Gastroenterology 2016;150:1305–1318

The Intestinal Microenvironment and FunctionalGastrointestinal Disorders

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Giovanni Barbara,1 Christine Feinle-Bisset,2 Uday C. Ghoshal,3 Javier Santos,4

Stepen J. Vanner,5 Nathalie Vergnolle,6 Erwin G. Zoetendal,7 and Eamonn M. Quigley8

1Department of Medical and Surgical Sciences, School of Medicine, University of Bologna, Italy; 2University of AdelaideDiscipline of Medicine, and National Health and Medical Research Council of Australia Centre of Research Excellence inTranslating Nutritional Science to Good Health, University of Adelaide Discipline of Medicine, Adelaide, South Australia;3Department of Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India;4Lab Neuro-Immune-Gastroenterology, Digestive System Research Unit, Department of Gastroenterology, Institut de RecercaVall d’Hebron, Hospital Vall d’Hebron, Barcelona, Spain; 5Gastrointestinal Diseases Research Unit, Queen’s University,Kingston, Ontario, Canada; 6INSERM, U1220, Toulouse, France; Université de Toulouse, UPS, Institut de Recherche en SantéDigestive, Toulouse, France; 7Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, WageningenUniversity, the Netherlands; and 8Lynda K and David M. Underwood Center for Digestive Disorders, Division ofGastroenterology and Hepatology, Houston Methodist Hospital and Weill Cornell Medical College, Houston, Texas

For decades, interactions between the enteric neuromus-cular apparatus and the central nervous system have servedas the primary focus of pathophysiological research in thefunctional gastrointestinal disorders. The accumulation ofpatient reports, as well as clinical observations, has belat-edly led to an interest in the role of various luminal factorsand their interactions with each other and the host in func-tional gastrointestinal disorders. Most prominent amongthese factors has been the role of food. As a consequence,although not always evidence-based, dietary interventionsare enjoying a renaissance in irritable bowel syndromemanagement. Not surprisingly, given its exploration in manydisease states, the gut microbiota has also been studied infunctional gastrointestinal disorders; data remain incon-clusive. Likewise, there is also a considerable body ofexperimental and some clinical data to link the pathogenesisof functional gastrointestinal disorders to disturbances inepithelial barrier integrity, abnormal enteroendocrinesignaling, and immune activation. These data providegrowing evidence supporting the existence of micro-organicchanges, particularly in subgroups of patients with func-tional dyspepsia and irritable bowel syndrome. However,their exact role in the complex pathophysiology and symp-tom generation of functional gastrointestinal disordersneeds to be further studied and elucidated, particularly withlongitudinal and interventional studies.

Abbreviations used in this paper: BA, bile acid; BAM, bile acid malab-sorption; CRF, corticotropin-releasing factor; EC, enterochromaffin cell;FD, functional dyspepsia; FGID, functional gastrointestinal disorder;FODMAP, fermentable oligosaccharides, disaccharides, mono-saccharides, and polyol; GI, gastrointestinal; 5-HT, 5-hydroxytryptamine,serotonin; IBS, irritable bowel syndrome; IBS-C, irritable bowel syndromewith constipation; IBS-D, irritable bowel syndrome with diarrhea; IBS-M,irritable bowel syndrome with mixed bowel habits; IFN, interferon; IL,interleukin; JAM, junctional adhesion molecules; MC, mast cell; NCGS,

Keywords: Microbiota; Bile Acids; Serotonin; Immune System;Food; Irritable Bowel Syndrome; Functional Dyspepsia.

lthough the focus of studies on the pathophysiology

nonceliac gluten sensitivity; NGF, nerve growth factor; PI, post-infectious;SCFA, short-chain fatty acids; SERT, serotonin reuptake transporter;SIBO, small intestinal bacterial overgrowth; TJ, tight junction; TLR, toll-likereceptor; TNF, tumor necrosis factor; TpH, tryptophan hydroxylase; ZO,zonula occludens.

Most current article

© 2016 by the AGA Institute0016-5085/$36.00

http://dx.doi.org/10.1053/j.gastro.2016.02.028

Aof functional gastrointestinal disorders (FGIDs) haslargely been on the enteric neuromuscular apparatus and itscentral connections through the gut�brain axis, the potentialimportance of the luminal environment was noted many de-cades ago in the first descriptions of FGID-type symptomsdeveloping de novo in the aftermath of an enteric infection.1

Clinical experience informed us of the importance of food asa symptom precipitant yet, up until very recently, little

research had been performed on interactionswith diet and/orthe products of digestion in the FGID sufferer. As the com-plexities of the human microbiota are increasingly under-stood, the possibility thatmicrobe-host interactions, includingimmune and metabolic responses, might be relevant to theFGIDs has emerged. How any one or a combination of theseluminal factors interact with each other and with the host is asubject of considerable research interest and putative path-ophysiological mechanisms have been postulated (Figure 1).

These will be explored further in this review. Caveatsthat might limit the outcomes of the current review must beacknowledged. Although we aim to refer to human studies,animal data could be mentioned when instrumental tobetter understand the role of microenvironmental factors inFGID. As most studies have been conducted in patientssuffering from functional dyspepsia (FD) and irritable bowelsyndrome (IBS), we will address other FGIDs only margin-ally. Pharmacological and other interventional approachesinvolving the intestinal microenvironment will not be sys-tematically reviewed here.

FoodThere is increasing recognition that dietary factors can

play a major role in the etiology and the pathogenesis of

Figure 1. Schematic representation of the putative interplay between luminal and mucosal factors in FGIDs. Microenviron-mental factors (eg, food, microbiota, bile acids) may permeate in excess through a leaky epithelial barrier, allowing amplifi-cation of signaling from the lumen to deeper mucosal and muscle layers, including overstimulation of the mucosal immunesystem. These factors may determine abnormal signaling to neural circuits (intrinsic primary afferent nerves and extrinsicprimary afferent nerves), which in turn may affect intestinal physiology and sensory perception.

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symptoms in both FD and IBS. Their impact could bemediated through direct interactions between dietarycomponents and mucosal receptors that may have beensensitized to these stimuli, or via down-stream events trig-gered by dietary components, such as the release of guthormones, changes in epithelial morphology, generation ofimmune responses, or altered signaling between the gut andthe brain.

Dietary factors that reportedly trigger symptoms includeeating patterns as well as specific foods and/or food com-ponents. Only a few small studies have evaluated the directeffects of administering specific foods or nutrients onsymptom provocation. No intervention studies have evalu-ated the impact of targeted dietary changes on symptomimprovement in FD.

Although patients with IBS have long associated theirsymptoms with food ingestion, a focused scientific andclinical interest in the potential role of food in IBS hasemerged only recently.

Role of DietNo major differences have been found in eating patterns

between FD patients and controls, although limited evi-dence suggests that patients eat fewer meals per week, andtend to eat more smaller meals/snacks, than controls.2

While up to 80% of patients report that fatty foods/meals induce their symptoms, and approximately 30%exclude fried foods to avoid symptoms, many other foods

are also reported to induce symptoms.3 Data on dietarynutrient composition in FD are limited and inconsistent,possibly because some patients modify their diets in anattempt to alleviate symptoms. The only available pro-spective study in FD patients noted trends toward lower fatand energy intakes and direct relationships between, onthe one hand, postprandial fullness and fat and energyintake and, on the other hand, bloating and fat intake.2

Wheat- and carbohydrate-containing foods have beenidentified as triggers for symptoms, and FD patientsfrequently report symptoms on exposure to milk and dairyproducts, although their role remains unclear. Data on fiberintake in FD are inconsistent.

