Gastroenterology 2016;150:1280–1291

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Fundamentals of Neurogastroenterology: Basic Science

Stephen J. Vanner,1 Beverley Greenwood-Van Meerveld,2 Gary M. Mawe,3

Terez Shea-Donohue,4 Elena F. Verdu,5 Jackie Wood,6 and David Grundy7

1Gastrointestinal Diseases Research Unit, Kingston General Hospital, Kingston, Ontario, Canada; 2Oklahoma Center forNeuroscience, Department of Physiology, VA Medical Center, University of Oklahoma, Health Sciences Center, Oklahoma City,Oklahoma; 3Department of Neurological Sciences, Pharmacology and Medicine Division, Gastroenterology and Hepatology,University of Vermont, Burlington, Vermont; 4Department of Medicine and Physiology, University of Maryland School ofMedicine, Baltimore, Maryland; 5Farncombe Family Digestive Health Research Institute, McMaster University, Health SciencesCenter, Hamilton, Ontario, Canada; 6Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio;7Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom

This review examines the fundamentals of neuro-gastroenterology that may underlie the pathophysiology offunctional GI disorders (FGIDs). It was prepared by aninvited committee of international experts and representsan abbreviated version of their consensus document thatwill be published in its entirety in the forthcoming bookand online version entitled Rome IV. It emphasizes recentadvances in our understanding of the enteric nervoussystem, sensory physiology underlying pain, and stresssignaling pathways. There is also a focus on neuro-immmune signaling and intestinal barrier function, giventhe recent evidence implicating the microbiome, diet, andmucosal immune activation in FGIDs. Together, these ad-vances provide a host of exciting new targets to identifyand treat FGIDs, and new areas for future research intotheir pathophysiology.

Keywords: Sensory Physiology; Enteric Nervous System; Neu-roimmune Signaling; Mucosal Barrier Function.

n the 8 years since the publication of Rome III there

Abbreviations used in this paper: CNS, central nervous system; CRF,corticotrophin-releasing factor; DC, dendritic cell; EC, enterochromaffin;ELA, early life adversity; ENS, enteric nervous system; FGID, functionalgastrointestinal disorder; HPA, hypothalamic-pituitary-adrenal; 5-HT,5-hydroxytryptamine; IBS, irritable bowel syndrome; ICC, interstitial cellsof Cajal; IEC, intestinal epithelial cells; IEL, intraepithelial lymphocytes; IL,interleukin; ILC, innate lymphoid cells; PAG, periaqueductal gray; PFC,prefrontal cortex; PRR, pattern recognition receptor; SERT, serotonin-selective reuptake transporter; TLR, Toll-like receptor; TRP, transientreceptor potential; VIP, vasoactive intestinal polypeptide.

Most current article

© 2016 by the AGA Institute0016-5085/$36.00

http://dx.doi.org/10.1053/j.gastro.2016.02.018

Ihas been rapid expansion in our understanding of thefundamentals of neurogastroenterology. What has fueledthis advance is the desire to integrate basic science researchwith clinical gastroenterology to better diagnose and treatfunctional gastrointestinal disorders (FGIDs). This researchcontinues to shed light on the complex hierarchy of neural,molecular, and cellular interactions that control gut func-tion. However, what recent research also has shown is thecomplex interaction between the host gut wall and theluminal microbial environment that is responsible forbalancing immune tolerance with protection against path-ogenic and antigenic material. Neuroimmune function andthe mechanisms that regulate mucosal barrier function,immune surveillance, innate and adaptive immunity, sen-sory signaling, and central nervous system (CNS) adaptationconsequently are the major themes for this review.

The Basis of Brain–Gut InteractionsThe GI tract has important barrier and immune functions

that interface with the luminal microbiota and protectagainst potential pathogenic and antigenic material. Integral

to these ostensibly conflicting functions is the ability tomonitor events in the gut wall and within the gut lumen toorchestrate reflexes that bring about appropriate patterns ofmotility, secretion, and blood flow to digest and absorb or todilute and expel. GI sensory mechanisms play a pivotal rolein triggering these reflexes by conveying sensory informa-tion to the enteric reflex circuits that provide local controland through afferent pathways to the CNS.

Pathways From Gut to BrainSensory information is conveyed from the GI tract to the

brainstem and spinal cord via vagal and spinal (splanchnicand pelvic) afferents, respectively. Most dorsal root ganglionneurons innervate somatic structures. It is estimated thatthe proportion of dorsal root ganglion neurons innervatingthe GI tract range between 3% and -7%. The dominance ofsomatic afferent input to the spinal cord and the conver-gence of visceral and somatic afferents on ascending spinalpathways accounts for the phenomenon of referred pain. Inaddition, afferent fibers from the colon and rectum mayconverge with fibers from other pelvic organs, contributingto cross-organ sensitization between gut, bladder, andreproductive organs that often complicates the clinicaldiagnosis of pelvic pain.1 The low density of innervation,convergence with somatic inputs, and viscerovisceralconvergence in the spinal cord can explain why gut paingenerally is localized poorly.

Subtypes of Visceral AfferentsGI afferent fibers terminate within the gut wall mainly as

bare nerve endings and are classified according to their

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terminal distribution as mesenteric, serosal, muscular,ganglionic (intraganglionic laminar endings), or mucosalendings.2 The location of these endings plays an importantrole in determining the functional properties of the afferent.Mucosal afferents respond to distortion of the mucosalepithelium and to luminal chemicals. Stretch or distension iseffective for stimulating endings in the muscle layers,ganglia, and serosa. These endings express an array ofmembrane receptors and ion channels that determineneuronal excitability, mechanosensitivity, and modulationby a host of chemical mediators within the GI milieu.Different populations of afferents respond over a range ofdistension volumes from innocuous (physiological) tonoxious levels that cause pain. Powerful contractions,especially against an obstruction, cause traction on themesentery and is especially painful.

