Mediators of Inflammation
Volume 2016 (2016), Article ID 1363818, 14 pages
http://dx.doi.org/10.1155/2016/1363818

Review Article

Neuroimmunomodulation in the Gut: Focus on Inflammatory Bowel Disease

Claudio Bernardazzi,¹ Beatriz Pêgo,¹ and Heitor Siffert P. de Souza1,2

¹Serviço de Gastroenterologia & Laboratório Multidisciplinar de Pesquisa, Hospital Universitário, Universidade Federal do Rio de Janeiro, 21941-913 Rio de Janeiro, RJ, Brazil
²D’Or Institute for Research and Education (IDOR), 22281-100 Rio de Janeiro, RJ, Brazil

Received 30 March 2016; Accepted 7 June 2016

Academic Editor: Marisa I. Gómez

Copyright © 2016 Claudio Bernardazzi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Intestinal immunity is finely regulated by several concomitant and overlapping mechanisms, in order to efficiently sense external stimuli and mount an adequate response of either tolerance or defense. In this context, a complex interplay between immune and nonimmune cells is responsible for the maintenance of normal homeostasis. However, in certain conditions, the disruption of such an intricate network may result in intestinal inflammation, including inflammatory bowel disease (IBD). IBD is believed to result from a combination of genetic and environmental factors acting in concert with an inappropriate immune response, which in turn interacts with nonimmune cells, including nervous system components. Currently, evidence shows that the interaction between the immune and the nervous system is bidirectional and plays a critical role in the regulation of intestinal inflammation. Recently, the maintenance of intestinal homeostasis has been shown to be under the reciprocal control of the microbiota by immune mechanisms, whereas intestinal microorganisms can modulate mucosal immunity. Therefore, in addition to presenting the mechanisms underlying the interaction between immune and nervous systems in the gut, here we discuss the role of the microbiota also in the regulation of neuroimmune crosstalk involved in intestinal homeostasis and inflammation, with potential implications to IBD pathogenesis.

1. Introduction

The enteric nervous system (ENS) constitutes a major autonomic division of the nervous system that provides the intrinsic innervation of the gut, capable of controlling different functions, such as motility, mucosal secretion and absorption, mucosal growth, local blood flow, and the immune function [1]. The ENS can be influenced by the central nervous system (CNS), establishing a two-way relationship treaded by the brain-gut axis. Actually, all basic gastrointestinal (GI) functions can be regulated by the ENS, but the coordination of the gut function and the maintenance of the homeostasis of the organism both require a communication between the GI tract and the CNS [2]. The ENS consists of two interconnected networks of ganglia and fibers encircling the GI tract, arranged in a peculiar way, which confers the ability to mediate its own reflexes. In this context, evidence shows that ENS can work independently of the CNS [3]. Because of these properties, ENS is a primary regulator of GI functions and has been referred to as a second brain in humans [3]. However, despite the ability of the ENS to regulate motility and secretion in an autonomous way, there are many connective links with the CNS, working in a bidirectional fashion [4].

Nerve cells located within the mucosa are in close proximity to immune cells, where they produce and respond to several common mediators [5, 6]. Upon ENS activation, mucosal immune cells expressing receptors for neurotransmitters can be stimulated to migrate, degranulate, differentiate, or secrete immunoglobulins, for example, [7–9]. Therefore, the communication between the ENS and the immune system within the mucosa participates in the control of major GI functions but can also be associated with pathological conditions, such as inflammatory bowel disease (IBD).

2. Enteric Nervous System

The ENS, commonly called “the little brain of the gut,” is a constituent of the peripheral nervous system which is composed by an intrinsic network containing enteric neurons cell bodies, intestinal cells of Cajal (ICC), interneurons and motor neurons, and enteric glial cells (EGC). The latter have been extensively studied in recent years and consist of small cells with stellate shape that is associated with neuron cell bodies and nerve fibers in intraganglionic connections. Evidence supports that EGC are very similar to astrocytes, not only morphologically, but also functionally [10]. All these elements are grouped into ganglia and interconnected by bundles of nerve processes forming plexuses, including the myenteric (Auerbach’s) and submucosal (Meissner’s) plexus.

