Enteric Microbiota Research Articles

Stress-related IBS

The gastrointestinal tract is exquisitely sensitive to different physical and psychological stressors. Irritable bowel syndrome (IBS) may be viewed as a disorder caused by stress-induced dysregulation of the complex interactions along the brain-gut-microbiota axis, which involves the bidirectional, self-perpetuating communication between the central and enteric nervous systems, utilising autonomic, psychoneuroendocrine, pain modulatory and immune signalling pathways. An overzealous stress response may significantly alter not only the sensitivity of the central and enteric nervous systems, but also other potentially important factors such as gut motility, intestinal mucosal permeability and barrier functioning, visceral sensitivity, mucosal blood flow, immune cell reactivity and enteric microbiota composition. Symptoms of these (mal)adaptive changes may include constipation, diarrhoea, bloating and abdominal pain, manifesting clinically as IBS. This article briefly reviews the current postulated stress-models of IBS.

Tu1800 Action Mechanism of Methane Gas Produced by Enteric Microbiota on Ileal Motor Function

Tu1800 Action Mechanism of Methane Gas Produced by Enteric Microbiota on Ileal Motor Function

O-004 Analysis of Chromatin and Transcriptional Profiles in Crohnʼs Disease Reveals Molecular Subclasses and Highlights Functional Regulatory Regions Implicated in Disease

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract, characterized by an inappropriate immune response to the enteric microbiota. Chromatin based assays have successfully identified cell type and disease-specific regions of DNA accessibility, and have been linked to differential transcription of target genes, but this approach has yet to be explored in CD. In this study we performed formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) to identify nucleosome-depleted regions associated with regulatory elements, and RNA-seq to quantify expression, to characterize disease-relevant changes in chromatin and establish links to putative target genes.Mucosal Biopsies for 21 CD and 12 non-IBD individuals were taken from macroscopically uninflamed portions of the ascending colon at time of surgery. We performed FAIRE-seq and RNA-seq on each tissue sample. Reads were aligned to personalized genomes constructed with genotypes assayed on the Illumina Immunochip platform. A genome-wide scan for differential regions and genes was performed using 2 sided t-tests on normalized read counts in 300bp regions (FAIRE-seq) and RPKM (RNA-seq).RNA-seq data analysis across all samples identified 2 distinct classes of patients associated with epithelial cell lipid transport (class I, n = 10, CD; n = 1, non-IBD) and maintenance of cellular and luminal pH (class II, n = 11, CD, n = 11, non-IBD). To unravel potential gene regulatory mechanisms we analyzed chromatin profiles using FAIRE-seq and histone annotations using tissue specific ChIP-seq data sets. Class I patients showed increased accessibility at distal sites marked for enhancer activity in small intestine, and reduced accessibility at sites marked as enhancers in colon, suggesting 2 distinct regulatory programs driving the expression of differential genes. This pattern was reversed in class II samples. In an analysis restricting to class II, which was characterized by normal colon enhancer activity, we found regulatory regions specific to CD and non-IBD, respectively, as well as 51 and 507 genes upregulated in the respective cohorts. Top ontologies of genes upregulated in CD included host response to bacteria and immune response. Differential regulatory genes were enriched near TSSs of differentially expressed genes, implying putative causal effect. In an integrative analysis, we identified 72 regulatory regions that associate with both disease status and expression of nearby genes. A retrospective analysis of patients in class I and II revealed that the majority of patients (10/11) in class I required post-operative anti-TNF therapy for disease recurrence compared to only 1/11 CD patients in the class II group.We used chromatin status as a novel marker of disease in Crohn's. We identified a subset of CD patients that are characterized at the chromatin level in the colon by enrichment of enhancer marks specific to small intestine, and loss of enhancer activity specific to colon. Among individuals with enhancer activity indicative of normal colon, we integrated data from well-described transcriptional markers with novel chromatin variables to identify molecular associations and interactions that affect disease predisposition. Despite small patient numbers we were able to use chromatin profiles to identify patients that required post-operative management with anti-TNF agents.