The majority of IBS patients associate ingestion of a widerange of foods with symptoms, particularly abdominalbloating and pain.4 Patients frequently report making di-etary adjustments, including reduced consumption of milkproducts, wheat products, alcohol, and certain fruits orvegetables that are high in poorly absorbed short-chaincarbohydrates and sugar alcohols (eg, onions) and anincreased intake of other fruits high in fermentable oligo-saccharides, disaccharides, monosaccharides, and polyols(FODMAPs; eg, grapes and pears).5 Data on such dietaryadjustments in IBS are not consistent. Many IBS patientsreport symptoms in response to wheat-containing products,reminiscent of the sensitivity to gluten that characterizesceliac disease, despite negative celiac serology and normalsmall intestinal morphology, a phenomenon that hasbeen termed nonceliac gluten sensitivity (NCGS). Subsets of

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patients also report symptoms after consumption of milkand dairy products, or spicy foods. The view that a lack ofdietary fiber was the main cause of IBS has been largelyrevised. While soluble fiber can have some beneficial effects,insoluble fibers, including bran, appear to be of neitherbenefit nor harm.

Provocation of SymptomsProspective studies in FD have only evaluated the effects

of fat on symptoms. While equicaloric high-fat and high-carbohydrate yogurt-based meals both increased FD symp-toms; pain, fullness, and nausea were greatest after thehigh-fat meal.3

Studies that manipulate dietary constituents providefurther insights. Ingestion of a high-FODMAP diet wors-ened symptoms (eg, abdominal pain, bloating, and exces-sive flatus) in IBS patients, compared with healthycontrols or a low-FODMAP diet. In addition, in patients ona low-FODMAP diet, blinded rechallenge with fructoseand/or fructan, but not glucose, exacerbated symptoms.6

The role of gluten in IBS remains uncertain. While onerecent study in patients with diarrhea-predominant IBS(IBD-D) and NCGS found an improvement in symptoms ona gluten-free diet, and their relapse when gluten wasreintroduced in a blinded fashion, another study was un-able to confirm gluten-specific, as distinct from FODMAP-related, effects on symptoms.7 In another study, exposureto gluten increased stool frequency and altered gut barrierfunction; mainly in IBS-D subjects who were humanleukocyte antigen DQ2 or DQ8 positive. While somestudies have found an improvement in symptoms on amilk- or dairy-free diet, these trials were often not blin-ded. Intolerance might also exist toward other compo-nents of milk. Acute ingestion of hot chili powder in acapsule with a meal increased abdominal pain and burningin IBS patients compared with healthy controls.

Potential MechanismsThe limited research that has been performed suggests

that symptoms generated by food ingestion in FD may bedue to exaggerated signals originating in the upper GI tract,including gastric hypersensitivity to distension, small in-testinal hypersensitivity to fat, and hypersensitivity to theeffects of gut hormones (particularly cholecystokinin), acid,capsaicin, and the products of colonic fermentation.3

Several factors could contribute to the pathophysiology offood-related symptoms. An enhanced phasic�colonic motorresponse to food ingestion and colonic hypersensitivity todistension can both contribute to a nonspecific increase inabdominal symptoms postprandially in IBS. FODMAPs areosmotically active and increasewater content in the intestinallumen. They are rapidly fermented to hydrogen, carbon di-oxide, methane, short-chain fatty acids (SCFAs), and lactate.Such responses could be exaggerated in IBS, the resultingdistension of the intestinal lumen may exacerbate visceralhypersensitivity. Gluten can cause amild immune response inIBS patients, associated with exaggerated responses inenteric and sensory nerves and compromised intestinal

barrier function.8 Some of the adverse reactions attributed to“gluten” might reflect a hypersensitivity to wheat or intoler-ance to FODMAPs. GI symptoms attributed to wheat (thelargest dietary fructan source) can also relate to FODMAPs,rather than gluten.7,8 Thus, the term wheat intolerance orsensitivity might be more appropriate than NCGS. A highprevalence of autoimmune disease among patients withwheat sensitivity has been described.9 Lipids can exacerbateIBS symptoms through modulation of distal gut motor func-tions and sensitivity. An increase in the density of sensoryfibers expressing transient receptor potential cation channel,subfamily V-1 receptors in IBS patients with visceral hyper-sensitivity can enhance transmission of pain signals,including those generated by spicy foods. Recently, a role fortransient receptor potential cation channel, subfamily V-4 hasalso been proposed as a possible pathway of pain trans-mission in patients with IBS (see Impact of Immune Activa-tion on Gut Sensorimotor Function).10

Translational ResearchProspective studies evaluating the effects of dietary in-

terventions in FD are urgently required.While recent studies have reported beneficial effects of a

low-FODMAP diet on symptoms,11 stool habits, and qualityof life in IBS, studies were small in size and further evidenceis required to determine whether a low-FODMAP diet isbetter than a standard diet in controlling symptoms.Furthermore, the observed effects on the reduction of fecalcommensal bifidobacteria, and detrimental effects on gutmicrobiota composition,12 require further investigation. Agluten-free diet improves IBS symptoms and reduces bowelfrequency and intestinal permeability.13,14 The gluten-specificity of these effects remains to be established.Comprehensive dietary counseling, including the adoptionof healthy eating habits, avoidance of foods rich in FOD-MAPs, insoluble fiber and artificial sweeteners, replacingwheat with spelt products, and the importance of ingestingdairy products, has been reported to be associated with asignificant reduction in IBS symptoms, including abdominalpain, diarrhea (in IBS-D) and constipation (in IBS-C), and amarked improvement in the quality of life.15

The Microbiota and its MetabolicInteractions

When food enters our intestine, the undigested compo-nents are utilized by the intestinal microbes, collectivelycalled the GI microbiota. The microbiota is dominated bybacteria belonging to the phyla Firmicutes, Bacteroidetes,and Actinobacteria. These microbes inhabit the various re-gions in the GI tract, of which the colon is most denselypopulated. The microbiota has a major impact, not only onprocesses that occur in the GI tract, but also on systemicfunctions, and thus plays a key role in our overall health.

Impact of Diet and Lifestyle on MicrobiotaIt is evident that lifestyle and diet are crucial de-

terminants of microbiota composition and function in

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humans. Comparative studies have demonstrated huge dif-ferences in microbiota composition between human pop-ulations in Western and those in developing countries andsuggested that these are based on lifestyle and long-termdietary pattern differences.16 Short-term dietary changeshave also been shown to impact the composition of themicrobiota. To date, such alterations have been shown inintervention studies that involve quite drastic changes indiets, while more subtle, short-term dietary interventionshave, in general, only a minor impact on microbiotacomposition.17 The impact of diet on the microbiota can bedirect, through changes in its composition or total energysupply, or indirect, via the induction of changes in intestinaltransit time or intraluminal pH. Of note, the impact of dieton the microbiota is also highly dependent on the intestinallocation. For example, the conversion of complex indigest-ible carbohydrates is the driving force for the microbiota inthe colon, while the microbiota in the small intestine islargely driven by the fast uptake and conversion ofsugars that are likely derived from digested dietarypolysaccharides.18

Microbiota Metabolism of Dietary Substratesin Functional Gastrointestinal Disorders

Carbohydrate. Complex dietary components can beconverted by the microbiota to a wide variety of metabo-lites that might involve cross-feeding and synthrophic in-teractions between individual microbes.17,19 Whichmetabolites are produced and in what quantities, isdependent on the dietary components. The fermentation ofcomplex carbohydrates, such as fibers and resistantstarches, results, in general, in the production of SCFAs,notably acetate, propionate, and butyrate; on a Westerndiet approximately 300 mmol SCFA are produced daily.Because SCFAs are fuels for our intestinal cells and serve assignaling molecules, they are considered as beneficial,particularly butyrate and propionate. Butyrate can be pro-duced by a wide variety of bacteria, and most well-knownare Faecalibacterium prausnitzii, Eubacterium rectale, Eu-bacterium hallii, and Roseburia intestinalis.19 Sources forbutyrate production include sugars, lactate, acetate, as wellas amino acids, such as lysine.20 This allows butyrate pro-ducers to engage in metabolic cross-feeding interactionswith organisms that convert complex food components.Propionate fermentation occurs via three distinct pathways,of which the succinate pathway is the most commonlyutilized route in the gut, mainly performed by Bacteroidesspp. and Veillonella spp.19 Acetate can be produced by awide variety of microbes in the gut from fermentation ofcarbohydrates in a so-called mixed fermentation withlactate or other SCFAs, such as propionate. Acetate may alsobe generated via reductive acetogenesis, the reduction ofcarbon dioxide with hydrogen, a process that is estimatedto be responsible for one third of total acetate production inthe intestine.21