The myenteric plexus extends from the upper esophageal to the external anal sphincter, situated between the longitudinal and the circular muscle layer. The submucosal plexus is restricted to the submucosa of the small and large intestines. In human, for instance, two ganglionated submucosal plexuses can be distinguished. Thereby, there is an internal submucosal plexus, which lies in the inner half of the submucosa, and also an external submucosal plexus, this one situated close to the inner border of the circular muscle layer [11] (Figure 1).

Figure 1: Schematic illustration showing the interaction between the central nervous system (CNS) and the enteric nervous system (ENS). The sympathetic and parasympathetic innervations interact with neurons in the gastrointestinal tract, passing the sympathetic innervations first through the celiac ganglion and the superior mesenteric ganglion (sup. mes. ganglion) and the inferior mesenteric ganglion (inf. mes. ganglion). The quadrant below the CNS and the ENS represents the intestinal mucosa and its myenteric and submucosal plexuses.

In the present review, the current knowledge and the clinical implication of ENS in IBD will be discussed.

3. Mediators of the Enteric Neurons System

Neurotransmitters are molecules produced by neurons that play a role in the transmission of information cell to cell, in maintaining stimulus of impulses, and act in the neuromuscular junction. When an action potential reaches the terminal button of a presynaptic neuron, a voltage-dependent calcium channel opens, resulting in the release of neurotransmitters in the synaptic cleft [12, 13]. These molecules, also known as neuropeptides, including acetylcholine, serotonin, substance P, corticotropin-releasing hormone, and vasoactive intestinal peptide (VIP), are distributed throughout the gut and participate in normal homeostasis as well as in inflammatory processes [14].

Acetylcholine (ACh) plays a role in both CNS and in ENS upon ligation with ACh receptors, ligand-dependent cation channels, of which the two major classes are the muscarinic and the nicotinic receptors [15, 16]. The activation of ACh receptors by binding to ACh determines the depolarization of the postsynaptic neuron and the initiation of a new action potential [17, 18].

Another neurotransmitter, abundant in intestinal neuroendocrine cells, is serotonin (5-hydroxytryptamine, 5-HT). About 95% of 5-HT in the human body is found in GI tract and its action influences luminal contents and secretions [19]. The most frequent component of the enteroendocrine cell population is the enterochromaffin cell (EC), estimated to contain 90% of the total intestinal 5-HT, while 10% is in enteric neurons [19–21]. Once released basolaterally, 5-HT can perform multiple functions, including action on primary intrinsic afferent neurons initiating peristaltic reflex, the stimulation of cholinergic neurons to release acetylcholine, resulting in smooth muscle contraction, and the stimulation of inhibitory nitrergic neurons to release NO, which results in smooth muscle relaxation [22]. In addition, 5-HT participates in potential mucosal protecting processes, stimulating active ion, mucus, and fluid secretion. The secretory effect of 5-HT is mediated by epithelial 5-HT2 receptors and neuronal 5-HT1P, 5-HT3, and 5-HT4 receptors [22]. In the GI tract, the abnormal secretion of 5-HT has been associated with various effects, such as nausea, vomiting, and alterations in the intestinal secretion and peristalsis [19], indicating that this neuroendocrine transmitter plays an important role in the regulation of gastrointestinal functions. Interestingly, the major source of 5-HT, EC cells, also expresses toll-like receptors, which make them capable of sensing microorganisms [23, 24].