There is a continuous barrage of information projectingfrom the gut to the CNS. Many afferent endings respond tolevels of distension that occur as part of normal digestionand these usually go unperceived. Instead, this informationis used in reflexes that control motility, secretion, bloodflow, and other aspects of GI function. In contrast, there areother afferents that respond only at high levels of stimulusintensity and function as nociceptors that mediate pain.Some afferents (so-called silent or “sleeping” nociceptors)are mechanically insensitive under normal circumstancesbut can be awakened in response to inflammation orinjury. In patients this process of sensitization can give riseto altered pain perception. In some cases, stimuli thatnormally are innocuous can cause pain (allodynia),whereas responses that are painful can become exagger-ated (hyperalgesia).

MechanotransductionMechanotransduction refers to the process by which

stimulus energy is interpreted by sensory nerve endings,leading to the generation of action potentials. There arespecific molecular mechanisms that underlie mechano-transduction. Moreover, the excitability of the afferentending is determined by various voltage-gated and calcium-dependent ion channels3 that set gain in the system, andthat can change according to external influences leading tohypersensitivity.

Sensory endings contain a variety of mechanosensitiveion channels that can convert the stimulus energy into ac-tion potentials. They respond to membrane deformation,causing channels to open or close, carrying ionic currentsinto or out of the nerve terminal to cause depolarization.Three main ion channel families have been identified asmechanosensitive: (1) the DEG/ENaC family that includesthe acid-sensing ion channels 1, 2, and 3; (2) the transient-receptor potential (TRP) channel family; and (3) the 2-porepotassium channel family that includes TREK-1 and TRAAK.Different combinations of these channels exist in differentpopulations of vagal, pelvic, and splanchnic afferents, sug-gesting a complex heterogeneity in sensory signaling.4

Another mechanism of mechanotransduction occurswhen a secondary sense cell releases mediators that act on

ionotropic or metabotropic receptors to stimulate sensoryendings. This indirect mechanism relies on close associationbetween afferent endings in the gut wall and various othercell types that are a source of these chemical ligands. Theseinclude mast cells, epithelial cells, enteroendocrine cells,macrophages, interstitial cells of Cajal (ICC), and entericneurons. Considerable attention has been paid to the role of5-hydroxytryptamine (5-HT) and adenosine triphosphate insensory signaling, especially in the context of post-inflammatory hypersensitivity.5

Luminal SensingSome vagal and pelvic afferent endings come into close

proximity to the mucosal epithelium, but never penetratethrough to the lumen. However, their proximity to the mu-cosa exposes them to chemicals absorbed across themucosal epithelium or released from enteroendocrine cellswhose apical membrane is exposed to luminal content. Thisis similar to the relationship seen between taste buds in themouth and gustatory afferents and as such provides amechanism by which mucosal afferents can taste luminalcontents. This is important for controlling digestive functionvia reflex effects on motility and secretion. However,nutrient detection also influences metabolic activity andenergy intake. The molecular basis for each modality ofgustatory taste has been identified. Strikingly, many of thesesame G-protein–coupled receptors and ion channels areexpressed within the GI tract. The cells expressing taste-receptor molecules in the GI mucosa have a characteristicmorphology, which is typified by the enterochromaffin (EC)cell.6 However, EC cells are just one of a diverse family ofenteroendocrine cells that are scattered diffusely in the GImucosa and whose mediators can act in a paracrine fashionon afferent fibers or diffuse into the blood stream for moredistant endocrine actions. Each type of enteroendocrine cellhas a characteristic distribution along the GI tract. Amongthe mediators released, cholecystokinin and glucagon-likepeptide-1 play important roles in reflex control of GI func-tion and in regulating food intake.

Peripheral SensitizationSensory neurons express a large array of receptors that

are activated by mediators released from various cellularsources within the gut wall. Neurotrophins, for example,play a role in axon guidance and remodeling of the sensoryinnervation after inflammation and injury. Their receptorsare expressed on different populations of GI sensory neu-rons. Both nerve growth factor and glial-derived neuro-trophic factor are important in the adaptive response tonerve injury and inflammation. Both also are possible me-diators underlying chronic pain. Increasing neurotrophinsignaling causes increased TRP channel expression (eg,TRPV1 and TRPA1), an increase in sodium channelexpression (NaV1.87), and a decrease in potassium channels.Any, or all of these, could contribute to the development ofhypersensitivity.8

Many other mediators are released during inflammation,injury, and ischemia, from platelets, leukocytes, lymphocytes,

Figure 1.Mechanismsunderlying sensitization.Luminal factors and medi-ators released in responseto ischemia, injury, andinflammation act on thesensory endings to drivesensitization. These pe-ripheral mechanisms arereinforced by centralmechanisms in the spinalcord and CNS. ATP,adenosine triphosphate;LIF, leukemia inhibitoryfactor; NGF, nerve growthfactor; PGE, prostaglandinE; TNF, tumor necrosisfactor. Modified fromGrundy and Brookes2 withpermission from Morganand Claypool.