A few human studies suggest a role for SCFAs in FGIDsand imply that nerves are involved. For example, areduction in abdominal pain in IBS patients administered

sodium butyrate was observed.22,23 It was speculated thatbutyrate reduced the hypersensitivity of intestinal mech-anoreceptors and altered neurotransmitter release,resulting in a reduction in luminal pressure and/or peri-stalsis. Others have observed higher levels of acetic acid,propionic acid, and total organic acids in IBS patients, withhigher acetic acid levels being associated with greater GIsymptoms.23,24

Carbohydrate fermentation also results in the produc-tion of hydrogen and carbon dioxide, which are the mainintestinal gases formed in the intestine by the microbiota.Whereas impaired handling of intestinal gases has beenconsistently described in IBS, the contribution of themicrobiota to this phenomenon is far from clear. Hydrogenis thought to inhibit fermentation, but can also serve as anenergy source for a variety of microbes, including meth-anogenic Archaea, reductive acetogens, and sulfate reduc-ers.17�19 The latter group produces sulfide, a toxiccomponent that is regarded as harmful to our health. Po-tential sources of the required sulfate include dietary com-ponents and host-derived substrates, such as mucin.Although the relative volumes of intestinally derived gasesexcreted in the breath have been used to relate FGIDsymptoms to microbial fermentation rates in situ in the gut,this extrapolation is fraught with problems due to cross-feeding between different microbial populations, such ashas been described here, resulting in altered relative con-centrations of intestinal gasses that together determine thetotal volume.

Relatively few data are available on qualitativechanges in gas composition in FGIDs. Increased methaneproduction in constipation-predominant IBS-C25 is wellknown, but it is unclear whether this is a cause or effect.One study correlated methane with a higher motility in-dex in IBS patients.25,26 Hydrogen sulfide signals throughmultiple pathways, including nerves, but human studiesare lacking.

Protein. Protein utilization requires protease activity,which is available in both humans and microbes. Althoughless frequently studied than carbohydrate fermentation,microbial fermentation of protein is, in general, consideredas potentially harmful to health, because amino acidfermentation can lead to toxic products, such as amines andammonia, as well as N-nitroso, indolic, sulfur, and phenoliccompounds.27 Potential sources of proteins for fermentationinclude diet and host-derived compounds. Although mostproteins are digested and taken up by the small intestine, ahigh-protein diet could lead to the arrival of significantprotein loads in the colon. Because microbes favor carbo-hydrate fermentation over protein fermentation, it has beenspeculated that low carbohydrate diets can promote proteinfermentation in the intestine.

A recent study showed that concentrations of fecalproteases were higher in IBS-D patients compared withhealthy controls, suggesting enhanced protein metabolismin the colon.28 Remarkably, most of these proteases were ofhuman origin. Nevertheless, it is conceivable that increasedprotease activity in the colon may lead to higher rates ofamino acid fermentation.

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Fat. Dietary fat content has also been negatively corre-lated with health status. In contrast to carbohydrates andproteins, however, fat is not believed to reach the colon andbe exposed to its microbiota in significant amounts becausemost is digested and absorbed in the small intestine. Oneindirect effect of dietary fat assimilation is its facilitation ofthe diffusion of bacterial components, such as lipopolysac-charide, across the epithelium, which could lead to low-gradeinflammation, such as some have described in IBS.29

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Microbiota Structure and FunctionalGastrointestinal Disorders

A recent report of the Rome Foundation on the micro-biota in FGIDs provided an excellent overview of theimportance of the microbiota in health and disease and,especially, in relation to FGIDs.30 Figure 2 provides a sche-matic representation on the role of the intestinal microbiotain conversion of dietary components and their potentialimpact on the pathophysiology of FGIDs. Several lines ofevidence suggest the involvement of the intestinal micro-biota in the pathogenesis of FGIDs in general and IBS inparticular: gastrointestinal (GI) infections are strong riskfactors for the development of FD and IBS (see Post-Infec-tious Functional Gastrointestinal Disorders); fecal micro-biota is substantially different in IBS and post-infectious(PI)-IBS compared with healthy controls, and showsreduced microbiota diversity30; innate and adaptive immu-nity directed to microbiota-derived molecules, including theexpression of toll-like receptors (TLRs) in the mucosa, theproduction of human b-defensin-2 and antibodies to bac-terial flagellin are substantially different in IBS comparedwith controls30; some evidence indicates the existence ofabnormal concentrations of fermentation end products,such as SCFAs, which might participate in symptom pro-duction in some patients with in IBS (see MicrobiotaMetabolism of Dietary Substrates in Functional Gastroin-testinal Disorders); one recent study showed that total SCFAlevel was significantly lower in IBS-C patients than in IBS-Dand IBS with mixed bowel habit (IBM-M) patients and

Figure 2.Overview of di-etary components andmetabolites produced inthe GI tract, and their as-sociation with irritablebowel syndrome or itssymptoms. *Increasedlevels in irritable bowelsyndrome patients. In-creases in methane (CH4)and hydrogen (H2) con-centrations contribute tobloating and distensionand intraluminal concen-trations of bile acids andproteases will promotediarrhea. BDFA, branched-chain fatty acid.

healthy controls; case�control studies show that systemicantibiotic use is a risk factor for de novo development ofFGIDs31; and in 43 randomized controlled trials, the relativerisk of IBS symptoms persisting with probiotics vs placebowas 0.79 (95% confidence interval: 0.70�0.89), with posi-tive effects on global IBS, abdominal pain, bloating, andflatulence scores.32

Nonetheless, major limitations still hamper the defini-tion of the role of the microbiota in FGIDs. Indeed, there isno consensus on the nature of the microbial signatures thatmay be consistently (either positively or negatively) corre-lated to FGIDs. These inconsistencies may relate to severalfactors, including methodological differences, variations insample sources, intrinsic variability between subjects, dif-ferences in subject selection and definition of study pop-ulations, overlap between the various FGIDs, and differencesin diet, therapy or other environmental exposures. It needsto be recognized that many studies described comparisonsbetween different groups of subjects on the basis of a singlefecal sample per subject, which only represents a snapshotof the microbiota and, as a result, such comparative analysescannot differentiate between cause, consequence, or coin-cidence. Given the large heterogeneity in the human popu-lation and the extent of microbial diversity, it is likely thatmany significant correlations are just coincidence. This may,in part, explain why there has been no consensus regardingwhether a specific microbe or groups of microbes is asso-ciated with a given FGID.30 Therefore, it is evident thatlongitudinal studies involving repeated sampling of themicrobiota will be crucial to differentiate cause fromconsequence or coincidence. Such studies could include in-terventions with dedicated diets or dietary supplements,specific pharmacological interventions, or novel therapies,such as fecal microbiota transplantation.