Substance P (SP) is localized in enteric nerves distributed throughout the gut and present in myenteric and submucosal plexuses [25, 26]. The effects attributed to SP, such as regulation of mucosal permeability [27], motility [28], secretion [29], epithelial cell proliferation [30], and inflammation [31], are initiated upon ligation with G-protein-coupled NK-1R, which is present in both the small and large bowel of animals and humans [32, 33]. In regard to intestinal inflammation, SP-NK-1R-induced proinflammatory signaling was shown to result in the production of a downstream cascade of proinflammatory molecules mediated by the activation of NF-kappa B [34, 35], or p38 mitogen-activated protein kinase [36]. It is interesting to note that NF-kappa B can also modulate NK-1R expression [37]. SP has been identified also in immune cells, such as dendritic cells, mononuclear phagocytes, and lymphocytes [28, 38–41], while NK-1R can be present in T and B cells, macrophages, dendritic cells, neutrophils, natural killer cells, and eosinophils [28, 39, 42–45]. Moreover, SP and NK-1R can promote inflammation by regulating intestinal angiogenesis through the increase in the expression of CCN1 (CYR61) [46], which is upregulated in the colon from UC patients [47].

Nitric oxide (NO) is regarded as a cellular signaling molecule, which can play different roles in the GI tract, such as participating in the maintenance of mucosal integrity and also regulating vascular tone and the mucosal blood flow [48]. NO is catalyzed by one of the isoforms of nitric oxide synthase (NOS), of which the GI tract expresses constitutively two: endothelial NOS (eNOS) and neuronal NOS (nNOS) [49]. An additional isoform is the inducible isoform (iNOS), which is upregulated in response to inflammation and other stimuli. The increase in NO concentration, in turn, results in the production of reactive oxygen species and consequently also potential oxidative stress [50]. In fact, increases in NO concentration have been associated with harmful effects in the GI tract, including human IBD and also experimental colitis [51]. Interestingly, the increase in both iNOS expression and NO production in patients with ulcerative colitis was shown to be secondary to enteroglial-derived S100B protein upregulation. This information unveils an unexpected mechanism by which the enteric glia mediates mucosal NO-dependent inflammatory responses [52]. The role of NO in intestinal homeostasis and inflammation is further reinforced by the fact that VIP is released from nerve terminals containing NOS. Such peptides are thought to comprise a nonadrenergic and noncholinergic nerve transmission circuit within the gut [53, 54].

4. Enteric Nervous System in the Pathogenesis of Inflammatory Bowel Disease

During the course of IBD, the marked immune cell infiltration and the activation of mechanisms that modulate cell turnover within the intestinal epithelium can lead to permeability changes with potential disruption of the intestinal barrier [55–57]. This milieu of changes, which in certain levels contributes to the pathogenesis of IBD, can further progress and ultimately affect also the morphology and function of the ENS [58]. Abnormalities such as axonal rupture of nerve fibers, damage of neuronal cell bodies, hyperplasia of EGC, and increase of axonal necrosis of gut nerves have been associated with IBD [59–62]. Villanacci et al., for example, observed differences in the number of neuronal cell bodies, ICC and EGC in patients with Crohn’s disease (CD) and ulcerative colitis (UC). Interestingly, abnormalities such as a reduction of enteroglia, found in noninflamed areas of the intestine, led to the hypothesis of a pathogenic role of the ENS in IBD [63]. Ohlsson et al. corroborated these findings and further demonstrated the presence of visceral ganglioneuritis, in addition to atrophy and vacuolar degeneration of ICCs in the small bowel of patients with CD [64]. On the other hand, in another study investigating the noninflamed tissue of CD patients, the transmitter colocalization patterns in rectal submucosal neurons by immunohistochemistry revealed an increase in the vasoactive intestinal polypeptide (VIP) population, extensive colocalization of choline acetyltransferase (ChAT) and NOS, and hypertrophied calcitonin gene-related peptide (CGRP) fibers [65], supporting the occurrence of adaptive alterations in the ENS in CD.

In the context of experimental colitis, enteric nervous abnormalities have also been reported in animal models. For example, results from a study using trinitrobenzene sulfonic acid- (TNBS-) induced colitis have shown that, in the beginning of inflammation, 20% of myenteric neurons are rapidly lost [66], and neuronal loss has been attributed to cell death induced by the inflammatory process consequent to TNBS-induced colitis and associated with infiltration of neutrophils [66, 67]. Hence, it is probable that the activation of immune cell-related molecules involved in IBD pathogenesis also might be responsible for the abnormalities of ENS.

5. Immune Cells Interaction with ENS

The presence of neuropeptides and neurotransmitter receptors on immune cells represents a strong indication of the integration between the ENS and the immune system.