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macrophages, mast cells, glia, fibroblasts, blood vessels,muscle, and neurons. Some mediators act directly on sensorynerve terminals and others act indirectly, causing release ofyet other agents from nearby cells. This “inflammatory soup”(Figure 1) contains amines, purines, prostanoids, proteases,cytokines, and so forth, which act on sensory nerve terminalsto increase sensitivity to both mechanical and chemicalstimuli (referred to as “plasticity”). Recent data have sug-gested that bacterial products also may drive afferentsignaling.9 Hypersensitivity is a feature of chronic pain statesand is considered to be a hallmark of FGIDs including irri-table bowel syndrome (IBS). Moreover, because theseafferents also trigger reflexes that coordinate gut function,sensitization also can cause hyper-reflexia or dysreflexia,leading to altered transit, resulting in diarrhea andconstipation.

Peripheral sensitization normally develops rapidly and isrelatively short-lived. However, in the presence of main-tained injury or inflammation, the sensitization can be pro-longed by changes in gene expression. These genes may alterthe expression of channels, receptors, or mediators in thesensory neuron.8 They also may modify the amount andpattern of neurotransmitters released by central nerve ter-minals in the brain and spinal cord. This alters the way thatsensory signals are processed within the CNS and contrib-utes to “central sensitization,”10 and may prolong hyper-sensitivity beyond the acute period of injury or inflammation.

Central SensitizationThese mechanisms can undergo plasticity in response to

injury and inflammation, leading to hypersensitivity andchronic pain states. These neurons transmit visceral signalsto ascending spinal pathways via glutamate and neuropep-tides. These transmitter mechanisms are up-regulated inresponse to inflammation and injury and contribute tohypersensitivity.

In the brain and spinal cord there are central neuro-plastic changes, termed central sensitization, that contributeto chronic pain. Within the dorsal horn of the spinal cord,there are 2 mechanisms that increase pain signals reachingthe brain: (1) increased synaptic transmission via glutamate,calcitonin gene-related peptide, and substance P ontoascending excitatory pathways, and/or (2) decreaseddescending inhibitory modulation. In the brain, sensitizationcan occur in the second-order spinal neurons, such as thethalamus, periaqueductal gray (PAG), parabrachial nucleus,and locus coeruleus. Increased signaling from those nucleithen can promote neuroplasticity, similar to long-termpotentiation mechanisms, that strengthen and/or add syn-aptic connectivity. The enhanced signaling then promotesabnormal processing of pain within the extended pain ma-trix (prefrontal cortex [PFC], anterior cingulate cortex,amygdala, insula), which can amplify the discomfort andnegative emotions associated with chronic visceral pain,11

and/or a decrease in the descending pain inhibitory sys-tem through the PAG and rostroventral medulla.12 Inparticular, the amygdala is a key nucleus that integratesnoxious visceral signals with anxiety/fear behaviors andhyperactivation could influence not only multiple nuclei inthe central pain matrix, but also descending brainstemnuclei that modulate GI function.13 Multiple clinical imagingstudies also have shown differences in function, connectiv-ity, and structure between IBS and healthy controls. Thus,central sensitization can promote chronic abdominal pain inIBS through remodeling of connections within both thebrain and spinal cord.

ENS NeurobiologyA universal perception of the enteric nervous system

(ENS) as a brain-in-the-gut implies that, similar to the brainand spinal cord, the ENS is assembled in a hierarchy ofneural organization.14,15 Output from the ENS determines

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moment-to-moment behavior of the gastrointestinalmusculature, secretory glands, and blood vasculature. Inte-gration of output to the muscles and secretory glands isreflected by coordinated patterns of motility and secretion,recognizable during clearly defined digestive states. Fivedifferent behavioral states are recognizable in the smallintestine: (1) physiological absence of motility; (2) post-prandial state with segmenting (mixing) motility integratedwith set-point feedback control of luminal pH and osmo-larity; (3) migrating motor complex in the interdigestivestate also integrated with set-point feedback control ofluminal pH and osmolarity; (4) a defensive state withcopious neurogenic hypersecretion and orthograde orretrograde power propulsion associated with urgency,diarrhea, and cramping abdominal pain; and (5) emeticprogram, which includes reversal of peristaltic propulsion inthe upper jejunum and duodenum to rapidly propel luminalcontents toward the open pylorus and relaxed antrum andcorpus. Coordinated neurogenic patterns of behavior in thelarge intestine are recognized as haustral formation, physi-ological absence of motility, defecatory power propulsionand defense that also is associated with urgency, diarrhea,and cramping lower abdominal pain.

Similar to the CNS, the ENS functions with chemicalsynaptic connections between sensory neurons, in-terneurons, and motor neurons. Interneurons are inter-connected synaptically into neural networks, which processinformation on the state of the gut, contain a library ofprograms for different patterns of behavior, and control theactivity of motor neurons. Motor neurons innervate themusculature, secretory glands, and blood vessels. Muscu-lomotor neurons initiate or inhibit the contractile activity ofthe musculature when they fire.15 Modulation of their firingfrequency, by input from interneuronal microcircuitry, de-termines minute-to-minute contractile strength. Secreto-motor neurons stimulate secretory glands to secretechloride, bicarbonate, and mucus,16,17 and determine theosmolarity and liquidity in the lumen. Neurogenic control ofbicarbonate secretion maintains a physiological pH set-pointin the lumen and accounts for some of the mucosal pro-tection against acid delivery from the stomach. A subset ofsecretomotor neurons simultaneously innervates bothsecretory glands and periglandular arterioles, and therebyenhance blood flow with secretion.

