. Methods We searched six electronic databases (MEDLINE, EMBASE, CINAHL, Cochrane Central Register of Controlled Trials, Web of Science’s Core collection and BIOSIS Preview) up to September 2016 and contacted authors for additional data. We included randomized controlled trials in which women with a singleton pregnancy received a probiotic, prebiotic or synbiotic intervention. Two independent reviewers extracted data using a piloted form and assessed the risk of bias using the Cochrane risk of bias tool. We used random-effects meta-analyses to pool the results. Results We identified 2574 publications, screened 1449 non-duplicate titles and abstracts and read 160 full text articles. The 49 publications that met our inclusion criteria represented 27 studies. No study used synbiotics, one used prebiotics and the rest used probiotics. Being randomized to take probiotics during pregnancy neither increased nor decreased the risk of preterm birth

Background The circadian clock is a highly conserved molecular pacemaker found in nearly every cell of the body. It consists of the genes BMAL1 and CLOCK that positively regulate CRY and PER, their negative regulators, resulting in a transcription/translation feedback loop that has a 24 hour cycle. This core clock mechanism drives the rhythmic expression of over 40% of the genome in a tissue-specific manner and therefore imposes 24 hour rhythms on many physiological processes. Shift work, which causes disruptions to the natural 24 hour physiological rhythms, has been shown to lead to an increased incidence of inflammatory bowel disease (IBD). Aims This study aims to characterize daily rhythms in inflammation and regeneration of the colon upon induction of acute colitis. We also aim to investigate the intestinal epithelial-specific effects of circadian clock disruption on overall disease progression. We hypothesize that the absence of a functional circadian clock eliminates proliferation rhythms of intestinal epithelial cells and disrupts the rhythms of inflammatory cytokines, thereby increasing the pathogenesis of IBD. Methods We tested the role of the clock in IBD using BMAL1+/+ (wild type) and BMAL1-/- (null mutant) mice. We also investigated the effects of the circadian clock specific to intestinal epithelial tissue using Vil+/+;BMAL1flox/flox (control) and VilCre/+;BMAL1flox/flox (conditional intestinal epithelial mutant) mice. Dextran Sulfate Sodium (DSS) was applied to induce acute colitis. Results We observed significantly decreased survival of BMAL1 circadian clock mutant mice when given colitis. A histology analysis indicates increased lesioning and overall inflammation in BMAL1-/- colon tissue. Disease activity and cytokine analyses reveal time-dependent severity in inflammatory response that is worse in BMAL1-/- mice. To test the circadian rhythms in intestinal regeneration of mice with IBD, we performed a 24 hour analysis comparing epithelial cell proliferation and cell death in colon tissue. We observed rhythmic expression of phosphor-histone H3 (a mitosis marker) in wild type mice which is eliminated in the BMAL1-/- lacking a circadian clock. Cell death which was measured by caspase 3 did not exhibit any differences between genotypes. Based on these results, we conclude that the loss of clock function leads to impaired regeneration during IBD, in part due to decreased and arrhythmic cell proliferation. Preliminary results in our VilCre/+;BMAL1flox/flox conditional intestinal epithelial mutant mice indicate that some of these effects may be epithelial-specific. Conclusions Our results support a critical role of the circadian clock in inflammatory bowel disease development. These data highlight that the circadian clock affects the regenerative abilities of intestinal epithelial cells. Funding Agencies CIHRChron’s and Colitis Canada, Ontario, University of Windsor

Background Abdominal pain is a common complaint in patients with chronic gastrointestinal disorders. Accumulating evidence suggests that gut microbiota is an important determinant of gut function, including visceral sensitivity. Germ-free (GF) mice have been shown to display visceral hypersensitivity, which normalizes after colonization. Sex also appears to play a key role in visceral sensitivity, as women report more abdominal pain than men. Thus, both gut bacteria and sex are important in the regulation of gut nociception, but the underlying mechanisms remain poorly understood. Aims To investigate the role of gut microbiota and sex in abdominal pain. Methods We used primary cultures of sensory neurons from dorsal root ganglia (DRG) of female and male conventionally raised (SPF) or germ-free (GF) mice (7–18 weeks old). To study the visceral afferent activity in vitro, calcium mobilization in DRG sensory neurons was measured by inverted fluorescence microscope using a fluorescent calcium probe Fluo-4 (1mM). Two parameters were considered i) the percentage of responding neurons ii) the intensity of the neuronal response. First, DRG sensory neurons were stimulated by a TRPV1 agonist capsaicin (12.5nM, 125nM and 1.25µM) or by a mixture of G-protein coupled receptors agonist (GPCR: bradykinin, histamine and serotonin; 1µM, 10µM and 100µM). We next measured the neuronal production of substance P and calcitonin gene-related peptide (CGRP), two neuropeptides associated with nociception, in response to capsaicin (1.25µM) or GPCR agonists (100µM) by ELISA and EIA, respectively. Results The percentage of neurons responding to capsaicin and GPCR agonists was similar in male and female SPF and GF mice. However, the intensity of the neuronal response was higher in SPF male compared to SPF female in response to capsaicin (125nM: p=0.0336; 1.25µM: p=0.033) but not to GPCR agonists. Neuronal activation was similar in GF and SPF mice of both sexes after administration of capsaicin or GPCR agonists. Furthermore, substance P and CGRP production by sensory neurons induced by capsaicin or GPCR agonists was similar in SPF and GF mice, regardless of sex. However, while the response to capsaicin was similar, the GPCR agonists-induced production of substance P was higher in SPF male mice compared to SPF females (p=0.003). The GPCR agonists-induced production of CGRP was similar in SPF male and female mice. Conclusions Our data suggest that at the level of DRG neurons, the absence of gut microbiota does not predispose to visceral hypersensitivity. The intensity of DRG neuronal responses to capsaicin and the GPCR agonists-induced production of substance P are higher in male compared to female mice, in contrast to previously published studies in various models of acute and chronic pain. Further studies are thus needed to investigate the role of sex in visceral sensitivity. Funding Agencies CIHR