5.1. T Cells

In normal conditions, mucosal T cells respond to different environmental challenges orchestrating the immune response in an adaptable fashion [68]. In IBD, such plasticity of T cells appears to be compromised, resulting in chronic inflammation [69]. Currently, in CD, the immune response has been regarded as a mixture of a T helper type 1 (Th1) and Th1/Th17 phenotypes [70], while in UC it comprises an atypical Th2 phenotype, with the addition of Th9 [71], and a less prominent Th17 response [72]. Recently, complex modulatory mechanisms reciprocally involving the ENS and the mucosal immune system have been recognized [73]. For instance, the vagus nerve appears to play an important role in this integrative process [74, 75] and, when stimulated, it acts as an anti-inflammatory promoter activating sympathetic neurons in the mesenteric ganglion that release noradrenalin, which activates T cells. These T cells, defined as memory cells, in turn, release acetylcholine (Ach) that inhibits proinflammatory cytokines from macrophages [76]. Particularly, in physiological conditions, T cells have been seldom seen in the proximity to ENS. Nevertheless, Sayani et al. demonstrated that, in the context of experimental intestinal inflammation, mucosal T cells increased, but being consistently excluded from ganglia. Such effect has been attributed to the expression of Fas-ligand (Fas-L) on enteric neurons, which appears to protect them against Fas-Fas-L-induced apoptosis, possibly further contributing to the resolution of inflammation [77]. Taken together, these evidences support the idea of a neural information system capable of controlling innate and adaptive immune responses.

Another interesting example of this integration is the evidence of hypothalamic-pituitary-adrenal axis regulation of intestinal inflammation [78], through the anti-inflammatory effects of glucocorticoids [79]. In IBD, the activated inflammatory cascade has been shown to affect GI motility and function [80], providing another indication of an intimate communication between ENS and the mucosal immune system (Figure 2).

Figure 2: Interaction between the enteric neuron system (ENS) and mucosal immune cells. Upon vagus nerve stimulation, the sympathetic innervation secretes neurotransmitters that can modulate immune cells and the inflammatory response. The intestinal microbiota also participates in the inflammatory response fine-tuning the interaction between the ENS and mucosal immune cells.

5.2. Macrophages

In the intestinal mucosa, resident macrophages are usually present in the lamina propria where they preferentially locate in the subepithelial area to constitute the first line of defense against potentially harmful external stimuli [81]. Nevertheless, macrophage subsets are distributed also below the epithelial layer, towards the submucosa and muscularis externa, exhibiting distinct phenotypes and probably specific functions [82].

In the gut, vagus nerve stimulation has anti-inflammatory properties, also known as cholinergic anti-inflammatory pathway, with influence on diverse immune-mediated disorders [83]. For example, vagus nerve activation by electrical stimulation and systemic nicotinic receptor agonists was shown to abate intestinal inflammation, by reducing the production of proinflammatory cytokines by macrophages [84]. Recently, the interaction between vagal efferents and intestinal macrophages has been investigated, but no clear evidence of direct modulation could be demonstrated. Hence, researchers proposed that vagal modulation of intestinal macrophages could actually be indirect, probably via cholinergic and nitrergic/VIPergic enteric neurons [85].

TNF-alpha, a prototypical Th-1-type of proinflammatory cytokine, associated with IBD and particularly with CD [86], can be modulated by the vagus nerve, through the inhibition of macrophage release. Notably, Wang et al. reported that the nicotinic acetylcholine receptor alpha 7 subunit (α7nAChR) is essential in acetylcholine inhibition of TNF-alpha production by macrophages [87], revealing a mechanism of neuromodulation of the immune response. In another study on the subject, the anti-inflammatory action of the vagus nerve in the intestine was shown to be dependent on its interaction with cholinergic myenteric neurons in intimate association with the muscularis macrophages. In addition, it has been suggested that resident macrophages expressing α7nAChR would probably be the ultimate intestinal target of such anti-inflammatory pathway [88].