Crohn's Disease: Evolution, Epigenetics, and the Emerging Role of Microbiome-Targeted Therapies.

Crohn's disease (CD) is a chronic, systemic, immune-mediated inflammation of the gastrointestinal tract. Originally described in 1932 as non-caseating granulomatous inflammation limited to the terminal ileum, it is now recognized as an expanding group of heterogeneous diseases defined by intestinal location, extent, behavior, and systemic extraintestinal manifestations. Joint diseases, including inflammatory spondyloarthritis and ankylosing spondylitis, are the most common extraintestinal manifestations of CD and share more genetic susceptibility loci than any other inflammatory bowel disease (IBD) trait. The high frequency and overlap with genes associated with infectious diseases, specifically Mendelian susceptibility to mycobacterial diseases (MSMD), suggest that CD may represent an evolutionary adaptation to environmental microbes. Elucidating the diversity of the enteric microbiota and the protean mucosal immune responses in individuals may personalize microbiome-targeted therapies and molecular classifications of CD. This review will focus on CD's natural history and therapies in the context of epigenetics, immunogenetics, and the microbiome.

Determination of American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

American ginseng is a commonly consumed herbal medicine in the United States and other countries. Ginseng saponins are considered to be its active constituents. We have previously demonstrated in an in vitro experiment that human enteric microbiota metabolize ginseng parent compounds into their metabolites. In this study, we analyzed American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Six healthy male volunteers ingested 1 g of American ginseng twice a day for 7 days. On day 7, biological samples were obtained and pretreated with solid phase extraction. The ginseng constituents and their metabolites were characterized, including 5 ginseng metabolites in plasma, 10 in urine, and 26 in feces. For the plasma, urine and feces samples, the levels of ginsenoside Rb1 (a major parent compound) were 8.6, 56.8 and 57.7 ng/mL, respectively, and the levels of compound K (a major metabolite) were 58.4 ng/mL, 109.8 ng/mL and 10.06 μg/mL, respectively. It suggested that compound K had a remarkably high level in all three samples. Moreover, in human feces, ginsenoside Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 were detected as the products of dehydration. Further studies are needed to evaluate the pharmacological activities of the identified ginseng metabolites.

Detection of Genotipically Related Multi-resistant Escherichia coli Isolates in Pig Feces and Carcasses

Background: Antimicrobial resistant bacteria are considered a hazard not only for the treatment of animal diseases but also for public health. Commensal bacteria, such as Escherichia coli are considered a good indicator of antimicrobial resistance in the population, because it is a gut inhabitant and thus undergoes constant pressure of selection by the administration of antimicrobials. Regarding the public health, it is important to evaluate if resistant bacteria carried in the intestinal content of slaughter pigs can be found on the surface of pre chill carcasses. Therefore, the aims of this study were to evaluate the frequency of antimicrobial resistance in E. coli isolated from feces and pig carcasses; and to assess if multi-resistant isolates from both sources were phenotypically and genotypically related.Materials, Methods & Results: Two sampling cycles were conducted in three pig slaughterhouses (A, B and C). In each cycle, samples were collected form: i. feces deposited on the pen floor of the lairage; ii. surface of carcasses at the prechill step. Samples were submitted to a protocol of isolation and confirmation of Escherichia coli. Isolates were grouped according to the origin: feces (n = 355); carcasses (n = 319); and evaluated for antimicrobial resistance by agar diffusion test. Ninety two isolates presenting multidrug resistance profile were analyzed by pulsed-field gel eletrophoresis (PFGE). Among the 674 isolates of E. coli, 7.4% were susceptible to all tested antibiotics while 79.5% (536/674) were multi-resistant. The most frequent resistance patterns were displayed to tetracycline (Tet, 85.9%), ampicillin (Amp, 73.0%), sulfonamide (Sul, 70.0%), florfenicol (Flo, 65.0%) and nalidixic acid (Nal, 58.9%). The most frequent multi-resistance profile among isolates from both origins was [AmpFloNalSulTet]. Multiresistant isolates originated from feces and carcasses displaying genotypically related pulsotypes (≥70% similarity) were found in all three slaughterhouses.Discussion: In agreement with other studies, E. coli isolated from pig feces and carcasses demonstrated a high frequency of antimicrobial resistance and multi-resistance. The most frequent resistance profiles included antimicrobials frequently used on farm as well as drugs that have been banned as feed additives some years ago in Brazil. The selection of resistant strains may be related to the selection pressiondue to the use of antimicrobials in the pig production chain as well as the co-selection of resistance mediated by genes located in common genetic elements. Therefore, the ban of an individual drug is not always associated with the immediate disappearance of the resistance phenotype in the bacteria population. The fact that most multi-resistant E. coli isolates from carcasses belonged to pulsotypes related to those originated from feces samples indicates that resistant E. coli isolates selected on farm may be able to survive the slaughter process and be found on the carcass. In this case, the possibility of those strains being able to reach the population through the consumption of pork products may have to be considered. This hazard has motivated the ban of antimicrobial use in animals in some countries. However, the ban of antimicrobials use on farm is a controversial issue, due to the economical losses that may result from this measure. Therefore, the prudent use of antimicrobials on farm should be encouraged and its influence in the multi-resistance profile of the enteric microbiota should be further studied.