Background & Aims Many patients with irritable bowel syndrome (IBS) perceive that their symptoms are triggered by wheat-containing foods. We assessed symptoms and gastrointestinal transit before and after a gluten-free diet (GFD) in unselected patients with IBS and investigated biomarkers associated with symptoms. Methods We performed a prospective study of 50 patients with IBS (ROME III, all subtypes), with and without serologic reactivity to gluten (antigliadin IgG and IgA), and 25 healthy subjects (controls) at a university hospital in Hamilton, Ontario, Canada, between 2012 and 2016. Gastrointestinal transit, gut symptoms, anxiety, depression, somatization, dietary habits, and microbiota composition were studied before and after 4 weeks of a GFD. HLA-DQ2/DQ8 status was determined. GFD compliance was assessed by a dietitian and by measuring gluten peptides in stool. Results There was no difference in symptoms among patients at baseline, but after the GFD, patients with antigliadin IgG and IgA reported less diarrhea than patients without these antibodies (P = .03). Compared with baseline, IBS symptoms improved in 18 of 24 patients (75%) with antigliadin IgG and IgA and in 8 of 21 patients (38%) without the antibodies. Although constipation, diarrhea, and abdominal pain were reduced in patients with antigliadin IgG and IgA, only pain decreased in patients without these antibodies. Gastrointestinal transit normalized in a higher proportion of patients with antigliadin IgG and IgA. Anxiety, depression, somatization, and well-being increased in both groups. The presence of antigliadin IgG was associated with overall reductions in symptoms (adjusted odds ratio compared with patients without this antibody, 128.9; 95% CI, 1.16–1427.8; P = .04). Symptoms were reduced even in patients with antigliadin IgG and IgA who reduced gluten intake but were not strictly compliant with the GFD. In controls, a GFD had no effect on gastrointestinal symptoms or gut function. Conclusions Antigliadin IgG can be used as a biomarker to identify patients with IBS who might have reductions in symptoms, particularly diarrhea, on a GFD. Larger studies are needed to validate these findings. ClinicalTrials.gov: NCT03492333 .

Background Dysbiosis may be defined as a change in the microbial composition or function that results in altered host function. Defensins are antimicrobial peptides, are part of innate immunity, and are important in host defense and maintaining homeostasis. Dysbiosis is a putative mechanism underlying the expression of many functional GI disorders like Irritable Bowel Syndrome (IBS) for which no biomarkers exist. Previous studies have revealed increased β-defensin (β-Def) levels in IBS patients, most likely due to changes in the microbiota. Aims We examined the hypotheses that: 1) Changes in β-Def are dependent on the manner in which dysbiosis is induced, and that 2) the direction of the change in β-Def depends on how dysbiosis was induced. Methods We used 4 models of experimentally induced dysbiosis to determine changes in fecal β-Def and to characterize the microbiota composition before and during the induction of dysbiosis. We used: 1) an antimicrobial cocktail (AC) in water; 2) a high-fat/ high-sugar diet (HFHSD); 3) a high salt diet (HSD) that we previously showed to induce a pro-inflammatory microbiota; and 4) mild restraint stress (MRS). All studies were performed in C57/BL6 mice except studies using MRS that were performed in NIH Swiss mice. In the AC or dietary studies, we employed a one-week intervention preceded by one-week baseline and recovery periods. In MRS studies, mice comparisons were made between a control and a stressed group. Stool samples were collected every 24 hours and were assayed for fecal β-Def levels analysis by an ELISA and microbial composition by 16S gene profiling. Results Exposure to AC or dietary change, but not MRS, resulted in significant decreases in fecal β-Def. Additionally, bacterial composition and diversity profiles were different in all mice except MRS mice (control vs. MRS males: p=0.414; control vs. MRS females: p=0.96). In contrast, mice exposed to the HSD revealed a significant increase in β-Def during treatment compared to baseline in both males (p=0.025) and females (p= 0.0019). The AC mice showed the largest changes and significant correlations between changes in β-Def levels and bacterial diversity (males: p=0.013, r=0.6; females: p=0.007, r=0.6) and richness (males: p=0.0008, r=0.70; females: p=0.003, r=0.62). However, no significant correlations were found between specific bacteria and β-Def levels in the HFHSD group. Conclusions We conclude that directional changes in fecal β-Def levels are dependent on the manner in which dysbiosis is induced. The use of β-Def as a biomarker requires comparisons with baseline levels obtained during remission in order to identify dysbiosis presence in microbiota-associated chronic GI conditions like IBS. Such an approach will identify patient subgroups that may benefit from microbiota-directed therapies. Funding Agencies CIHR