Interaction of the ENS with ICC18 is a major determinantof each of the motility programs stored in its library. Elec-trically conducting junctions (gap junctions) connect smoothmuscle fibers one to another to form a functional electricalsyncytium. Action potentials propagate from muscle fiber tomuscle fiber in 3 dimensions and trigger a contraction asthey enter each neighboring muscle fiber. ICC are non-neuronal pacemaker cells that also connect one to anotherto form electrical syncytial networks that extend around thecircumference and throughout the longitudinal axis of thesmall and large intestine. The ICC networks generate elec-trical pacemaker potentials (also called electrical slow waves)that spread via gap junctions into the intestinal circularmuscle, where they depolarize the muscle to action potentialthreshold and thereby trigger contractions.

The functional characteristics of the circular muscle as aself-excitable electrical syncytium implies that ICC networksshould continuously evoke contractions that spread in 3dimensions throughout the entire syncytium, which is ineffect the entire length of the intestine. Nonetheless, in thenormal bowel, long stretches of intestine are found in a stateof physiological ileus. Attention to the functional electricalsyncytial properties of the musculature suggests thatinhibitory musculomotor neurons and control of their ac-tivity by the integrative microcircuits in the ENS haveevolved as a mechanism that determines when ongoing slowwaves initiate a contraction, as well as the distance anddirection of propagation after the contraction starts.

Overall, a normal ENS is essential for a healthy boweland absence of irritating symptoms, such as those associ-ated with Rome-based diagnostic criteria for FGIDs. Anyneuropathic change in the ENS most likely will result in asymptomatic bowel. Functional propulsive motility and itsintegration with specialized secretory functions cannotwork in the absence of the ENS, as underscored in theaganglionic terminal segment of Hirschsprung’s disease andautoimmune ENS denervation of the lower esophagealsphincter in achalasia.

ENS Neuroplasticity inPathophysiological Conditions

Gut functions are altered under various pathophysio-logical conditions, and it has become increasingly clear thatalterations in the intrinsic reflex circuits of the gut areinvolved. Over the past decade, much progress has beenmade toward determining what elements of the circuits arealtered, the mechanisms of these alterations, which changespersist after recovery from inflammation, and the effects ofneuroplasticity on propulsive motility.

Mucosal Serotonin SignalingOne mechanism of activating enteric neural reflex cir-

cuits is the release of 5-HT from EC cells in the intestinalmucosa.19 Serotonin released from EC cells activatesintrinsic enteric reflexes and also sends signals related todigestive reflexes, satiety, and pain to the CNS via vagal andspinal afferents. Serotonin signaling is terminated by reup-take into epithelial cells, all of which express the serotoninselective reuptake transporter (SERT) on their basal surface.A consistent feature of mucosal 5-HT signaling in theinflamed bowels of human beings and experimental animalsis a decrease in SERT expression.19 This has been shown inulcerative colitis and diverticulitis in human beings, and alsoin diarrhea-predominant and constipation-predominant IBS.The effects of decreased SERT expression are likely to becomparable with those related to serotonin-selective–receptor inhibitor use, with increased mucosal 5-HT avail-ability resulting in alterations in gut reflexes.

Decreased SERT expression in the inflamed bowel is likelyto involve the actions of the proinflammatory cytokines, tu-mor necrosis factor a, and interferon g.20 The contributingfactors for decreased SERT in IBS have not been identified,

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but it may involve a genetic predisposition, given that certainpolymorphisms of the SERT gene are associated withdecreased SERT expression. It also is possible that alteredSERT expression in IBS develops as a compensatory responseto altered gut function; however, SERT expression is notaltered in opiate-induced constipation.21

Impact of Enteric Neuroplasticityon Gut Functions

Inflammation is associated with changes along the ENSreflex circuitry that include increased 5-HT availability, hy-perexcitability of AH (sensory) neurons, interneuronal syn-aptic facilitation, and suppressed purinergic neuromusculartransmission22 (Figure 2). It is highly likely that these alter-ations lead to changes in neurogenic secretory and motorfunctions in the bowel, but the nature of the changes probablydiffers between secretory and motor responses. Neurogenicsecretion can be activated by 5-HT release from EC cells, andinvolves a 2-neuron reflex circuit consisting of an AH neuronand an S neuron. With increased 5-HT availability, AH neuronhyperexcitability, and a strengthening of synaptic signals tothe secretomotor (S) neurons, it is likely that secretion isenhanced. One potential pitfall in this scheme is that 5-HTreceptors on the processes of AH neurons could becomedesensitized by increased exposure to 5-HT.

The effects of neuroplastic changes on motility are moreconvoluted than secretion because the reflex circuitry ismore complicated, involving an excitatory signal passingupstream from a given site and an inhibitory signal passingdownstream. For an unequivocal set of signals to be trans-mitted, there cannot be much noise in the system. Thisquiescent background state is disrupted in the inflamedcolon by increased 5-HT availability in the lamina propriaand by increased spontaneous activity of AH neuronsthroughout the inflamed regions. This results in an overlap

of contradictory ascending and descending signals at a givensite, and a decrease in the ability of the ENS to generate thepressure gradient that result in propulsive motility, result-ing in a form of pseudo-obstruction. Experimentally,increasing AH neuron excitability in normal colons disruptsmotility whereas suppressing hyperexcitability of AH neu-rons in inflamed preparations improves motility.23

Furthermore, when the inhibitory junction potential isprotected and AH neuron activity is attenuated in trini-trobenzene sulfonic acid–inflamed colons, propulsivemotility is restored to its control velocity.24 These findingsunderscore the delicate balance of enteric neural signaling,especially as it relates to motor functions.