Bile AcidsBile acids (BAs) play a central and critical role in the

digestion and absorption of fat and fat-soluble vitamins,and a highly efficient enterohepatic circulation ensures the

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conservation of secreted BAs; the primary means of BAconservation being active absorption via the apicalsodium-dependent ileal BA transporter located on theapical surface of ileal enterocytes. BA absorption andsecretion are closely linked through a feed-back loop,which involves a number of receptors and mediators thatultimately impact on the rate-limiting enzyme in BA syn-thesis (Figure 3).33

BAs have a variety of physiological effects of relevance tothe FGIDs; on motility, intestinal secretion, membranepermeability, and visceral sensation,34 and act as importantsignaling molecules with effects well beyond the GI tract. AsBAs repress bacterial growth in the intestine, the develop-ment of microbial enzyme pathways capable of deconju-gating and transforming BAs is an important adaptiveresponse by commensal bacteria.35 In contrast, antibacterialand mucosal immune-stimulating effects of BAs play animportant role in the prevention of small intestinal bacterialovergrowth (SIBO).36

Human physiological studies suggest a role for luminalBA signaling to enteric nerves in causing altered smallbowel motility and increased sigmoid and rectal motility. InIBS-D, it was estimated that as many as 10% of patients

Figure 3. Schematic representation of the interactions betweenof germ-free and normally colonized animals. The scheme7a-hydroxylase and levels of taurine-conjugated b-muricholic aof sterol 12a-hydroxylase and cholic acid levels are similar in gmuricholic acid is a natural antagonist of the farnesoid X receplimiting enzyme cholesterol 7a-hydroxylase in germ-free animreduction in taurine-conjugated b-muricholic acid. This leads tothus up-regulating the fibroblast growth factor 15 (FGF15), whicaddition, the microbiota affects intestinal bile acid metabolism aC4, cholesterol-4; CYP7A1, cholesterol 7a-hydroxylase; CA, chfibroblast growth factor 15; b-MCA, b-muricholic acid; TCA, talithocholic acid; CYP8B1, sterol 12a-hydroxylase; TbMCA, taurinfrom Sayin et al.120

malabsorb BAs37,38 and infusion of BAs in the colondisproportionately stimulated motility compared with con-trols. In idiopathic BA malabsorption (BAM),39 phase3�induced neurogenic secretions were increased in thejejunum, and prostigmine increased the colonic motilityindex, implying involvement of the enteric nervous sys-tem.40 Genetic variants of the G protein�coupled bile acidreceptor (TGR5), found on multiple cells, including entericnerves, have also been linked to transit time in patients withFGIDs.38 Altered metabolism of BAs by colonic bacteriamight also be involved, as constipation and increased transittime correlated with a reduction of colonic BAs, possibly theresult of bacterial sulfation.41

Epithelium and Mucosal BarrierThe intestinal luminal-mucosal interface represents the

first location where toxic and immunogenic particles facethe scrutiny of the mucosa-associated immune system. Lossof molecular and functional integrity of the epithelial barriercould lead to activation of mucosal immune responses andset in motion events that are closely related to the originand clinical manifestations of several FGIDs.

the microbiota and bile acids as illustrated by a comparisonshows increased activity and expression of cholesterol

cid in germ-free mice. In contrast, the expression and activityerm-free and normally colonized mice. Taurine-conjugated b-tor (FXR), which, in turn, may elicit reduced inhibition of rate-als mice. In contrast, normally colonized animals show aincreased activation of farnesoid X receptor in enterocytes,

h in turn suppresses cholesterol 7a-hydroxylase in the liver. Innd increases their excretion. BACS, bile acyl-CoA synthetase;olic acid; FGFR4, fibroblast growth factor receptor 4; FGF15,urine-conjugated cholic acid; DCA, deoxy cholic acid; LCA,e-conjugated b-muricholic acid. Reproduced with permission

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Molecular StructureThe apical junctional complex keeps enterocytes tightly

sealed and regulates paracellular permeability.42 Thiscomplex is composed of tight junctions (TJs), adherensjunctions, and desmosomes. Intracellular (zonula occludens[ZO]-1, ZO-2, and ZO-3, and cingulin) and surface-membraneproteins (occludin, claudins, and junctional adhesion mole-cules [JAMs]) are major components of TJs.42 Adherensjunctions are mainly composed of e-cadherin, catenin, andactin filaments.42 Occludin seems to regulate the integrity ofTJs, while claudins determine their strength, size, and ionselectivity, and JAMs their construction and assembly.43 Allare linked to actomyosin fibers by members of the ZO familyand, in this way, control the opening/closing of TJs at par-acellular spaces.44 Zonulin transactivates the epithelialgrowth factor receptor via proteinase-activated receptor 2activation and reversibly regulates intestinal permeability.

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Intestinal Permeability and Barrier DysfunctionThe passage of molecules across the epithelium takes

place mainly via two distinct routes: the paracellularpathway, which allows small molecules (<600 Da) to diffusethrough TJs, and the transcellular pathway, which facilitatesthe transit of larger particles via the processes of endocy-tosis or exocytosis. Rapid changes in permeability usuallyoccur via myosin light-chain kinase-mediated cytoskeletoncontraction and endocytosis of TJ proteins. In contrast, moresustained changes in permeability involve the transcrip-tional modulation of TJ proteins, epithelial cell apoptosis,and ultrastructural alterations in the epithelium.42

Acute stress either reduces net water absorption or in-creases jejunal secretion in healthy subjects through theparasympathetic nervous system and mast cell (MC) acti-vation.45 In addition, higher background levels of stresshave been related to decreased water secretion in healthyfemale volunteers exposed to cold pain stress.46 Strongerstresses, like abdominal surgery, GI infections, hemorrhagicshock or intensive exercise, increase intestinal permeability.Corticotropin-releasing factor (CRF) enhances transcellularuptake of macromolecules in the human colon via CRF-R1and CRF-R2 receptors on subepithelial MCs.47 Acute psy-chological stress also increases small intestinal permeabilityin humans; peripherally administered CRF reproduces thiseffect and MC stabilization blocks the effects of both stressand CRF.48

In-depth reviews on the role of physiological and path-ophysiological stimuli controlling the gut barrier have beenpublished recently.42,49 Vasoactive intestinal polypeptideregulates chloride secretion, mucin release, and paracellularpermeability, partly through a direct effect on ZO-1. Sub-stance P stimulates the release of pro-inflammatorycytokines and vasoactive mediators by macrophages,eosinophils, and MCs, contributing to chloride secretion,increased intestinal permeability, and vascular leakiness.Nerve growth factor has been involved in nerve- andMC-mediated stress-induced barrier dysfunction. Both pro-gesterone and estradiol have been shown to reduce chloridesecretion in intestinal epithelial cells, whereas estradiol

reinforced epithelial permeability and up-regulated JAM-Aand occludin expression. Other mediators, including CRF,leptin, and cholecystokinin, may increase permeability,while insulin-like growth factor, ghrelin, KdPT andglucagon-like peptide 2, may decrease intestinalpermeability.42,49

Various strains of Vibrio cholera, Clostridium difficile, andtoxin-producing strains of Escherichia coli have been shownto enhance intestinal permeability through direct TJdisruption, the production of toxins or proteases, and theactivation of the inflammatory cascade. In contrast, pro-biotics promote barrier integrity by increasing occludin,claudin 3, and ZO-1 and ZO-2 expression.50

Interferon (IFN)-gamma and tumor necrosis factor(TNF)�a induce barrier dysfunction through myosinlight-chain kinase and claudin-2 up-regulation and down-regulation of occludin. Many other cytokines and pro-teases have effects on barrier function, including interleukin(IL)-3, IL-4, IL-17, IL-22, and IL-26, IFN-alfa, IFN-beta, andtransforming growth factors�a, and �b.51

The impact of nutritional factors on the intestinal barrierhave been reviewed recently.49 In predisposed individuals,gluten and other specific food components can lead toincreased intestinal permeability through the zonulinpathway and MC-mediated enhancement of both passageroutes. Whey proteins can improve barrier function by atransforming growth factor�b-mediated increase in intes-tinal claudin-4 expression. Other nutritional products, suchas glutamine, butyrate, arginine, fatty acids, and prebiotics,have been shown, to some extent, to exert a protective effecton the intestinal barrier.