Background Increasing evidence suggests that gut microbiota play a key role in gastrointestinal (GI) tract function. We have previously shown that fecal microbiota transplantation diarrhea predominant IBS patients into germ-free mice induces faster GI transit, increased permeability and innate immune activation. However, it is unknown whether gut dysfunction is induced by microbiota from patients with chronic constipation. Aims Here, we investigated the role of the intestinal microbiota in the expression of severe slow transit constipation in a patient with previous C difficile infection and extensive antibiotic exposure. Methods Germ-free (GF) mice (14 weeks old) were gavaged with diluted fecal content from the patient with constipation (PA) or a sex and age-matched healthy control (HC). 12 weeks later, we assessed gut motility and GI transit using videofluoroscopy and a bead expulsion test.. We then investigated intestinal and colonic smooth muscle isometric contraction in vitro using electric field stimulation (EFS), and acetylcholine (Ach) release was assessed by superfusion using [3H] choline. Histological changes were evaluated by H&E and immunohistochemistry. Results Mice with PA microbiota had faster whole GI transit (score 18.9 ± 0.9 (N=9) than mice with HC microbiota (15.4 ± 1.0, N=10, p=0.032), with markers located mainly in the distal small bowel and cecum. However, bead expulsion from the colon was significantly longer in PA mice (420.8 s ± 124.6 s, N=9) than in HC mice (82.6 s ± 20.0 s, N=10, p=0.026). This delayed colonic transit was likely due to colonic retroperistalsis visualized videofluoroscopically by retrograde flow of barium in the right colon of PA mice. There was no difference between the two groups in small intestinal or colonic tissues in Ach release or contractility induced by carbachol or KCl,. EFS caused transient biphasic relaxation and contraction in small intestine and colon, with the colonic contraction being stronger in the PA group. Microscopic tissue analysis showed disruption of the interstitial cells of Cajal (ICC) network and increased lymphocyte infiltration in colonic mucosa and submucosa in PA mice. Conclusions These results indicate that the microbiota is a driver of delayed colonic transit in a patient whose constipation started following extensive antibiotic exposure for C. difficile infection. The observed dysmotility pattern was not due to lower muscle contractility but likely caused by immune mediated changes in the ICC network. Funding Agencies CIHR

Background Major depressive disorder (MDD) affects approximately 4.4% of the global population. Despite its high prevalence, little is known about the mechanisms underlying this disorder. Recent studies in both humans and rodents have suggested that the intestinal microbiota may play a role in depression. Altered microbiota composition has been found in a subset of MDD patients. Preclinical studies have suggested that fecal microbiota transplant using pooled MDD patient samples can induce depressive-like behaviour in rodents. We have previously shown that the use of different microbiota donors with irritable bowel syndrome results in the induction of different phenotypes in recipient mice. Thus, we have hypothesized that pooling microbiota samples abrogates features that are unique to individual donors. Aims (1) Investigate whether the transfer of individual MDD patient microbiota can induce depressive-like behaviour in germ-free (GF) mice (2) Identify features of individual MDD patient microbiota that are associated with the depressive-like phenotype Methods GF NIH Swiss mice of both sexes (min. n=10 per group, total n=110) were colonized with either fecal microbiota from a single donor, MDD patient (MDD1-4) or matched healthy control (HC1-4), or pooled fecal microbiota from MDD1-4 or HC1-4. Mouse behaviour was assessed, using the open field test, three chamber sociability assay, tail suspension test, and sucrose preference test. Stool samples were collected throughout the experiment for 16S rRNA gene sequencing. Results Mice colonized with microbiota from patient MDD1 exhibited depressive-like behaviour, as assessed by the sucrose preference test and sociability assay, when compared to mice colonized with HC1 microbiota. This was not true for mice colonized with individual microbiota from the other three patients (MDD2-4) or with pooled MDD microbiota. Comparative analysis of the 16S data revealed a significant difference in Faith’s Phylogenetic Diversity between MDD1 microbiota and pooled MDD microbiota. Four bacterial species were found to be significantly associated with the depressive-like phenotype in mice: Bacteroides acidifaciens, Bacteroides ovatus, unclassified species of Phascolarctobacterium (Veillonellacae family), and Eggerthella lenta. The relative abundances of these species did not differ significantly between the two pooled groups. Conclusions Microbiota from some, but not all, MDD patients can induce a depressive-like phenotype in GF mice. The ability to induce depressive-like behaviour in GF mice is lost when microbiota from multiple patients is pooled. Specific bacterial species may be responsible for the successful transfer of the depressive-like phenotype to mice. Funding Agencies NIH

Background Abdominal pain is a common complaint in patients with chronic gastrointestinal disorders. Its treatment is of limited efficacy as the pathophysiology is largely unknown. Accumulating evidence suggests that gut microbiota is an important determinant of gut function, including visceral sensitivity. Germ-free (GF) mice have been shown to have altered pain signaling, which normalizes after colonization. Sex also appears to play a key role in visceral sensitivity, as abdominal pain is diagnosed predominantly in female patients. Thus, both gut bacteria and sex are important in the regulation of gut nociception, but the underlying mechanisms remain poorly understood. Aims To investigate the role of gut microbiota and sex in abdominal pain. Methods We used primary cultures of sensory neurons from dorsal root ganglia (DRG) of female and male conventional mice (SPF) or germ-free (GF) mice, age 7–18 weeks. To study the visceral afferent activity in vitro, calcium mobilization in DRG sensory neurons was measured by inverted fluorescence microscope using a fluorescent calcium probe Fluo-4 (1mM). Two parameters were considered: i) percentage of responding neurons and ii) intensity of neuronal response. First, DRG sensory neurons were stimulated by a TRPV1 agonist capsaicin (12.5nM, 125nM and 1.25µM) or by an agonist mix of G-protein coupled receptors (GPCR: bradykinin, histamine and serotonin; 1µM, 10µM and 100µM). We next cultured Escherichia coli JM83 (E. coli) and Enterobacter aerogenes (E. aer) overnight in LB and LDMIIG medium, respectively. Bacterial supernatant of 1010 CFU/mL was diluted to 1% in Krebs Ringer solution to treat DRG neurons from GF/SPF mice and calcium mobilization was measured. Results The percentage of neurons responding to capsaicin and GPCR agonist was similar in SPF male and SPF female. In contrast, the intensity of the neuronal response was higher in SPF male compared to SPF female mice in response to capsaicin (125nM p Conclusions Our data suggest that at the level of DRG neurons from healthy mice, female sex and the absence of gut microbiota do not predispose to visceral hypersensitivity. In fact, the intensity of neuronal responses to capsaicin appear to be higher in DRGs from male mice. Furthermore, we show that metabolites from certain bacteria can activate sensory neurons. Thus, further studies are needed to investigate the role of gut microbiota and sex in visceral sensitivity Funding Agencies CIHR