The Influence of Prebiotics on Neurobiology and Behavior.

Manipulating the intestinal microbiota for the benefit of the brain is a concept that has become widely acknowledged. Prebiotics are nondigestible nutrients (i.e., fibers, carbohydrates, or various saccharides) that proliferate intrinsic, beneficial gut bacteria, and so provide an alternative strategy for effectively altering the enteric ecosystem, and thence brain function. Rodent studies demonstrating neurobiological changes following prebiotic intake are slowly emerging, and have thus far revealed significant benefits in disease models, including antiinflammatory and neuroprotective actions. There are also compelling data showing the robust and favorable effects of prebiotics on several behavioral paradigms including, anxiety, learning, and memory. At present, studies in humans are limited, though there is strong evidence for prebiotics modulating emotional processes and the neuroendocrine stress response that may underlie the pathophysiology of anxiety. While the mechanistic details linking the enteric microbiota to the central nervous system remain to be elucidated, there are a number of considerations that can guide future studies. These include the modulation of intestinal endocrine systems and inflammatory cascades, as well as direct interaction with the enteric nervous system and gut mucosa. Our knowledge of gut microbiome-brain communication is steadily progressing, and thorough investigations validating the use of prebiotics in the treatment of neuropsychiatric disorders would be highly valued and are encouraged.

The microbiota and gut-brain axis

The ability of gut microbiota to communicate with the brain and hence modulate behavior is an emerging novel concept in health and disease. The enteric microbiota interacts with the host to form essential relationships that govern homeostasis. Although enteric bacterial fingerprint of each individual is quite unique, there appears to be a certain balance that confers individual’s health benefits. A developing number of studies demonstrated that the microbiome of the human digestive tract might have had an effect on the elements of the focal anxious framework (CNS), through recognized pathways called the gut–brain axis. Recent data showed that the human microbiome ecosystem interfered with the brain’s development, central signaling systems, and behavior. It has been proposed that the disruption of the human microbiome may contribute to the etiology and course of some psychiatric disorders. Therefore, a decrease in the desirable gastrointestinal bacteria would lead to deterioration in gastrointestinal, neuroendocrine, immune functioning and consequently an illness. This review article presents an overview about the main pathways of the gut-brain axis and consequences of stress to the individual components.

Obeticholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats

In advanced cirrhosis, gut bacterial translocation is the consequence of intestinal barrier disruption and leads to bacterial infection. Bile acid abnormalities in cirrhosis could play a role in the integrity of the intestinal barrier and the control of microbiota, mainly through the farnesoid X receptor. We investigated the long-term effects of the farnesoid X receptor agonist, obeticholic acid, on gut bacterial translocation, intestinal microbiota composition, barrier integrity and inflammation in rats with CCl4-induced cirrhosis with ascites. Cirrhotic rats received a 2-week course of obeticholic acid or vehicle starting once ascites developed. We then determined: bacterial translocation by mesenteric lymph node culture, ileum expression of antimicrobial peptides and tight junction proteins by qPCR, fecal albumin loss, enteric bacterial load and microbiota composition by qPCR and pyrosequencing of ileum mucosa-attached contents, and intestinal inflammation by cytometry of the inflammatory infiltrate. Obeticholic acid reduced bacterial translocation from 78.3% to 33.3% (p<0.01) and upregulated the expression of the farnesoid X receptor-associated gene small heterodimer partner. Treatment improved ileum expression of antimicrobial peptides, angiogenin-1 and alpha-5-defensin, tight junction proteins zonulin-1 and occludin, and reduced fecal albumin loss and liver fibrosis. Enteric bacterial load normalized, and the distinctive mucosal microbiota of cirrhosis was reduced. Gut immune cell infiltration was reduced and inflammatory cytokine and Toll-like receptor 4 expression normalized. In ascitic cirrhotic rats, obeticholic acid reduces gut bacterial translocation via several complementary mechanisms at the intestinal level. This agent could be used as an alternative to antibiotics to prevent bacterial infection in cirrhosis.

Paneth cell α-defensins and enteric microbiota in health and disease.

Antimicrobial peptides are major effectors of innate immunity of multicellular organisms including humans and play a critical role in host defense, and their importance is widely recognized. The epithelium of the intestine is the largest surface area exposed to the outer environment, including pathogens, toxins and foods. The Paneth cell lineage of intestinal epithelial cells produces and secretes α-defensin antimicrobial peptides and functions in innate enteric immunity by removing pathogens and living symbiotically with commensal microbiota to contribute to intestinal homeostasis. Paneth cells secrete α-defensins, HD5 and HD6 in humans and cryptdins in mice, in response to bacterial, cholinergic and other stimuli. The α-defensins have selective activities against bacteria, eliciting potent microbicidal activities against pathogenic bacteria but minimal or no bactericidal activity against commensal bacteria. Therefore, α-defensins regulate the composition of the intestinal microbiota in vivo and play a role in homeostasis of the entire intestine. Recently, relationships between dysbiosis, or abnormal composition of the intestinal microbiota, and diseases such as inflammatory bowel disease and lifestyle diseases including obesity and atherosclerosis have been reported. Because α-defensins regulate the composition of the intestinal microbiota, Paneth cells and their α-defensins may have a key role as one mechanism linking the microbiota and disease.

O USO DA MICROBIOLOGIA COMO FERRAMENTA PARA A CONSERVAÇÃO DE AVES AMEAÇADAS: DADOS PRELIMINARES PARA O PAPAGAIO-DE-CARA-ROXA, Amazona brasiliensis (Aves: Psittacidae) NO PARANÁ

Amazona brasiliensis, também conhecido popularmente como papagaio-de-cara-roxa, é uma espécie de Psittacidae endêmica da Mata Atlântica distribuída entre o litoral sul de São Paulo e o litoral Norte de Santa Catarina. Este estudo foi concentrado no estado do Paraná, visando uma prévia caracterização do perfil sanitário natural em filhotes da população por meio de análises microbiológicas. O estudo epidemiológico de uma espécie ameaçada inicia-se com a determinação dos agentes infecciosos comuns na população, que provavelmente co-evoluíram com a espécie e representam baixo risco aos espécimes de vida livre. Do total de colônias isoladas quase 75% foram positivas para a família Enterobacteriaceae. Normalmente, a microbiota entérica de psitacídeos é composta por micro-organismos Gram-positivos, porém a alta porcentagem de Gram-negativas isoladas pode ser explicada pela diferença entre a composição microbiana de adultos e filhotes. Seria interessante um estudo mais detalhado para uma eventual comparação entre possíveis sinais clínicos e micro-organismos presentes em cada indivíduo amostrado.