Ethanol promotes separation of ZO-1 proteins, disas-sembly of actin and myosin filaments and myosin light-chainkinase activation. Nonsteroidal anti-inflammatory drugs,methotrexate, tacrolimus, omeprazole, and corticosteroidscan also enhance intestinal permeability, but heparin,vitamin D, and larazotide can decrease permeability.

Mucosal Barrier and FunctionalGastrointestinal Disorders

There is little information on the status of mucus pro-duction in IBS other than isolated reports on the potentiallybeneficial effects of probiotics or mesalazine on mucusquality and production, higher levels of trefoil factor 3 in theurinary IBS proteome, and increased expression of genesinvolved in the production of mucin 20 in the colon of IBS.

Enhanced intestinal permeability has been reported in FDand in subsets of patients with IBS (Supplementary Table 1)and linked to alterations in JAM-A, ZO-1, e-cadherin,claudins, and occludin (Supplementary Table 2), and thesechanges were associated with MC activation and clinicalmanifestations.

Enteroendocrine SystemThe enteroendocrine system, the largest endocrine

organ, constitutes 1% of the gut epithelium. Fourteen cellpopulations, including enteroendocrine cells and enteric

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nerves, produce transmitter substances that signal toneighboring cells (paracrine), distant targets via thevascular system (endocrine) or through intrinsic/extrinsicnerves (neurocrine). These effector targets, in turn, controlgut motility, secretion, sensation, absorption, vascular tone,microcirculation, immunity, and cell proliferation (Figure 4,Supplementary Table 3).52�54

Serotonin Metabolism and ReceptorsSerotonin (5-hydroxytryptamine [5-HT]), is a paracrine/

neurocrine amine primarily contained in the gut (95%) andonly minimally in the brain (5%). Serotonin is synthesizedfrom tryptophan, in enterochromaffin cells (EC) (90%) andautonomic nerves (10%).55,56 Synthesis and release ofserotonin involves conversion of dietary tryptophan to5-hydroxy-L-tryptophan (catalyzed by tryptophan hydroxy-lase [TpH], isoforms, TpH1 [in gut] and TpH2 [inbrain]),56�58 granular packaging by vesicular monoaminetransporter 159 and release, mainly determined by bowelwall distension, mucosal stroking, food, amino acids, hypo-or hyper-osmotic solutions, glucose, galactose, adenosine,cholera toxin, and chemotherapeutic agents.59,60 SCFA,which may be produced in increased amounts by intestinalmicrobiota fermentation of carbohydrate substrates, canalso promote the release of serotonin.59 An alternativemetabolic pathway leads to the production of kynurenicacid, and not serotonin, from tryptophan, and results inreduced serotonin synthesis.61 The serotonin reuptaketransporter (SERT) terminates serotonin action (Figure 4).57

Figure 4.Mechanism of action of serotonin and its re-uptake in5-HT, serotonin.

In the gut, serotonin stimulates intrinsic primaryafferent neurons, which synapse in the myenteric plexuswith ascending and descending inter-neurons to evokemotility- and secretion-induced reflexes and also transmitinformation to the brain.55,57,62

Serotonin Metabolism in FunctionalGastrointestinal Disorders

SERT hyperactivity may lead to increased reuptake ofserotonin, hence reducing the effects of the amine on targettissues. In contrast, hypofunction of SERT may increaseserotonin concentrations, leading to gut hypercontractility,hypersensitivity, diarrhea, and pain.57,60 Accordingly, in celllines, infection with enteropathogenic E coli reduced SERTactivity.63 The SERT protein is encoded by a gene on chro-mosome 17q11 and is composed of 14 exons encoding 630amino acids. Insertion/deletion of 44 base pairs in the 5-HT-transporter-gene-linked polymorphic region, leading toreduced SERT expression, has been reported in IBS-D.64 The5-HT-transporter-gene-linked polymorphic region (S/L)was more common than the S/S polymorphism in FD,particularly in the postprandrial distress syndrome.65 In anIndian study, solute carrier family 6 (neurotransmittertransporter), member 4 (SLC6A4) polymorphism and higherlevels of 5-HT were associated with IBS, particularly PI-IBSand IBS-D.66 The homozygous S genotype (reducing SERTexpression) was more common in IBS-D.64 A meta-analysison 25 studies, including 3443 IBS patients and 3359 con-trols, showed that the 5-HT-transporter-gene-linked

IBS. ACh, acetylcholine; GABA, gamma amino butyric acid;

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polymorphic region mutation was associated with IBS-C, butnot with IBS-D and IBS-M, and, particularly, among EastAsians.67 More studies are needed to clarify this issue.

The implications of serotonin in FGID have beenreviewed previously.59,68 Data suggest that subgroups ofpatients with FGIDs show altered serotonergic signaling(Figure 4). Accordingly, the epigastric pain syndrome sub-type may have higher basal serotonin levels.65 In contrast,some data suggest that subsets of patients with FD mayhave low basal and postprandial plasma levels of seroto-nin.69 Studies suggest that IBS-D is associated with elevated,and IBS-C with reduced, serotonin plasma levels.59,70 In onestudy, both serotonin and kynurenic acid levels were lowerin the duodenal mucosa and higher in plasma in IBS, thancontrols, suggesting a contribution from the kynurenic acidpathway.61 As chili ingestion increases FGID symptoms andgranisetron, a 5-HT 3-receptor antagonist, prevents it;serotonin is suggested as the mediator of chili-inducedsymptoms.71 Although a low-FODMAP diet has beenshown to improve IBS symptoms,12 data on its effect onbrain and gut serotonin levels are lacking.

In PI-IBS (see Post-Infectious Functional GastrointestinalDisorders), altered EC cell numbers have been reported.60,72

PI-IBS patients had higher rectal mucosal serotonin thannon�PI IBS-D and non-diarrheal IBS. The enteroendocrineand immune systems are widely interconnected, as sug-gested by the proximity of immune cells to EC cells.59,68

Furthermore, immune cells, including B and T lympho-cytes, monocytes, macrophages, and dendritic cells, expressserotonergic receptors and MCs; macrophages and T cellssynthesize serotonin from tryptophan.59 Serotonin ischemotactic for dendritic cells, MCs, and eosinophils andmay participate in the recruitment of these immune cells in

Figure 5. Post-infectious IBS and re

the intestinal mucosa (see Mucosal Immune Activation).59

Low-grade inflammation, such as has been detected inFGIDs can, in turn, contribute to altered serotonin synthesisand reuptake through changes in SERT expression.73,74 Th1responses generate IFN-gamma and TNF-a, which inhibitSERT; Th2 responses, such as occur in parasitic infestations,stimulate IL13, which increases EC numbers and TNF-a and,therefore, inhibit SERT.75

Efficacy for serotonergic agents, such as 5-HT3 receptorantagonists (alosetron, cilansetron, and ondansetron),including a large multicenter trial on ramosetron in femaleIBS-D patients with promising results,76 and 5-HT4 agonists(cisapride, tegaserod, and prucalopride) in the treatment ofIBS-D and IBS-C, respectively, also provide evidence for arole for serotonin in the pathogenesis of FGIDs.56,57

Immune System and Neuro-ImmuneInteractionsPost-Infectious Functional GastrointestinalDisorders

The observation that FD and IBS can develop after anepisode of acute infectious gastroenteritis supports theinvolvement of the immune system in the pathophysiologyof FGIDs. The mean incidences of PI-FD and PI-IBS afterinfection with diverse pathogens (bacteria, parasites, orvirus) are 9.6% and 10%, respectively, with an overall oddsratio of 2.5 for the presence of an FGID at 6 months postinfection compared with controls.32,77 Risk factors for PI-IBSinclude the severity and duration of the acute infection,female sex, psychological comorbidity (eg, hypochondriasis,neuroticism, depression, adverse live events, perceived

lated risk factors. RR, relative risk.