BACKGROUND: Accumulating evidence suggests that mast cells play an important role in the pathogenesis of Irritable Bowel Syndrome (IBS). Colonic mast cells are increased in patients with IBS, and their products, including histamine and tryptase are putative mediators of visceral hypersensitivity in IBS. AIMS: To investigate the effect of gut microbiota on mast cells in a humanized mouse model of IBS. METHODS: Germ-free NIH Swiss mice were colonized with fecal microbiota from two IBS patients (non-constipation) (n=24 mice/patient), and one healthy control (HC) (n=8). Fecal bacteria and their supernatants were collected from each mouse. Intestinal tissues were taken for immunohistochemistry and RNA scope assay. Mouse bone marrow derived mast cells (BMMC) or peritoneal mast cells (PMC) were obtained from additional mice and co-cultured with 10% bacteria or 10% bacterial supernatant for 4 hours. We studied mast cell degranulation (β-hexosaminidase assay), chemotaxis, adherence, histamine levels (ELISA), and expression of histamine receptors (H1R, H2R, H4R) and chemokines (CXCL12) using real time PCR. RESULTS: Tryptase positive mast cells and tryptase positive area were higher in the colon of IBS mice compared to HC mice. RNA scope demonstrated direct interactions between colonic mast cells and bacteria in mice with IBS but not HC microbiota. Microbial supernatant from IBS mice did not alter β-hexosaminidase and histamine release from BMMC. However, microbiota from IBS mice induced BMMC degranulation compared to microbiota from HC mice. Furthermore, bacterial supernatant from IBS mice increased BMMC chemotaxis and adherence. Expression of H2R, H4R and CXCL12 mRNA was higher in PMC stimulated by bacterial supernatant from IBS mice, compared to those from HC mice. CONCLUSIONS: Microbiota and microbial products from mice with IBS microbiota induce mast cell degranulation, enhance mast cell chemotaxis and adherence, and increase expression of H2R, H4R and CXCL12. Our results suggest that IBS microbiota and their products may induce mast cell infiltration and activation, likely resulting in gut dysfunction and symptom generation. FUNDING AGENCIES: CIHRNIH

BACKGROUND: Wheat-related disorders involve a wide spectrum of conditions, triggered by the ingestion of gluten-containing cereals. The induction of gluten-specific immune responses in celiac disease is well established. However, the contribution of gluten and/or non-gluten proteins in the generation of symptoms in other wheat-related disorders is controversial. Amylase trypsin inhibitors (ATIs) are pest-resistant molecules in modern wheat with TLR4-activating capacities. AIMS: We investigated the role of ATIs in the generation of gut barrier dysfunction and dysmotility in wild-type mice as well as in the severity of gluten-induced immunopathology in genetically predisposed mice. We also determined the metabolic capacity of core microbial groups to degrade ATIs, and examined how this impacts ATIs or gluten-induced gut dysfunction. METHODS: To determine if ATIs could induce gut dysfunction in non-genetically susceptible mice, C57BL/6 or Myd88-/- mice were placed on a wheat free diet (WFD) or ATI diet for 1 week. To study the adjuvant effect of ATIs, NOD/DQ8 mice sensitized with cholera toxin and gliadin received a diet containing: 1) ATIs with no gluten (ATIs), 2) gluten depleted of ATIs (G-ATIs), or 3) depleted gluten supplemented with ATIs (G+ATIs), for 2 weeks. Non-sensitized NOD/DQ8 (controls) received a WFD for 2 weeks. To study the metabolic capacity of microbes to degrade ATIs in vivo, Lactobacillus strains isolated from the human intestine and with different hydrolytic capacity against ATI were pooled and supplemented to mice by oral gavage daily during dietary intervention. CD3(+) intraepithelial lymphocytes (IELs) were measured by immunohistochemistry, intestinal permeability by Ussing chambers, gene expression by Nanostring Technology and 16S rRNA profiling of cecal microbial content via Illumina MiSeq. RESULTS: We show that ATIs not only induce innate immune responses through the TLR4-MD2-CD14 pathway, but also barrier dysfunction in C57BL/6 mice in the absence of overt mucosal damage. When combined with gluten in mice with a permissive celiac genetic background (NOD-DQ8 transgenic), ATI exacerbate gluten-induced immunopathology increasing intestinal permeability, IEL proliferation and upregulation of pro-inflammatory genes. We also demonstrate that ATIs can be degraded by certain commensal bacteria such as Lactobacillus, thereby reducing their immune-stimulatory capacity in mouse models. CONCLUSIONS: ATIs may play a role in wheat-related disorders. Microbiome-modulating strategies based on the use of strains with specific ATIs-degrading capacity may be effective in patients with wheat-sensitive disorders. FUNDING AGENCIES: CAG, CIHR