Prebiotic administration normalizes lipopolysaccharide (LPS)-induced anxiety and cortical 5-HT2A receptor and IL1-β levels in male mice

The manipulation of the enteric microbiota with specific prebiotics and probiotics, has been shown to reduce the host’s inflammatory response, alter brain chemistry, and modulate anxiety behaviour in both rodents and humans. However, the neuro-immune and behavioural effects of prebiotics on sickness behaviour have not been explored. Here, adult male CD1 mice were fed with a specific mix of non-digestible galacto-oligosaccharides (Bimuno®, BGOS) for 3weeks, before receiving a single injection of lipopolysaccharide (LPS), which induces sickness behaviour and anxiety. Locomotor and marble burying activities were assessed 4h after LPS injection, and after 24h, anxiety in the light–dark box was assessed. Cytokine expression, and key components of the serotonergic (5-Hydroxytryptamine, 5-HT) and glutamatergic system were evaluated in the frontal cortex to determine the impact of BGOS administration at a molecular level. BGOS-fed mice were less anxious in the light–dark box compared to controls 24h after the LPS injection. Elevated cortical IL-1β concentrations in control mice 28h after LPS were not observed in BGOS-fed animals. This significant BGOS×LPS interaction was also observed for 5HT2A receptors, but not for 5HT1A receptors, 5HT, 5HIAA, NMDA receptor subunits, or other cytokines. The intake of BGOS did not influence LPS-mediated reductions in marble burying behaviour, and its effect on locomotor activity was equivocal. Together, our data show that the prebiotic BGOS has an anxiolytic effect, which may be related to the modulation of cortical IL-1β and 5-HT2A receptor expression. Our data suggest a potential role for prebiotics in the treatment of neuropsychiatric disorders where anxiety and neuroinflammation are prominent clinical features.

Colon cancer chemopreventive effects of baicalein, an active enteric microbiome metabolite from baicalin.

Baicalin is a major constituent of Scutellaria baicalensis, which is a commonly used herbal medicine in many Asian countries. After oral ingestion, intestinal micro-biota metabolism may change parent compound's structure and its biological activities. However, whether baicalin can be metabolized by enteric microbiota and the related anti-cancer activity is not clear. In this study, using human enteric microbiome incubation and HPLC analysis, we observed that baicalin can be quickly converted to baicalein. We compared the antiproliferative effects of baicalin and baicalein using a panel of human cancer cell lines, including three human colorectal cancer (CRC) cell lines. In vitro antiproliferative effects on CRC cells were verified using an in vivo xenograft nude mouse model. Baicalin showed limited antiproliferative effects on some of these cancer cell lines. Baicalein, however, showed significant antiproliferative effects in all the tested cancer cell lines, especially on HCT-116 human colorectal cancer cells. In vivo antitumor results supported our in vitro data. We demonstrated that baicalein exerts potent S phase cell cycle arrest and pro-apoptotic effects in HCT-116 cells. Baicalein induced the activation of caspase 3 and 9. The in silico modeling suggested that baicalein forms hydrogen bonds with residues Ser251 and Asp253 at the active site of caspase 3, while interactions with residues Leu227 and Asp228 in caspase 9 through its hydroxyl groups. Data from this study suggested that baicalein is a potent anticancer metabolite derived from S. baicalensis. Enteric microbiota play a key role in the colon cancer chemoprevention of S. baicalensis.

Osteopontin Deficiency Accelerates Spontaneous Colitis in Mice with Disrupted Gut Microbiota and Macrophage Phagocytic Activity.