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stress, negative illness beliefs78), smoking, and being a childat the time of the infection79 (Figure 5). The pathogenesis ofPI-FGIDs is multifactorial and involves both pathogen andhost factors.78 In PI-IBS, the colorectal mucosa showsincreased infiltration of macrophages, MCs, and intra-epithelial lymphocytes, as well as PYY-containing enter-oendocrine cells.72,78 The association of the TNF-a SNPrs1800629 with PI-IBS supports the hypothesis of a geneticpredisposition possibly contributing to increased epithelialpermeability and an inability to resolve an acute inflam-matory process.80

Mucosal Immune ActivationNumerous studies have shown increased numbers of

mucosal immunocytes (ie, MCs, eosinophils, and T cells) inadult and pediatric patients with FD and IBS. Severalprecipitating factors have been claimed, including foodallergy, an abnormal microbiota, BAM, and increased in-testinal permeability. The magnitude of the inflammatoryresponse is several-fold less than that seen in acuteinflammation in inflammatory bowel disease. The wideoverlap with healthy controls, possible geographic and di-etary variation, and lack of methodological standardizationmight explain the failure of some studies to confirm thepresence of increased immune cells in FGIDs. The nature ofthe inflammatory process is also different from that seen inacute GI inflammation in inflammatory bowel disease, withno involvement of neutrophils or frank tissue distortion.30,81

Eosinophils, usually linked to allergic reactions, have beenassociated with postprandial distress syndrome and earlysatiety.82,83 Increased MC numbers have been detected inthe stomach and duodenum of patients with FD,83 in theesophagus of patients with noncardiac chest pain,84 andthroughout the gut in IBS-D and IBS-C,85 particularly in fe-males, in PI-FD and PI-IBS (for review, see Barbara et al81).Genetic factor, such as the TNFa rs1800629 genotype,80

have also been implicated. Microbial molecular pattern-mediated activation of innate immunity suggests a patho-genic contribution of the gut microbiota.86 TLRs areexpressed on human submucosal and myenteric neuro-ns87–89 and altered TLR expression has been observed inIBS tissues. For example, TLR4 expression in colonicmucosal biopsies from IBS patients was increased, particu-larly in those with alternating-type IBS.90 TLRs 5 and 2were also up-regulated, while TLRs 7 and 8 were down-regulated.86,90 Biopsy studies have provided evidence ofepithelial permeability changes in IBS patients and bacterialproteases may play a role.91 Thus, in some IBS patients,there may be increased expression of TLRs and/or adisruption of the mucosal barrier and increased bacterialtranslocation resulting in increased TLR signaling and/or anabnormal immune response to luminal microbes.86,90

Impact of Immune Activation onGut Sensorimotor Function

Supernatants obtained after incubation of mucosalbiopsies from IBS subjects contained increasedamounts of histamine, serotonin, polyunsaturated fatty acid

metabolites,81 and proteases, including tryptase andtrypsin.10 The exact source of proteases remains unclear;they may originate from mucosal MCs, gut bacteria,92 orpancreatic secretions.28 In adoptive transfer experiments,biopsy supernatants from IBS subjects evoked abnormalfunctional responses in enteric and sensory nerves ofrecipient rodents93–95 and human tissues.96 These effectswere at least partly related to immune and endocrine fac-tors, including proteases, histamine, and serotonin.73,93,95

Application of biopsy supernatants to human or rodenttissues suggests that, in IBS, serine proteases, or poly-unsaturated fatty acid metabolites, act respectively onproteinase-activated receptors93 and transient receptorpotential cation channel, subfamily V-4,10 to mediatevisceral pain. In addition to these acute effects, a recentstudy suggested that the chronic release of immune me-tabolites could affect the structure of mucosal neural net-works in IBS, that is, increased neuronal density andoutgrowth, as well as increased expression of MC nervegrowth factor in the colonic mucosa of patients with IBScompared with controls.97 Mucosal supernatants of patientswith IBS evoked increased neurite growth and expression ofGAP43 (a key neuronal growth protein) when applied toprimary cell cultures of rat myenteric plexus or to neuro-blastoma cell line SH-SY5Y cultures.97

Probiotics can have beneficial effects in IBS, through themodulation of immune function. Indeed, Bifidobacteriuminfantis 35624, but not strains of Lactobacillus salivarius,was able to reduce a systemic proinflammatory cytokineprofile along with symptom improvement.98

Immune Activation and SymptomsAlthough several studies have demonstrated intestinal

immune activation in FGIDs, reports of correlations withsymptoms have been limited. Correlations were found be-tween colonic MC density close to nerves and abdominalpain and bloating, and between bowel habit dissatisfaction,global IBS symptoms, and circulating T cells.85,99,100 Inaddition, immune activation featuring increased small bowelhoming T cells has been associated with the intensity ofpain, nausea, and vomiting in FD.101 Mucosal MCswere associated with fatigue and depression, suggestingthe potential role of psychological factors in thebrain�gut�immune axis in IBS.

Implications for ManagementDiet and Food Components

Food has long been recognized as an important precip-itant of symptoms in FD and IBS. Exaggerated GI and colonicelectrophysiological and motor responses to food ingestionhave been extensively documented and a variety of hy-potheses have been advanced to explain these responses,particularly in IBS. This process has also generated variousdiagnostic strategies, which will be briefly reviewed here.

Food allergy. Although up to 20% of the populationand much higher proportions of IBS patients are convincedthat they are allergic to certain foods, and this is a major

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contribution to their problem, food allergy, traditionallydenoted by an activation of IgE-mediated antibodies to afood protein, has not been linked convincingly to IBSpathogenesis and the status of IgG-based testing remainsunclear.102 Although confocal endomicroscopy studiessuggest the existence of rapid morphological and functionalchanges in the epithelium and immune system of the smallintestine after challenge with foods to which the patientsreported intolerance, and symptomatic improvement wererecorded following avoidance of these foods, this approachseems to be too cumbersome to be applied on a largescale.103

Food intolerance. The contribution of lactose maldi-gestion to IBS depends on the prevalence of lactose maldi-gestion in the population studied. Furthermore, subjectivereports of lactose intolerance correlate poorly with formaltests of lactose malabsorption, rendering such tests oflimited value in the evaluation of IBS.104 Lactose intoleranceshould be identified in patients with FGID and milk productsavoided accordingly.

Although IBS subjects appear, both subjectively andobjectively, to be intolerant of fructose,105 formal tests offructose malabsorption failed to discriminate between IBSsubjects and healthy controls.106 Again, it does not seempossible to recommend testing or dietary fructose elimina-tion alone as an evidence-based treatment strategy for IBSor other FGIDs.

Sorbitol intolerance has also been reported in IBS and itis likely that sorbitol has an additive effect to fructose, withfurther exacerbation of symptoms.106 Here again rates ofintolerance and malabsorption do not tally, thereby, limitingthe value of diagnostic testing or dietary advice.

Gluten intolerance/sensitivity. The relationshipsbetween celiac disease, “gluten-sensitivity,” and IBS remainunclear, with various studies reporting increased107 orexpected108 rates of celiac disease among IBS subjects. Thestatus of that entity which has come to be referred to asNCGS109 is particularly unclear. Other than excluding celiacdisease, and providing evidence of intolerance after double-blind challenge, there are no currently validated diagnosticmethods for diagnosing this entity.