Neuroimmune Cross-TalkNo perfect animal model exists for investigating the

neurophysiological basis of altered motility in FGIDs, butone approach that has been used is to determine whatinflammation-induced neuroplastic changes persist beyondthe recovery of inflammation. This approach is obviouslyrelevant to postinfectious IBS, but in the past decade anumber of studies have shown that IBS is accompanied by adetectable increase in immune cells and inflammatory me-diators in the mucosal layer. Furthermore, many inflam-matory bowel disease patients show IBS-like symptomsafter resolution of their macroscopic inflammation. There-fore, inflammation-induced changes in neuronal functioncould be a contributing factor in IBS and refractory in-flammatory bowel disease, but these changes in neuronalexcitability and synaptic strength would not be detectablewith current diagnostic techniques. Several inflammation-induced changes in the ENS, including AH neuron hyper-excitability, do persist beyond recovery of inflammation,25,26

supporting the possibility that long-term changes in entericcircuitry could contribute to FGIDs.

Figure 2. Diagram show-ing inflammation-inducedchanges in the propulsivemotor circuitry of thecolon. Modified fromMawe22 with permissionfrom Journal of ClinicalInvestigation.

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Neuroimmune FunctionFor many years, the contribution of immune cells to the

pathogenesis of FGIDs largely was ignored. Recent evidencederived from patient and animal studies, however, hasshown the untapped therapeutic potential of the mecha-nisms involved in the cross-talk among immune cells,epithelial cells, smooth muscle, enteric nerves, and their rolein the generation of symptoms in FGIDs.

The intestine is a unique compartment containingenteric neurons and a large number of regionally distrib-uted resident immune cells. The expression of receptors forneurotransmitters on immune cells, and receptors for im-mune mediators on neurons/nerves, provides a foundationfor neuroimmune interactions (Figure 3). Immune cellssynthesize and release mediators that alter neuronal activitythough neural expression of receptors for pathogen- anddamage-associated molecules and for cytokines generatedby resident and infiltrating cells. Immune cells also releaseclassic neurotransmitters, fostering the concept of the“neuroimmune synapse.” Neuroimmune cross-talk isinvolved in proinflammatory and anti-inflammatory neuralreflexes and is important for the full development of the gutimmune system and maintenance of mucosal homeostasis.Amplification of the bidirectional communication amongepithelial cells, innate and adaptive immune cells, and ENSprovides a bridge to the adaptive immune response tophysiologic or pathogenic stimuli.

Cells Involved in Neuroimmune InteractionsIntestinal epithelial cells. Intestinal epithelial cells

(IECs) express pattern recognition receptors (PRRs),including membrane-spanning Toll-like receptors, intracel-lular nucleotide oligomerization domain-like receptors, andretinoic acid-inducible gene 1–like receptors, all of whichrespond to pathogen-derived signals to promote tissue-specific innate immunity. Epithelial-derived cytokines (eg,interleukin [IL]25, thymic stromal lymphopoietin) activate

Figure 3. Nerves expressreceptors for immune cellmediators. Immune medi-ators bind to receptors onnerves and can result ineither excitation or inhibi-tion of gut function. PAR,protease activated recep-tor; TNF, tumor necrosisfactor; TRP, transient re-ceptor potential; TTX-s,tetrodotoxin sensitive Naþ

channels.

receptors on resident immune cells to initiate immune re-sponses. IECs also amplify immune responses by producingchemokines, such as IL8, that recruit immune cells. Immunemediators binding to IEC receptors affect function through avariety of signaling pathways or through activation oftranscription factors that control expression of specificgenes. The close association of epithelial cells, nerves, andimmune cells greatly facilitates their interactions.

Intraepithelial lymphocytes. Intraepithelial lympho-cytes (IELs) are a heterogeneous population of T-cellsubtypes—distinct from peripheral T cells—that are inter-spersed among IECs. IELs express surface markers that playa role in their migration and retention in the mucosalcompartment and generate molecules, allowing them totether epithelial cells. In human beings, the majority of IELsare found in the proximal small intestine where they areimportant in mucosal tolerance and in maintenance ofbarrier function through the production of cytokines thataffect permeability.27

Innate lymphoid cells. Innate lymphoid cells (ILCs)are a recent discovery, arise from a poorly defined precur-sor pool, and generate cytokines identified with polarizedadaptive immune responses (T-helper [Th]1, Th2, or Th17).Unlike T cells, ILCs lack antigen receptors and are notinvolved in immune memory,28 but are important for theinitiation of host immune responses. ILC fate is modulatedby the cross-talk among epithelial cells, luminal factors, andother immune cells, implicating them in both protective andinappropriate immune responses.29

Dendritic cells. Intestinal dendritic cells (DCs) shapeadaptive immune responses to harmful or infectious intra-luminal stimuli through acquisition of luminal antigens andmigration to mesenteric lymph nodes to present these an-tigens to naive T cells. Sensory neuropeptides participate inthe recruitment of DCs during neurogenic inflammation, andactivation of vasoactive intestinal polypeptide (VIP) re-ceptors inhibits the migration of mature DCs to sites ofinflammation, inducing a more tolerogenic phenotype. DCs

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express nicotinic and dopaminergic receptors that shiftfunction toward production-specific profiles of cytokinesand this neuromodulation may play a role in inflammatoryGI pathologies.