BackgroundOsteopontin (OPN) is a multifunctional protein expressed in a variety of tissues and cells. Recent studies revealed increased OPN expression in the inflamed intestinal tissues of patients with inflammatory bowel disease (IBD). The role of OPN in the pathophysiology of IBD, however, remains unclear.AimsTo investigate the role of OPN in the development of intestinal inflammation using a murine model of IBD, interleukin-10 knock out (IL-10 KO) mice.MethodsWe compared the development of colitis between IL-10 KO and OPN/IL-10 double KO (DKO) mice. OPN expression in the colonic tissues of IL-10 KO mice was examined by fluorescence in situ hybridization (FISH) analysis. Enteric microbiota were compared between IL-10 KO and OPN/IL-10 DKO mice by terminal restriction fragment length polymorphism analysis. The effect of OPN on macrophage phagocytic function was evaluated by phagocytosis assay.ResultsOPN/IL-10 DKO mice had an accelerated onset of colitis compared to IL-10 KO mice. FISH analysis revealed enhanced OPN synthesis in the colonic epithelial cells of IL-10 KO mice. OPN/IL-10 DKO mice had a distinctly different enteric bacterial profile with a significantly lower abundance of Clostridium subcluster XIVa and a greater abundance of Clostridium cluster XVIII compared to IL-10 KO mice. Intracellular OPN deletion in macrophages impaired phagocytosis of fluorescence particle-conjugated Escherichia coli in vitro. Exogenous OPN enhanced phagocytosis by OPN-deleted macrophages when administered at doses of 1 to 100 ng/ml, but not 1000 ng/ml.ConclusionsOPN deficiency accelerated the spontaneous development of colitis in mice with disrupted gut microbiota and macrophage phagocytic activity.

A Combination Supplement of Fructo- and Xylo-Oligosaccharides Significantly Abrogates Oxidative Impairments and Neurotoxicity in Maternal/Fetal Milieu Following Gestational Exposure to Acrylamide in Rat.

Prebiotic oligosaccharides are demonstrated to confer a wide spectrum of physiological benefits during pregnancy. In view of this, focused attempts are being directed towards understanding their role as modulators of brain chemistry and behavior. Epidemiological studies have identified that exposure to neurotoxins during prenatal/early life can profoundly impact neurodevelopment/function. In this context, we have tested the hypothesis that a combination of prebiotic supplements during gestation has the propensity to attenuate acrylamide (ACR) induced oxidative impairments, mitochondrial dysfunction and neurotoxicity in maternal and fetal brain of rats. To achieve this, pregnant dams given oral supplements of a combination of fructo- and xylooligosaccharides (FOS+XOS, 3g/kg/day) during gestation days (GD 0-19) were exposed to ACR (200ppm in drinking water, GD 6-19). The behavioral analysis revealed that ACR dams fed prebiotics displayed higher exploratory behavior in the open field test. The prenatal evaluation showed that ACR-induced decrements of placental/fetal weights were markedly restored with prebiotic feeding. Prebiotics significantly offset markers of oxidative stress, restored enzymic antioxidants, cholinergic and mitochondrial function in the maternal and fetal brain. Concomitantly, prebiotics restored ACR-induced depletion in the levels of dopamine and γ-aminobutyric acid in the maternal cortex that positively correlated with cecal bacterial numbers. Collectively, these data suggest that prenatal prebiotic oligosaccharide supplements protect developing brain against oxidative stress-mediated neurotoxicity. While the underlying mechanism/s by which prebiotics abrogate the impact of neurotoxicants in the developing brain merits further studies, we speculate that it may be mediated predominantly through attenuation of oxidative stress and proliferation of enteric microbiota.

Deciphering the pathogenesis of NSAID enteropathy using proton pump inhibitors and a hydrogen sulfide-releasing NSAID.

The small intestine is a significant site of ulceration and bleeding induced by nonsteroidal anti-inflammatory drugs (NSAIDs). The pathogenesis is poorly understood. The present study explored the roles of bile, bacteria, and enterohepatic circulation to NSAID enteropathy, using both a conventional NSAID (naproxen) and a gastrointestinal-safe naproxen derivative (ATB-346), as well as proton pump inhibitors (PPIs). Rats were treated orally with naproxen or equimolar doses of ATB-346 over a 5-day period, with or without PPI administration, and intestinal damage was quantified. The cytotoxicity of bile from the rats was evaluated in vitro. Biliary excretion of naproxen and ATB-346 was determined. The impact of the NSAIDs and of PPIs on the composition of the intestinal microbiota was examined by deep sequencing of 16s rRNA. Naproxen caused significant intestinal damage and inflammation, whereas ATB-346 did not. Naproxen, but not ATB-346, dose dependently increased the cytotoxicity of bile, and it was further increased by PPI coadministration. Whereas biliary excretion of naproxen was significant in naproxen-treated rats, it was greatly reduced in rats treated with ATB-346. The enteric microbiota of naproxen-treated rats was distinct from that in vehicle- or ATB-346-treated rats, and PPI administration caused significant intestinal dysbiosis. The increase in cytotoxicity of bile induced by naproxen and PPIs may contribute significantly to intestinal ulceration and bleeding. Some of these effects may occur secondary to significant changes in the jejunal microbiota induced by both naproxen and PPIs.