The Microbiota and its Metabolic InteractionsSmall intestinal bacterial overgrowth, fecal and

colonic mucosal microbiota. The status of SIBO in IBSremains highly controversial. Two factors contributing tovariations in prevalence of SIBO among IBS subjects havebeen the test modality and diagnostic criteria used to di-agnose SIBO.110 Although some patients with SIBO maypresent with IBS-type symptoms, it does not appear thatSIBO is a major contributor to the pathogenesis of IBS, ingeneral.111 The lactulose breath test has shown poor diag-nostic performance to detect SIBO. While the glucose breathtest performs slightly better, routine testing for SIBO cannotbe currently recommended.30

Although abnormalities in the fecal and colonic micro-biota have been identified among IBS subjects and microbialsignatures associated with certain demographic and etio-logical features in IBS, a fecal or mucosal microbial signal

diagnostic of IBS or of an IBS subtype or subpopulation hasyet to be validated.30 Approaches aimed at modifying themicrobiota, mainly with probiotics and nonabsorbable an-tibiotics, are now widely applied in clinical practice,particularly in patients with IBS, however, several questionsremain to be elucidated, including, type of probiotics,dosage, relevant subgroups, therapeutic gain over placebo,treatment and retreatment schedules, as well as mode ofaction.30

Bile acids. Abnormalities in fecal BAs, as well as inserum markers of BA synthesis, have been reported in asubgroup of IBS-D,112 and BAM may be responsible for asignificant proportion of those with IBS-D.113 The 23-seleno-25-homo-tauro-cholic acid test, the most widelyemployed and validated test for the diagnosis of BAM, is notuniversally available. Alternate approaches include themeasurement of fecal BAs, or serum levels of 7 a-hydroxy-4-cholesten-3-one (C4), or a therapeutic trial of a bile saltsequestering agent.34,41

Epithelium and Mucosal BarrierThe usefulness of measures of barrier integrity (such as

the lactulose-to-mannitol excretion ratio) in the diagnosis orassessment of FGIDs has not been established,114 nor havethe diagnostic role of ex vivo approaches on biopsies(Ussing chambers) and assays for molecular markers orsurrogates of altered permeability (eg, endotoxin, anti-lipopolysaccharide antibodies, bacterial lactate, butyrateproduction, and hemolysin test).49,114 The use of endoscopicendomicroscopy detecting rapid functional/structuralmucosal changes after challenge with food allergens,although attractive, remains to be confirmed in futurestudies.103 Strategies to modify mucosal permeabilityinclude the use of probiotics and dietary interventions,although there is still uncertainty on the potentialbenefits.49

Enteroendocrine SystemAlthough changes in basal or stimulated levels of a

number of enteric hormones and neurotransmitters (suchas postprandial levels of 5-HT) have been described in IBSand other FGIDs, and manipulations of 5-HT metabolismhave been shown to provoke symptoms, none have achievedthe status of a diagnostic test. The use of drugs acting onserotonin agents remains a field of interest, now generatingnew potential therapeutic approaches for IBS-D with olderproducts with a new indication (eg, ondansetron)115 ornewer products (eg, ramosetron)76 being tested in largeclinical trials.

Immune System and Neuro-Immune InteractionsThat the engagement between luminal contents on one

hand, and the microbiota and the immune system on theother hand, might be relevant to the pathogenesis of IBS issuggested by studies documenting the up-regulation of im-mune biomarkers and various members of the Toll receptorfamily in this disorder. However, given the variability inresults between studies, it is not possible at this time to

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employ measures of the mucosal or systemic responses inthe diagnosis of an FGID or in the delineation of a subgroupthereof.116 Two recent large placebo-controlled studies inIBS patients showed that mesalazine was not clinically su-perior over placebo, although both studies suggestedthat subgroups, including PI-IBS, showed sustainedresponses.117,118

With respect to gases released in the process of bacterialfermentation, it should be noted that with some, but notcomplete, consistency, the detection of methane in thebreath has been linked to the predominance of constipationin IBS.119 The therapeutic implications of these findingsremain unclear.

Conclusions and Future DirectionsWhile the role of food and dietary components is, at last,

attracting the attention it has long deserved, many questionspersist. While dietary studies are challenging, only large-scale, prospective studies using validated instruments canprovide much needed information on the food habits ofFGID subjects and also address such questions as to whatextent dietary preferences in FGID subjects reflect thesubconscious exclusion of items to which they are intol-erant. Of the multiple factors that have been proposed,intolerance to poorly absorbed dietary carbohydrates hasemerged as a major contributor in IBS, with the status oftrue food allergy and gluten sensitivity remaining unclear.Different mechanisms may be relevant to various subjectsor subject groups. Long-term studies of dietary in-terventions will be important by defining, not just theirbeneficial effects on symptoms, but also identifying anynegative consequences.

Although some tantalizing findings have been reported,studies of the gut microbiota and the host immune responsein FGIDs have yielded variable and sometimes conflictingresults: there is a need for longitudinal studies that includefunctional profiling of the microbiota, its metabolites(including gases and SCFAs) and related immunologicalresponses in well-characterized IBS populations. Such in-formation will be critical to guiding therapeutic strategiesthat aim to modulate the microbiota, its products, and/orthe immune response.

The signal transduction mechanisms that generate re-sponses to BAs need to be delineated, that is, whether theyare receptor-mediated or generated by paracrine ornonspecific effects. Above all, in relation to BAs, the currentpaucity of human studies needs to be addressed. Further-more, microbiota�BA interactions in FGIDs need to beexplored.

While functional and molecular alterations of factorsinvolved in intestinal permeability might explain IBS path-ophysiology and symptom generation, and could yieldinnovative biomarkers valuable in both diagnosis andassessment of therapeutic response in FGIDs, furtherstudies using validated and preferably noninvasive markersare needed to more precisely define the mechanisms andfunctional consequences of these alterations and their pri-macy in a given FGID.

Well-designed studies on large numbers of patients andcontrols evaluating the enteroendocrine system, includingchanges in serotonergic responses, are also lacking in pa-tients with FGIDs. Dietary, behavioral, pharmacological, andgut-microbiota�directed manipulations of enteroendocrineresponses are likely to be important approaches to themanagement of these disorders.

Supplementary MaterialNote: The first 50 references associated with this article areavailable below in print. The remaining references accom-panying this article are available online only with the elec-tronic version of the article. To access the supplementarymaterial accompanying this article, visit the online versionof Gastroenterology at www.gastrojournal.org, and athttp://dxdoi.org/10.1053/j.gastro.2016.02.028.

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Reprint requestsAddress requests for reprints to: Giovanni Barbara, MD, Department of Medicaland Surgical Sciences, St Orsola-Malpighi Hospital, Via Massarenti, 9, Building#5, I-40138 Bologna, Italy. e-mail: giovanni.barbara@unibo.it; fax: (39) 051345864.

AcknowledgmentsThe authors would like to thank Maria Raffaella Barbaro for editing themanuscript.

Conflicts of interestThe authors disclose no conflicts.

(IBS:HC)

Proportion ofabnormal

values (IBS)Diagnosticcriteria

19:10 42% (8/19) Rome III

54:22 39% (21/54) Rome III

93:52 ns Rome II

93:52 ns Rome II

15:15 ns Rome II

15:16 ns Rome II

31:12 23% (7/31) Rome I

93:52 ns Rome II

14:15 ns Rome II

15:12 ns Rome II

20:30 ns Rome II

13:5 77% (10/13) Rome II

34:15 ns Rome III

14:15 ns Rome II

13:5 ns Rome II

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Supplementary Table 1.Evidence of Altered Intestinal Permeability in IBS.