T cells. As central constituents of the adaptive immuneresponse, T cells are natural targets of the nervous system.Specific cell markers subdivide populations of effector Tcells into different phenotypes including cytotoxic (CD8þ),T helper (CD4þ), memory (CD4þ or CD8þ, CD45RO), andregulatory (CD25þ). Some effector cells are retained anddifferentiate into resident tissue memory cells, which areresponsible for rapid responses to subsequent antigenicstimuli. T cells express receptors for neurotransmittersincluding 5-HT, dopamine, norepinephrine, glutamate, andacetylcholine (muscarinic and nicotinic). Their ability torelease acetylcholine and produce choline acetyltransferaseallows them to function as a non-neuronal cholinergicsystem.30

Mucosal mast cells. Mucosal mast cells reside inhealthy gut and are important in the transition from innateto adaptive immunity. They release both preformed andnewly synthesized mediators including proteases, hista-mine, prostaglandins, 5-HT, cytokines, and chemokines thatdepend on the phenotype, which is influenced by themicroenvironment. The nature and timing of mediatorrelease is determined by the type of receptors activated andthe strength and duration of the stimuli. There is a well-documented anatomic and functional interaction betweenmucosal mast cells and nerves.

Macrophages. Macrophages are the largest populationof mononuclear phagocytes in the gut. Mucosal macro-phages respond to luminal contents and to specific IEC-derived mediators. Macrophages associated with smoothmuscle are implicated in inflammation- and infection-induced changes in gut motility.31 Macrophage activationby Th1 cytokines leads to the development of the proin-flammatory classically activated phenotype, whereas Th2cytokines promote the development of the anti-inflammatory alternatively activated phenotype. Macro-phages express nicotinic and muscarinic receptors foracetylcholine and are in close contact with cholinergicneurons. Activation of these receptors enhances or inhibitsmacrophage phagocytosis and modulates production of cy-tokines. Macrophages also express a- and b-adrenergic re-ceptors as well as receptors for 5-HT, substance P, VIP,adenosine, and a number of proteases that activateprotease-activated receptor 1 and protease-activatedreceptor 2.

Immune Modulation of IntegratedNeural Responses

Activation of vagal nerves has beneficial effects thatinclude inhibition of proinflammatory cytokines and atten-uation of tissue injury. Recent evidence has shown ananatomic and functional interaction between macrophagesin the muscularis externa of the intestine with vagal efferentfibers synapsing on cholinergic, nitric oxide, and VIP-containing neurons in the ENS.32 Sympathetic nerves

innervate gut-associated lymphoid structures and modulatethe responses of immune cells expressing adrenergic re-ceptors. Proinflammatory actions of sympathetic nerves aremediated by a2-adrenergic receptors whereas anti-inflammatory effects are mediated by the b3-adrenergicreceptor. Catecholamines bind with higher affinity to a

than to b receptors, so the distance from the source of theimmune cells that express both receptors can influence theresponse.

Neurogenic inflammation is a response triggered byserine proteases, elaborated by enteric pathogens, mastcells, and neutrophils. Cleavage of protease-activated re-ceptor 2 on extrinsic primary afferents sensitizes TRPchannels and releases proinflammatory sensory neuropep-tides such as substance P and calcitonin gene-related pep-tide. There is also a neuronal and nerve fiber hyperplasia ininflammation that also may contribute to the severity of theresponse.33

Interaction of the Epithelial Barrier andthe ENS With Gut Luminal Content

The intestinal epithelial barrier plays a critical role in themaintenance of homeostasis within the gut and there isgrowing evidence that alterations in this barrier may be animportant factor in the pathogenesis of FGIDs. The epithe-lium, along with underlying immune structures in the lam-ina propria, plays a pivotal role in controlling the hostimmune response to luminal antigens. These luminal factorsalso may signal directly or indirectly, through the host im-mune response, to other effector systems including the ENS.These complex interactions, if dysregulated, can lead to gutdysfunction and symptom onset.

Intestinal Barrier StructureThe intestinal barrier (Figure 4) consists of a single layer

of epithelial cells that physically separates the host from theintestinal lumen.34 IECs are bound together by the epithelialapical junctional complex, comprised of tight junctions,adherens junctions, and other membrane complexes con-taining the membrane proteins nectin and junctional adhe-sion molecule. Overlying the apical side of epithelial cells isa mucus layer primarily produced by goblet cells, antimi-crobial peptides, and immunoglobulins.35

Intestinal Barrier FunctionWater and ion transport. The gut is capable of

handling approximately 9 L of fluid per day, absorbedmainly by the small intestine. This fluid movement involvesboth absorptive and secretory processes, which can occurthrough the paracellular or the transcellular route. Theparacellular pathway involves water movements coupled tonutrient absorption (solvent drag), whereas the trans-cellular route involves the passage of water through apicaland basolateral membranes of epithelial cells by passivediffusion, cotransport with ions and nutrients, or throughaquaporins.

Figure 4. Intestinal barrierstructure and function.Adapted with permis-sion from Natividadet al.46 MAMP, microbe-associated molecularpattern; PRR, patternrecognition receptors.

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Antigen sampling. Immune sampling of luminal con-tent is constant and key to mount an appropriate immuneresponse. Peyer’s patches are overlaid with specializedepithelial cells called microfold cells, through which anti-gens are transported and exposed to antigen-presentingcells in the lamina propria. Direct sampling may occurthrough extension of dendrites by specialized dendriticcells. This process mainly occurs in the ileum, whereas inthe upper small intestine sampling may be more dependenton changes in paracellular permeability.

Immune defense. Gastric and intestinal secretions,and peristalsis, aid in digestion and immune defense byflushing microbes and toxins. The outer layer of mucus inthe colon traps and contains large numbers of bacteriawhereas the inner layer is maintained relatively sterile, inpart by antimicrobial proteins (defensins, cathelicidins,proteases, and C-type lectins) produced by various enter-ocytes, Paneth cells, and innate immune cells. Some anti-microbial proteins are expressed constitutively and othersare dependent on intestinal microbial colonization. Epithe-lial cells also transport IgA produced by B cells into the gutlumen. IgA deficiency is associated with increased pene-tration of bacteria into host tissues.36 Epithelial cells alsoare armed with antigen detection and immune signalingmechanisms, and in some cases can even act as antigen-presenting cells for neighboring IELs.