BACKGROUND: Anxiety disorders are the most prevalent psychiatric conditions within primary care, affecting up to 29% of people across their lifetime. Generalized Anxiety disorder (GAD) is frequently comorbid with major depressive disorder, resulting in greater functional impairment. Gut microbiota have been shown to modulate brain chemistry and function, possibly also playing a role in the genesis of anxiety. Bacteria can produce, or interact with the host metabolism of neuroactive substances, including classical neurotransmitters and trace amines, like octopamine, which despite found in trace concentrations in the mammalian brain, can significantly affect CNS function. Trace amines alter catecholamine release, reuptake and biosynthesis, and modulate dopamine and serotonin metabolism. AIMS: We investigated whether microbiota from patients with GAD and an absence of immune activation can induce altered behaviour in gnotobiotic mice and whether this is accompanied by changes in trace amines levels. METHODS: Germ-free NIH Swiss mice (n=34) were colonized with microbiota from either a GAD patient (n=17) with moderate anxiety, comorbid depression, and low serum and fecal octopamine, or an age and sex-matched healthy control (HC) (n=17). Immune activation/inflammation profiles of human donors was assessed from tissue and plasma cytokines, CRP and β-defensin-3 levels. Microbiota profiles were assessed via 16S rRNA based Illumina. Three weeks post-colonization, mouse behaviour was assessed by standard psychometric tests. Emotionality z-scores were calculated to provide a robust integrated behavioural assessment. Fecal b-defensin-3 was measured via ELISA. After sacrifice, brain BDNF and GDNF expression will be assessed by immunofluorescence, and gene expression by qPCR. RESULTS: There were no differences in fecal b-defensin levels between mice with GAD and HC microbiota. However, GAD mice exhibited greater anxiety and depressive-like behavior compared to HC mice in the digging, marble-burying, step-down and tail suspensions tests. Behavioural z-scoring across all six standard psychometric tests showed a significant increase in group emotionality score means of GAD-colonized mice compared to HC-colonized mice (p= 0.02; Figure 1). CONCLUSIONS: Our results suggest that GAD microbiota has the ability to induce anxiety and depressive-like behavior, by mechanisms independent of the immune system, likely by altered production of trace amines. This needs to be confirmed however by full metabolomics analysis of stool and tissues samples, which is currently underway. Behavioural z-scoring across all six standardized psychometric tests showed a significant increase in group emotionality score means of GAD-colonized mice compared to HC-colonized mice (p= 0.02) FUNDING AGENCIES: NIH

Background & Aims Wheat-related disorders, a spectrum of conditions induced by the ingestion of gluten-containing cereals, have been increasing in prevalence. Patients with celiac disease have gluten-specific immune responses, but the contribution of non-gluten proteins to symptoms in patients with celiac disease or other wheat-related disorders is controversial. Methods C57BL/6 (control), Myd88–/–, Ticam1–/–, and Il15–/– mice were placed on diets that lacked wheat or gluten, with or without wheat amylase trypsin inhibitors (ATIs), for 1 week. Small intestine tissues were collected and intestinal intraepithelial lymphocytes (IELs) were measured; we also investigated gut permeability and intestinal transit. Control mice fed ATIs for 1 week were gavaged daily with Lactobacillus strains that had high or low ATI-degrading capacity. Nonobese diabetic/DQ8 mice were sensitized to gluten and fed an ATI diet, a gluten-containing diet or a diet with ATIs and gluten for 2 weeks. Mice were also treated with Lactobacillus strains that had high or low ATI-degrading capacity. Intestinal tissues were collected and IELs, gene expression, gut permeability and intestinal microbiota profiles were measured. Results In intestinal tissues from control mice, ATIs induced an innate immune response by activation of Toll-like receptor 4 signaling to MD2 and CD14, and caused barrier dysfunction in the absence of mucosal damage. Administration of ATIs to gluten-sensitized mice expressing HLA-DQ8 increased intestinal inflammation in response to gluten in the diet. We found ATIs to be degraded by Lactobacillus, which reduced the inflammatory effects of ATIs. Conclusions ATIs mediate wheat-induced intestinal dysfunction in wild-type mice and exacerbate inflammation to gluten in susceptible mice. Microbiome-modulating strategies, such as administration of bacteria with ATI-degrading capacity, may be effective in patients with wheat-sensitive disorders.

BACKGROUND: The mechanisms underlying IBS are poorly understood but growing evidence suggests that immune activation and the microbiota may be involved. We recently demonstrated that a diet low in fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAP) improves symptoms in IBS patients and this is correlated with decreased mast cell activation and changes in the microbiota composition. Thus, FODMAPs may be a useful tool to evaluate IBS mechanisms. AIMS: To investigate the interplay of host’s gut function, intestinal microbiota and immune factors in response to FODMAPs in the humanized IBS mouse model. METHODS: Germ-free NIH Swiss mice were colonized with fecal microbiota from two IBS patients (non-constipation) with a high (P1) or low (P2) urinary histamine level (n=20 mice /patient), suggesting differing levels of immune activation. Mice were assigned to a custom-made low or high FODMAP diet (LF and HF, respectively). Three weeks later, GI transit (beads study), cecal volume (CT scan) and intestinal permeability (FITC-Dextran) were assessed. Animals were then euthanized and neuronal excitability assessed by patch clamp recordings of DRG neurons (action potential rheobase) exposed to colonic supernatants and changes in mechanosensitivity of single unit afferent recordings in mouse distal colon from mice receiving LF or HF diets. RESULTS: P1 mice receiving a HF diet had slower GI transit (p=0.02), increased cecal volume (p=0.004), indicative of increased gas content, increased permeability (FITC-Dextran: p=0.04), and lower weight gain (p=0.04) compared to P1 mice on a LF diet. In electrophysiological studies, the rheobase was decreased (24%; p=0.003) and mechanosensitivy increased (p=0.06) in the HF compared to the LF mice. The decreased rheobase was blocked by a H1 or a PAR2 antagonist. In contrast, P2 mice receiving the HF diet displayed modest increase in cecal volume (p=0.02) but no differences in GI transit, permeability or weight compared to those on the LF diet. The rheobase was modestly decreased (17%, p