FindingsMagnitudeof change Site Methods Clinical subtype N

Intestinal permeabilityIncreased ns small intestine and

colon5h and 24h urinary recoveries of orally

administered sugars (lactulose/mannitol urinary ratio)

D-IBS

ns ns 24h urinary recoveries of orallyadministered sugars (lactulose/mannitol urinary ratio)

D-IBS

þþ proximalgastrointestinaltract

3h urinary recoveries of orallyadministered sugars (sucrose/lactulose urinary ratio)

FAP/IBS (children)

þ colon 3h urinary recoveries of orallyadministered sugars (sucralose/lactulose urinary ratio)

FAP/IBS (children)

þþþ small intestine 5h urinary recoveries of orallyadministered 51Cr-EDTA

PI-D-IBS but not C-IBS

þþ small intestine 5h urinary recoveries of orallyadministered 51Cr-EDTA

D-IBS þ PI-D-IBS

þþþ small intestine 6h urinary recoveries of orallyadministered sugars (lactulose/mannitol urinary ratio)

PI-IBS

Unchanged - small intestine 3h urinary recoveries of orallyadministered sugars (lactulose/mannitol urinary ratio)

FAP/IBS (children)

- colon 24h urinary recoveries of orallyadministered polyethylene glycols orsugars (lactulose/mannitol urinaryratio)

IBS

- colon 24h urinary recoveries of orallyadministered 51Cr-EDTA

PI-D-IBS þ C-IBS

Increased in response totryptase

þ rectum HRP diffusion through the rectal biopsiesin Ussing chambers

D-IBS

Increased paracellularpermeability

þþ colon FITC diffusion through the colon biopsiesin Ussing chambers

IBS

þ colon FITC diffusion through the colon biopsiesin Ussing chambers

C-IBS þ D-IBS þ A-IBS

Increased in response toNSAIDs

ns colon 24h urinary recoveries of orallyadministered sugars (lactulose/mannitol urinary ratio)

IBS

Decreased transepithelialresistance

þþ colon FITC diffusion through the colon biopsiesin Ussing chambers

IBS

Supplementary Table 1.Continued

FindingsMagnitudeof change Site Methods Clinical subtype N (IBS:HC)

Proportion ofabnormal

values (IBS)Diagnosticcriteria

Epithelial barrier integrityDecreased tight junction

protein occludinþþ colon mucosa WB D-IBS but neither C-IBS

nor A-IBS50:33 32% (6/19) ;

18% (9/50total IBS)

Rome III

þþ colon mucosa WB (no change in qRT-PCR) IBS 25:18 ns Rome IIDecreased tight junction

protein claudin-1þþ colon mucosa WB (no change in qRT-PCR) D-IBS but neither C-IBS

nor A-IBS50:33 53% (10/19) ;

36% (18/50total IBS)

Rome III

Decreased tight junctionprotein zonulaoccludens-1

þþþ colon mucosa qRT-PCR IBS 21:12 ns Rome IIþþ colon mucosa WB IBS 50:33 ns Rome III

NOTE. Data from Matricon J, Meleine M, Gelot A, et al. Review article: Associations between immune activation, intestinal permeability and the irritable bowel syndrome.Aliment Pharmacol Ther 2012;36:1009-31.

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Supplementary Table 2.Molecular alterations in patients with IBS and FD2

Molecular alterations Localization Clinical subtype

Reduced ZO-1 expression Cecum IBS-A and IBS-DReduced E-cadherin expression Cecum IBS-ADecreased occludin expression Colon IBS-CReduced JAM-A expression Cecum IBSDecreased ZO-1 expression Small intestine IBS-DDecreased ZO-1, claudin-1 and occludin expression Rectosigmoid IBS-DDecreased ZO-1 and occludin expression Rectosigmoid IBS-DIncreased claudin-2 expressionReduced occludin phosphorylation and enhanced redistribution

from the member to the cytoplasmIncreased myosin kinase expressionReduced myosin phosphatase expressionEnhanced phosphorylation of myosin

Jujunum IBS-D

Reduced ZO-1 expressionZO-1 redistribution from the TJ to the cytoplasm

Jejunum IBS-D

Decreased occludin and claudin-1 expression Descending colon IBS-DAltered subcellular distribution of occluding and claudin-1 Descending colon IBS-C and IBS-DDecreased occludin expression Descending colon IBSReduced ZO-1 expression Colon IBSReduced ZO-1 expressionDecreased occludin expressionReduced phosphorylation of serine / threonine residues (p-OCLN)Reduced b-catenin expressionReduced E-cadherin expressionReduced desmocollin-2 and desmoglein-2 expression

Duodenum FD

NOTE. Data from Martinez C, Gonzalez-Castro A, Vicario M, et al. Cellular and molecular basis of intestinal barrier dysfunctionin the irritable bowel syndrome. Gut Liver 2012;6:305-315.

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Supplementary Table 3.Summary of Major Chemical Messengers Controlling Gastrointestinal Function

Name Chemical nature Cell of origin Action Mechanism of actionKnown abnormalities in

patients with IBS

Serotonin 5-hydroxytryptamine Enterochromaffin cells andenteric neurones

Increases motility andvisceral sensation

Paracrine and neurocrine Increased and reducedactivity in IBS-D andIBS-C

Nitric oxide (NO) Gas Enteric nitrinergic nerves Relaxation of gut andvascular smoothmuscles

Inhibitory neurotransmitter Alters gut motility

Vasoactive intestinalpeptide (VIP)

28 amino acid (AA) peptide Enteric neurones Relaxation of gut andvascular smoothmuscles

Inhibitory neurotransmitter Higher levels in IBS thanhealthy controls

Ghrelin 28 AA peptide Oxyntic cells of stomach Stimulation of gastric andintestinal motility

Endocrine Lower density of ghrelincells in IBS-C andhigher in IBS-D

Neurotensin 13 AA peptide Intestinal N cell and entericneurones

Induces ileal brake, colonicmotility, pancreaticsecretion and inhibitsgastric secretion

Endocrine

Substance P 11 AA peptide Enteric neurones Smooth musclecontraction andinhibition of gastric acidsecretion

Neurotransmitter Increased substance Pcontaining nerves inIBS than controls

Somatostatin 14 and 28 AA peptides Intestinal D cells and entericneurones

Inhibits digestive secretionand post-prandial gutmotility

Paracrine and endocrine Reduced D cell density inIBS-C and IBS-D

Neuropeptide Y (NYY) 36 AA peptide Enteric neurones Decreases gut motility anddigestive secretion

Neurotransmitter Lower NYY in IBS-D thanIBS-C

Gastric inhibitory peptide(GIP)

42 AA peptide Small intestinal cells Inhibits gastric acidsecretion

Endocrine Reduced GIP cell density inIBS-C and IBS-D

Peptide YY (PYY) 36 AA peptide Intestinal H/L cell Reduces gut motility anddigestive secretion

Endocrine and paracrine Reduced PYY cell densityin IBS-C and IBS-D

Secretin 27 AA peptide Intestinal S cell Reduced gastric, small andlarge bowel motility

Endocrine Reduced S cell density inIBS-D, but not in IBS-C

Enteroglucagon 69 AA peptide Intestinal L cell Inhibits gastric, pancreaticsecretion and mediatesileal brake

Endocrine

Cholecystokinin (CCK) 8, 33, 39, 58 AA peptides Intestinal I-cell and neurones Delay gastric but enhancegallbladder and gutmotility

Endocrine andneurotransmitter

Reduced CCK cell densityin IBS-D, but not inIBS-C

Galanin 30 AA peptide Enteric neurones Inhibits gastric, pancreaticand intestinal secretion

Neurotransmitter

Motilin 22 AA peptide Intestinal M cell Induces migrating motorcomplex

Reduction in IBS thanhealthy control

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Supplementary Table 3.Continued

Name Chemical nature Cell of origin Action Mechanism of actionKnown abnormalities in

patients with IBS

Gastrin 17 and 34 AA peptide Gastric G-cell Stimulates gastric acidsecretion

Endocrine

Pancreatic polypepdide(PP)

36 AA peptide Intestinal PP cell Relaxation of gallbladderand stimulation of gutmotility

Endocrine Higher in IBS-D andreduced in IBS-C

NOTE. Data from: El-Salhy M, Gundersen D, Gilja OH, et al. Is irritable bowel syndrome an organic disorder? World J Gastroenterol 2014;20:384-400; Miller LJ.Gastrointestinal hormones and receptors. Philadelphia, Pa: Lippincott-Williams and Wilkins, 1999; Furness JB, Rivera LR, Cho HJ, et al. The gut as a sensory organ. NatRev Gastroenterol Hepatol 2013;10:729-740.

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