Molecular Mechanisms of Interactions Betweenthe Intestinal Barrier and Luminal Antigens

A key process in innate recognition of microbial antigensis mediated by pattern recognition receptors (PRRs), whichinclude Toll-like receptors (TLRs), nucleotide binding oligo-merization domain-like receptors, RNA helicases, C-type

lectin receptors, and cytosolic DNA sensors. These receptorsrecognize evolutionary conserved pathogen-associatedmolecular patterns expressed by various microorganisms,and shared by symbiotic microorganisms. Both epithelial andimmune cells express PRRs. Upon activation, PRRs triggersequential activation of intracellular signaling pathways andlead to induction of a range of cytokines and chemokines thatpromote immune and physiological responses. PRR signalingalso facilitates the differentiation of T cells and B cells toestablish antigen-specific adaptive immunity.

Critical intestinal barrier adaptations occur in responseto microbial signals after gut colonization.37 In germ-freeanimals, expression of antimicrobial peptides is negligible,low levels of IgA are secreted, the composition of the mucusis altered, TLR expression is reduced, and zonula occludens1 proteins are diminished. After bacterial colonization, thereis expansion of the lamina propria, along with increased cellproliferation and increased expression of innate microbialrecognition receptors.

Interaction of the Intestinal BarrierWith Luminal Content

Gut microbiota. A key strategy of the mammalian in-testine in maintaining homeostasis is to regulate the inter-action between luminal microbiota and the intestinalepithelial cell, as well as immune surveillance cells in thebarrier. However, disruption of the epithelial barrier, forexample, during acute gastroenteritis, could allow signalingby the microbiota directly to the immune system in thelamina propria and possibly directly to enteric and nerveterminals of dorsal root ganglia neurons. Multiple TLRs alsoare found on enteric and autonomic neurons and TLR acti-vation can affect their excitability.

Figure 5. Chronic psychological stress plays a significant role in the pathophysiology of IBS. CRF, corticotropin releasingfactor. Reproduced from Barbara et al45 with permission from the Journal of Neurogastroenterology Motility.

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Food components. There is increasing recognitionthat luminal food components induce symptoms in manypatients with FGIDs.38 Enhanced signaling resulting fromaltered intestinal barrier and/or exaggerated neuroimmuneresponses could underlie these actions. One importantcomponent is sensitivity to wheat-containing diets in someIBS patients. This sensitivity could be related to glutenbecause the gluten-derived peptide P31-43 increased IL15-positive cells from biopsy specimens from celiac patientsand induced stress markers on epithelial cells. Other studieshave shown that gliadin, the storage protein in gluten, in-creases permeability, and studies in animal models of glutensensitivity have shown that gliadin or gluten can increasepermeability, which then leads to increased uptake ofmicrobiota antigens that further amplify the immune andfunctional responses to gluten. Other components in wheatare capable of inducing innate immune responses that couldlead to gut dysfunction. Amylase trypsin inhibitors thatprotect the grain from pests can activate the TLR-4 pathway.Interestingly, amylase trypsin inhibitor reactivity has beenimplicated in allergy and Baker’s asthma. Whether amylasetrypsin inhibitors alter the epithelial barrier and play a rolein a proportion of wheat-related IBS symptoms remainsunknown, but may explain some allergic reactions to wheatcomponents that potentially could lead to mast celldegranulation and symptoms. Another important compo-nent is the carbohydrates and smaller amounts of protein

that are not absorbed in the small intestine. These aremetabolized by colonic bacteria to short-chain fatty acidsand intestinal gases. Studies suggest fermentable substancescan cause colonic epithelial cells to express receptors forthese short-chain fatty acids, potentially altering the prop-erties of enteric neurons, leading to changes in motility andsecretion. Fermentable food components also produce anumber of gases, including H2, CH4, and CO2, which couldproduce symptoms as a result of the gas-induced distensionof the colon and secondary activation of neural reflexes. H2Sgas, produced by sulfur-reducing bacteria in the intestinallumen, also has been implicated in the regulation of gutfunction, including secretion, motility, and nociceptivesignaling. Finally, attention recently has centered on bileacids, particularly in diarrhea-predominant IBS patients, inwhom such acids may induce alterations in intestinalphysiology, by signaling to the epithelium, immune cells,blood vessels, smooth muscle, ENS, and autonomic nerves.

StressAlthough the etiology of FGIDs is unknown, there is

compelling evidence that psychological and physicalstressors play an important role (Figure 5). It is a generallyaccepted hypothesis that dysfunction of the bidirectionalcommunication between the brain and the gut, in partthrough activation of the principal neuroendocrine stress

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system, namely the hypothalamic–pituitary–adrenal (HPA)axis, plays a role in the symptomatology of IBS. The HPA axisis activated by stress, causing the release of corticotropinreleasing factor (CRF) from the paraventricular nucleus ofthe hypothalamus into the hypophyseal portal circulation tobind in the anterior pituitary. Adrenocorticotropic hormonethen is released from the pituitary into the systemic circu-lation to cause the synthesis and release of the glucocorticoidcortisol (corticosterone in rats) from the adrenal cortex.Clinical studies have implicated HPA axis dysregulationbased on multiple reports of increased cortisol levels andexaggerated HPA responses to stressors in IBS patients.