BACKGROUND: The detailed mechanisms underlying IBS are poorly understood. Gut microbiota appears to play an important role in IBS pathogenesis, but little is known about its interaction with specific dietary components. We have previously shown that some patients with IBS improve symptoms when on low FODMAPs diet, which was associated with low urinary histamine. AIMS: To investigate mechanisms underlying IBS symptom generation due to the interaction of a high fermentable diet and the intestinal microbiota. METHODS: Germ-free NIH Swiss mice were colonized with fecal microbiota from two IBS patients (non-constipation), with either a high (HH) or low (LH) urinary histamine level (n=24 mice/patient), and one healthy control (HV) (n=8). Specific-pathogen free (SPF) mice (n=8) were included as additional controls. Mice were assigned to a custom-made low or high fermentable carbohydrate diet (LF and HF, respectively). GI transit (beads study), cecal volume (CT scan), intestinal permeability (FITC-Dextran) and gut microbiota composition (Illumina sequencing) were assessed. Neuronal excitability by patch clamp recordings of DRG neurons (action potential rheobase) exposed to colonic supernatants and changes in mechanosensitivity of single unit afferent recordings in mouse distal colon were measured. RESULTS: Diet significantly altered gut microbiota composition and diversity only in HH colonized mice. All mice on HF diet (HV, HH, LH, and SPF) had increased cecal volume (Diet effect: p

BACKGROUND: Patients with inflammatory bowel disease (IBD) frequently suffer from psychiatric comorbidities, mainly anxiety and depression. Infliximab, an anti-TNFα antibody, is commonly used for treatment of colitis. There are anecdotal reports of IBD patients having improvement in mood only hours after infliximab infusion, well before intestinal mucosal healing could occur. AIMS: To evaluate the immediate effects of infliximab infusion on intestinal and extra-intestinal symptoms and immune markers in IBD patients. METHODS: IBD patients attending the McMaster University and Charlton infusion clinics were enrolled in the study. Patients were examined at baseline (day 0), 1 day, and 7 days after the infliximab infusion. We assessed anxiety and depression (HAD and STAI-Y scales), disease specific quality of life (SIDBQ), as well as disease activity scores using the Harvey Bradshaw Index (HBI) or the Mayo Score. Serum C-reactive protein (CRP), kynurenine, tryptophan, and serotonin levels were measured at each visit. RESULTS: We enrolled 41 IBD patients (Crohn’s disease = 35, Ulcerative Colitis = 5, indeterminate colitis = 1; median age 33 yrs, median disease duration 9 yrs). Disease activity decreased at day 1 compared to baseline (p=0.01), but this was not sustained at day 7. Anxiety scores (HADS-A) decreased significantly at day 1 (p=0.006), and remained lower at day 7 (p=0.009). An improvement in quality of life (SIBDQ) was seen only at day 7 (p=0.017). The decrease in anxiety scores strongly correlated with the improvement in quality of life (SIBDQ r=-0.81; p

BACKGROUND: Interactions between microbiota and gastrointestinal (GI) function may be evident from the early stage of life. Microbiota contributes to myenteric nerve development and normal gut function maintenance. Earlier studies suggested that germ-free or antibiotics treated mice have decreased gastrointestinal transit. Also, abnormal gut microbiota is common in patients with altered GI motility, like IBS. However, the underlying mechanism are not fully understood. AIMS: Here, we investigated the role of microbiota in GI motility in vivo and vitro. METHODS: Specific-pathogen-free (SPF) mice and germ-free (GF) mice (12–16 weeks old) were used. Gastric emptying was assessed by videofluoroscopy and GI transit was assessed by SHAPE study using metallic beads, as described previously. Mice were then sacrificed and samples from jejunum, ileum and mid-colon were taken. Isometric contraction was assessed after electric field stimulation (EFS) and pharmacological treatments. Acetylcholine (Ach) release was assessed by superfusion using isotope [(3)H]. Expression of genes related to neuromuscular function was assessed by Illumina, immunostaining was also performed. RESULTS: There were no significant difference in gastric emptying rates between SPF and GF mice, which were 26.4±4.1 % (n=10) and 23.1±3.1 % (n=11), respectively. However, GI transit (total scores) was faster in SPF mice compared to GF mice, with 13.6±1.7 (n=10) and 5.4±0.2 (n=11) respectively. Beads were mainly located in the cecum in SPF mice, but in GF mice, most of them remained in the proximal small intestine. In contractility studies, carbachol induced similar level of contraction in SPF or GF tissues, both in the small bowel and colon. In SPF tissues, the neural stimulation caused a transient contraction in jejunum, and biphasic relaxation and contraction in ileum and colon. In GF tissues, neural stimulation caused similar response in ileum and colon, however, in jejunum, it caused biphasic response with a significant relaxation followed by a contraction. No difference was seen in EFS-induced Ach release between SPF and GF tissues. CONCLUSIONS: Microbiota appears to be essential in the establishment of normal small intestinal motility. Germ-free mice have altered jejunal motility, likely due to a non-cholinergic neural response. Gene expression study and immunostaining are underway to reveal the underlying mechanism. FUNDING AGENCIES: None