Th17 cells give rise to Th1 cells that are required for the pathogenesis of colitis

Th17 cells reactive to the enteric microbiota are central to the pathogenesis of certain types of inflammatory bowel disease. However, Th17 cells display substantial developmental plasticity, such that some progeny of Th17 cell precursors retain a predominantly IL-17A(+) phenotype, whereas others extinguish IL-17 expression and acquire expression of IFN-γ, giving rise to "Th1-like" cells. It remains unclear what role these subsets play in inflammatory bowel disease. Using a Th17 transfer model of colitis, we found that IFN-γ-deficient Th17 cells retained an IL-17A(+) phenotype and were unable to induce colitis in recipients. Development of disease required the transition of a subset of Th17 precursors to Th1-like cells and was contingent on the expression of both Stat4 and T-bet, but not the IL-12 or IFN-γ receptors. Moreover, Th17 cells could provide "help" for the development of pathogenic Th1 cells from naïve precursors. These results indicate that Th17 cells are potent mediators of colitis pathogenesis by dual mechanisms: by directly transitioning to Th1-like cells and by supporting the development of classic Th1 cells.

TLR5 expression in the small intestine depends on the adaptors MyD88 and TRIF, but is independent of the enteric microbiota

In our recent article Hörmann and coworkers have reported a role for epithelial cell-intrinsic TLR2 signaling for proliferation and renewal of the small intestinal epithelium. In this study, MyD88 and TRIF expression in the small intestine were affected by gut microbiota. Here, we report that in contrast to TLR2 and its co-receptor TLR1, TLR5 transcripts are not changed by presence of gut microbiota nor regulated through TLR2 or TLR4. Similar to TLR2 also TLR5 depends on MyD88 and TRIF adaptors. Our results indicate that TLR adaptor molecules could be determinants of TLR expression in the small intestine.

Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2.

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2(-/-)) mice were used to investigate the role of this receptor in E. faecalis-induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2(-/-) mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2(-/-) mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2(-/-) mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2.

Genetic Variation in SLC30A2 and Associated Alterations in Enteric Microbiota in Preterm Infants

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of preterm infants. Defects in Paneth cells (PC), which are specialized secretory cells that release antimicrobial peptides, cytokines, and zinc (Zn) to maintain enteric microbiota, are associated with NEC. We recently found that the Zn transporter ZnT2 (SLC30A2) is critical for PC function and that non‐synonymous genetic variation (GV) in SLC30A2 occurs in ~35% of the population. Here we aimed to determine if a relationship exists between GV in SLC30A2 and the composition of enteric microbiota in preterm infants. DNA and fecal samples were collected from 20 infants (mean gestational age 33 weeks). The entire coding region of SLC30A2 and fecal genomic DNA were sequenced. 74,332 sequences were retrieved and data revealed that the fecal microbial community was dominated by Enterobacteriaceae (44%). Three unknown species in the Enterobacteriaceae family strongly correlated (p&lt;0.05) with a region of dense GV in the C‐terminus, after accounting for multiple testing via FDR correction. These species were each approximately 5‐fold more abundant in infants with GV than in those without. Recent studies highlight the importance of establishing normal microbiota after birth, especially in preterm infants. Because several members of Enterobacteriaceae are known pro‐inflammatory opportunistic pathogens, our study suggests that GV in SLC30A2 affects the establishment of normal microbiota, which could potentially increase risk for enteric infections.