Multiple lines of evidence have shown activation ofcentral mechanism(s) resulting in colorectal hypersensitiv-ity, involving descending facilitation from the brain toinduce remodeling of colorectal responsiveness via sensiti-zation of spinal dorsal horn neurons. Brainstem regionsresponsible for the modulation of descending inhibitorypain signals are modulated by both pain and stress. The PAGreceives excitatory signaling from the PFC and inhibitorysignaling from the amygdala. The rostroventral medullareceives not only direct nociceptive information from thespinoreticular pathway but also integrated pain and stresssignals from the amygdala and PAG. In addition, the locuscoeruleus and amygdala form a circuit that can potentiateboth endocrine and autonomic stress responses. Centralstructures regulating affective and sensory processesincluding the amygdala, insula, cingulate, and PFC showenhanced activation in IBS patients. In animal models and inIBS patients, imaging studies have shown that limbic regionsregulating sensory processing and emotion, including theamygdala, show greater responsiveness to visceral stimu-lation. The amygdala is an important limbic structureinvolved in the potentiation of the HPA axis, with diffuseconnections to pain-modulatory networks, and has beenimplicated in visceral sensitivity and aberrant HPA activityobserved in IBS patients.13 The amygdala is sensitive tocorticosteroids but, in contrast to the hippocampus and PFC,the amygdala facilitates behavioral, neuroendocrine, andautonomic responses to stress. Thus, this altered balance instress modulation induced by amygdala hyperactivity mayrepresent an essential aspect of alterations in GI motorfunction, colonic permeability, and colorectal sensitivityapparent in IBS. In support, increasing amygdala cortico-sterone in rats by stereotaxically implanting corticosteronemicropellets onto the central nucleus of the amygdala cau-ses a persistent increase in the sensitivity to visceral stimulias well as inducing anxiety-like behavior.39 These findingssuggest that in IBS patients exposed to chronic stress,increased amygdala activation dysregulates the HPA axis.

Clinical observations have suggested that abdominalpain and altered bowel habits are more common in females,and that menstrual cycle–linked differences are observed insymptom reporting. Differences in CNS processing ofvisceral information is a potential explanation becausestudies using positron emission tomography imaging havesuggested that gender differences in regional brain re-sponses to rectal pressure exist in IBS patients. Differencesin pain sensitivity may be the result of cyclic hormonal

changes in females, however, the mechanisms are poorlyunderstood. Effectively abolishing the activational role ofovarian hormones via ovariectomy reverses the effects ofearly life adversity (ELA) on adult visceral pain hypersen-sitivity in female rats, whereas reintroduction of ovarianhormones via a subcutaneous estradiol pellet was sufficientto induce visceral hyperalgesia.40 These data provide sup-port that ovarian hormones play a prominent role inmaintaining the persistent effects of ELA on increased painsensitivity in human beings and rodent models. Studies ofneonatal maternal separation to induce ELA also haveshown features of the IBS phenotype including motilityabnormalities, colonic hypersensitivity, and enhanced gutpermeability.40

Remodeling of the epigenome by the environment orchronic stress may result in long-term changes in geneexpression.41 A recent study showed the importance of his-tone acetylation in stress-induced visceral pain by showingthat direct administration into the brain of a histone deace-tylase inhibitor reversed visceral hypersensitivity induced bystress or activation of the amygdala with corticosterone.42,43

In another study, exposure to ELA was associated with CRFpromoter hypomethylation and an increase in CRF tran-scriptional responses to stress in adulthood suggesting thatneonatal stress causes long-lasting epigenetic changes in theCRF expression within the HPA axis.44

In summary, stress plays an important role in functionalbowel disorders with recent evidence from experimentalmodels showing that chronic adult stress or early life stresscan recapitulate IBS phenotypes, and provide new insightsinto the underlying mechanisms of IBS.

Conclusions and Future DirectionsThis review highlights many advances in our under-

standing of the cellular and molecular mechanisms under-lying GI physiological and pathophysiological systems thatmay play a role in FGIDs. Examples of important themes andresearch questions for future research are as follows:

1. Sensory mechanisms underlying sensitization ofnociceptors and visceral hypersensitivity: which me-diators act to sustain signaling in specific patients andare there critical pathways? When and how are cen-tral (CNS) and peripheral mechanisms (ENS/auto-nomic nervous system) dominant?

2. Barrier function regulating intestinal permeability,tight junction proteins, and microbiome signaling:which pathways are involved in FGIDs and when?Which mechanisms regulate them?

3. Neuroimmune function regulating immune mediatorsand bidirectional neural signaling: which immunecells are activated and which mediator(s) are mostimportant? What role do the vagal anti-inflammatory/sympathetic proinflammatory and central pathwaysplay?

4. ENS preprogrammed synaptic networks and neuro-plasticity: can peripheral (eg, microbiome) or central

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(eg, stress) pathways switch ENS networks to changesymptoms (eg, alternating diarrhea and con-stipation)? When and how does ENS neuroplasticityunderlie FGIDs?

5. Psychological stress and the HPA axis/autonomicnervous system response: how does it lead to visceralhypersensitivity and alter gut function in FGIDs?

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Reprint requestsAddress requests for reprints to: Stephen J. Vanner, MD, GastrointestinalDiseases Research Unit, 76 Stuart Street, Kingston General Hospital,Kingston, Ontario, Canada. e-mail: vanners@hdh.kari.net; fax: (613) 544-3114.

AcknowledgmentsAll authors contributed to the organization and writing of the review.

Conflicts of interestThe authors disclose no conflicts.

FundingSupported by Canadian Institute of Health Research grant 110986 (S.V.);National Institutes of Health grant DK62267 (G.M.); National Institutes ofHealth grant R01-DK083418 (T.S.-D.); and the European Community FP7Program (D.G.).