BACKGROUND: Dysbiosis is defined as alterations or instability in gut microbial composition resulting in altered host function. It is triggered by many environmental factors and has been implicated in the pathogenesis of the Irritable Bowel Syndrome (IBS). Recent focus on the putative role of intestinal microbiome generating low grade inflammation and immune activation in IBS has increased the need for a marker of dysbiosis. β-defensins (β-def) are inducible antimicrobial peptides secreted by diverse cell types in response to pathogen encounter or pro-inflammatory cytokines. Studies in IBS patients show that dysbiosis results in skewing of innate mucosal immunity toward a proinflammatory response (increased β-def levels) in the absence of macroscopic signs of inflammation. Our lab recently demonstrated that colonizing germ-free mice with IBS microbiota results in IBS-like changes in gut function and increase in colonic β-def expression in recipient mice. AIMS: To investigate whether fecal β-def levels constitute a reliable biomarker of dysbiosis. METHODS: We induced dysbiosis using different protocols including single antibiotic (Neomycin), an antibiotic cocktail (ABC) or a high-fat/high-sugar diet (HF/HSD). Mice (n=20) were divided into 3 groups and all received 1 week of treatment, preceded and followed by 1-week long baseline and recovery periods. The single antibiotic was administered to C57/BL6 ASF mice, the ABC was given to C57/BL6 SPF mice and another group of C57/BL6 SPF mice received HF/HSD. Stool samples were collected every day for 16S rRNA gene profiling, qPCR analysis of total bacterial load and fecal β-def levels analysis by an ELISA. RESULTS: SPF mice receiving the ABC showed a significant decrease in β-def levels (p

Gut immune system, microbiota and diet appear to play important roles in irritable bowel syndrome (IBS). Increased levels of colonic mast cell (MC) located in close proximity to mucosal nerves were reported in IBS patients, correlating with increased abdominal pain. We showed recently that IBS patients’ beneficial response to a low-fermentable diet was associated with changes in gut microbiota and urinary histamine. Here we investigated the interaction between microbiota, diet and MC in a humanized IBS mouse model. Germ-free mice, receiving custom-made low (LF) or high (HF) fermentable diets, were colonized with fecal microbiota from two IBS patients, with high (HH) or low (LH) urinary histamine (n = 24 mice/patient), and one healthy control (n = 8). HH mice on HF-diet (HHHF) had increased c-kit + MCs in both jejunum and colon compared to HH mice on LF-diet (HHLF), which was associated with increased visceral sensitivity assessed by electrophysiology. Colonic MC number in HHHF mice negatively correlated with the relative abundance of several bacterial genera. Tryptase + area and MCs were higher in the jejunum of HHHF compared to HHLF mice. Furthermore, most MCs were in close proximity to PGP9.5 + nerve fibers. In the jejunum, the density of PGP9.5 + and substance P + nerves was higher in HHHF mice. In summary, MCs may play a key role in host-microbiota-diet interactions in a subset of IBS patients, contributing to increased abdominal pain perception.

Background Changes in hygiene and dietary habits, including increased consumption of foods high in fat, simple sugars, and salt that are known to impact the composition and function of the intestinal microbiota, may explain the increase in prevalence of chronic inflammatory diseases. High salt consumption has been shown to worsen autoimmune encephalomyelitis and colitis in mouse models through p38/MAPK signaling pathway. However, the effect of high salt diet (HSD) on gut microbiota and on intestinal immune homeostasis, and their roles in determining vulnerability to intestinal inflammatory stimuli are unknown. Here, we investigate the role of gut microbiota alterations induced by HSD on the severity of murine experimental colitis. Results Compared to control diet, HSD altered fecal microbiota composition and function, reducing Lactobacillus sp. relative abundance and butyrate production. Moreover, HSD affected the colonic, and to a lesser extent small intestine mucosal immunity by enhancing the expression of pro-inflammatory genes such as Rac1, Map2k1, Map2k6, Atf2, while suppressing many cytokine and chemokine genes, such as Ccl3, Ccl4, Cxcl2, Cxcr4, Ccr7. Conventionally raised mice fed with HSD developed more severe DSS- (dextran sodium sulfate) and DNBS- (dinitrobenzene sulfonic acid) induced colitis compared to mice on control diet, and this effect was absent in germ-free mice. Transfer experiments into germ-free mice indicated that the HSD-associated microbiota profile is critically dependent on continued exposure to dietary salt. Conclusions Our results indicate that the exacerbation of colitis induced by HSD is associated with reduction in Lactobacillus sp. and protective short-chain fatty acid production, as well as changes in host immune status. We hypothesize that these changes alter gut immune homeostasis and lead to increased vulnerability to inflammatory insults. Electronic supplementary material The online version of this article (10.1186/s40168-018-0433-4) contains supplementary material, which is available to authorized users.

Irritable bowel syndrome (IBS) is a common disorder characterized by altered gut function and often is accompanied by comorbid anxiety. Although changes in the gut microbiota have been documented, their relevance to the clinical expression of IBS is unknown. To evaluate a functional role for commensal gut bacteria in IBS, we colonized germ-free mice with the fecal microbiota from healthy control individuals or IBS patients with diarrhea (IBS-D), with or without anxiety, and monitored gut function and behavior in the transplanted mice. Microbiota profiles in recipient mice clustered according to the microbiota profiles of the human donors. Mice receiving the IBS-D fecal microbiota showed a taxonomically similar microbial composition to that of mice receiving the healthy control fecal microbiota. However, IBS-D mice showed different serum metabolomic profiles. Mice receiving the IBS-D fecal microbiota, but not the healthy control fecal microbiota, exhibited faster gastrointestinal transit, intestinal barrier dysfunction, innate immune activation, and anxiety-like behavior. These results indicate the potential of the gut microbiota to contribute to both intestinal and behavioral manifestations of IBS-D and suggest the potential value of microbiota-directed therapies in IBS patients.

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.

Cognitive decline in elderly people often derives from the interaction between aging-related changes and age-related diseases and covers a large spectrum of clinical manifestations, from intact cognition through mild cognitive impairment and dementia. Epidemiological evidence supports the hypothesis that modifiable lifestyle-related factors are associated with cognitive decline, opening new avenues for prevention. Diet in particular has become the object of intense research in relation to cognitive aging and neurodegenerative disease. We reviewed the most recent findings in this rapidly expanding field. Some nutrients, such as vitamins and fatty acids, have been studied longer than others, but strong scientific evidence of an association is lacking even for these compounds. Specific dietary patterns, like the Mediterranean diet, may be more beneficial than a high consumption of single nutrients or specific food items. A strong link between vascular risk factors and dementia has been shown, and the association of diet with several vascular and metabolic diseases is well known. Other plausible mechanisms underlying the relationship between diet and cognitive decline, such as inflammation and oxidative stress, have been established. In addition to the traditional etiological pathways, new hypotheses, such as the role of the intestinal microbiome in cognitive function, have been suggested and warrant further investigation. AuthorCount:4

BACKGROUND: Depression and anxiety have overlapping etiologies and their pathophysiology remains largely unknown. Accumulating data suggest that gut microbiota modulates brain function and behaviour and may play a role in the pathophysiology of depression and anxiety AIMS: To investigate whether gut microbiota from patients with Generalized Anxiety Disorder (GAD) induces anxiety-like behaviour in gnotobiotic mice and whether this is accompanied by changes in brain chemistry and immune markers. METHODS: Germ-free NIH Swiss mice (n=15) were colonized with microbiota from either a patient with GAD and history of depression (n=7) or from an age and sex-matched healthy control (HC) (n=8). Both the GAD patient (female, 19 years) and healthy control (female, 20 years) were well characterized and selected based on their Depression, Anxiety, and Stress Scale (DASS) scores from a pool of participants of an ongoing clinical study. Three weeks post-colonization, the mice underwent behavioural assessment using standard psychometric tests. Stool β-defensin levels were measured by ELISA. Microbiota profiles were assessed by 16S rRNA based Illumina analysis. Lastly, BDNF expression in brain samples was measured by immunofluorescence and neural gene expression by Nanostring gene assay. RESULTS: GAD-colonized mice had a distinct microbiota profile from that of HC-colonized mice and each group clustered around their respective human donor. Fecal β-defensin levels were higher in the GAD patient than the HC (109.5ng/ml vs 18.01ng/ml). Similarly, mouse fecal β-defensin levels were significantly higher in GAD-colonized mice than in HC-colonized mice (M= 62.26 vs M= 29.10, p < 0.005). Mice colonized with GAD microbiota exhibited significantly greater anxiety and depressive-like behaviour than HC-colonized mice, as they spent less time in the center of the open field arena, buried a greater number of marbles, spent a greater time digging and remained more time immobile. GAD-colonized mice had increased BDNF expression in the amygdala but decreased expression in the hippocampus. GAD-colonized mice also exhibited differential expression of neural genes in both the amygdala and hippocampus. CONCLUSIONS: Our results suggest that GAD microbiota can induce anxiety and depressive-like behaviour, accompanied by elevated immune markers. This is associated with altered neural gene and BDNF expression in brain regions involved in emotional processing. Altogether, our data suggest that gut microbiota may contribute to the pathophysiology of anxiety and depression, at least in a subset of patients with signs of gut immune activation. These findings may lead to the development of novel biomarkers and treatments for mood disorders. FUNDING AGENCIES: CIHRNIH

BACKGROUND: Growing evidence suggests that immune activation and gut microbiota are involved in the pathophysiology of IBS. We have recently demonstrated that a diet low in fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAP) improved symptoms in IBS patients and this was associated with lower urinary histamine and changes in the microbiota composition. A high FODMAP diet in germ-free mice colonized with IBS patient microbiome modulated motility, permeability, and visceral sensitivity. However, the underlying mechanisms are unclear. AIMS: To investigate the interactions of host’s immune system and intestinal microbiota in response to FODMAPs in the humanized IBS mouse model. METHODS: Germ-free NIH Swiss mice were colonized with fecal microbiota from two IBS patients (non-constipation) with a high (HH) or low (LH) urinary histamine (n=24 mice/patient), and one healthy control (HV) (n=8). SPF mice (n=8) were included as additional controls. Mice were assigned to a custom-made low or high FODMAP diet (LF and HF, respectively). Microbiota composition was analysed by 16S rRNA gene sequencing. At sacrifice, intestinal tissues were collected for immunohistochemistry (c-kit staining) and immunofluorescence (tryptase, PGP9.5, and Substance P) detection of mast cell and nerves. RESULTS: HH colonized mice receiving HF diet had increased number of mast cells (c-kit positive cells) in both small intestine and colon compared to HH mice on a LF diet (Effect of diet x microbiota: p=0.041, F(1,32)=4.51 for small intestine, and p=0.029, F(1,30)=5.25 for colon). No differences were observed in the colon and small intestine of LH colonized mice. Immunofluorescent staining of colonic and small intestinal sections revealed high number of tryptase positive mast cells in HH colonized mice on HF diet. Moreover, the majority of the mast cells were in close proximity of nerve fibers (PGP9.5 positive) (Fig 1). In HH colonized mice, colonic mast cell numbers were negatively correlated with the relative abundance of Adlercreutzia spp, Oscillospira and Ruminococcus spp. Similarly, in the small intestine of HH colonized mice, mast cell numbers negatively correlated with the relative abundance of Blautia, Ruminococcus, and Lactobacillus spp. CONCLUSIONS: A high fermentable diet alters intestinal microbiota composition only in mice colonized with microbiota from a patient with high urinary histamine. These changes are associated with increased numbers of mast cells in both the colon and small intestine. Furthermore, mast cells seem to closely interact with neurons, likely contributing to the functional changes observed in these mice. FUNDING AGENCIES: CIHRNIH