5.3. Neutrophils

Like T cells, neutrophils are rarely observed in submucosal and myenteric plexuses in normal conditions, but during chronic active IBD these inflammatory cells accumulate and infiltrate the mucosa, contributing to the tissue injury [89] and possibly also affecting the ENS.

In experimental colitis in rats, induced by dinitrobenzene sulfonic acid (DNBS) administration, Sanovic et al. demonstrated a significant neuronal reduction in the inflamed segments in the first 24 hours, with less than half of neurons remaining by days 4 to 6 and thereafter, when inflammation had diminished. The neuronal damage was associated with the early accumulation of neutrophils and eosinophils within the ganglia, an effect more prominent in the submucosal ganglia [90]. In another experimental study with DNBS-induced colitis, mice treated with anti-neutrophil antibody had a significant attenuation of tissue damage and a greater number of neurons compared to nontreated colitic mice [67]. In conjunction, these data suggest that neutrophils might participate in the loss of ENS neurons during inflammatory intestinal conditions, including IBD.

5.4. Eosinophils

In the past three decades, relevant evidences point to an important role of eosinophils in IBD [91–94]. Smyth et al., studying different clinical stages of IBD, demonstrated that the major basic protein (MBP, a cationic protein released by eosinophils, which can be cytotoxic in high concentrations), as well as eosinophils, localizes to nerves and ganglia in the mucosa of patients with refractory disease [95]. Moreover, an increased expression of eotaxin-3 and ICAM-1, molecules involved in tissue eosinophilia and leukocytes transmigration, respectively, was detected in the same mucosal location, in the vicinity of nerves and ganglia [96]. In refractory CD patients, eosinophils have been found close to nerves within the smooth muscle layer. Furthermore, eosinophils localized specifically to SP and ChAT nerves, in CD mucosa, suggesting an indirect role for eosinophils also as mediators of smooth muscle contraction and gut motility [95]. Eosinophils have also been implicated in the pathophysiology of UC, and the involvement in mucosal inflammation and destruction was suggested to be associated with SP innervation and the neurokinin-1 receptor (NK-1R) expression, most marked in areas of mucosal accumulation of eosinophils [97, 98].

5.5. Mast Cells

Mast cells play an important role in innate and adaptive immune responses by regulating the allergic reaction and defense against pathogens. Their growth and proliferation are regulated by cKit ligand stem cell factor (SCF), nerve growth factor (NGF), IL-3, IL-4, IL-9, and IL-10 [99]. Under physiological conditions, mast cells are present in the mucosa, submucosa, and the circular muscle layer [100] and play a role in allergic diseases by releasing proteases, cytokines, and chemokines [101].

Increased mast cell numbers are observed in the proximity of mucosal enteric nerve fibers in a model of visceral hypersensitivity in rats [102]. In a model of food allergy in mice, Lee et al. observed that nerve fibers expressing the neurotransmitter CGRP (calcitonin gene-related peptide) were increased and colocalized to mucosal mast cells in the colonic mucosa [103]. The proximity of mast cells to mucosal enteric nerve fibers has also been described in adult patients with irritable bowel syndrome (IBS) [104], a disease in which clinical manifestations notably overlap with IBD [105]. In an animal model of IBS, for instance, Barbara et al. showed that mucosal mast cells are capable of exciting nociceptive visceral sensory nerves, suggesting their implication in visceral hypersensitivity in IBS [106]. Ileal segments of patients with CD were shown to host a marked number of mast cells displaying piecemeal degranulation associated with ICC, in the muscularis. Of note, various types of injury were described in ICC, probably due to direct contact with mast cells and the chronic release of their potentially cytotoxic granule contents [107].

6. Purinergic Receptors

Purinergic receptors, also known as purinoceptors, are transmembrane receptors including the P1 and P2 subtypes. P2 is composed of two subforms, namely, P2Y and P2X. P2Y, and its variants, is a G-protein coupled receptor, while P2X, and its variants, is a ligand gate ion channel [108]. P2X7 is the most studied purinoceptor, and it has been implicated in the induction of caspase activity, cytokine secretion, and cell death. The ligand for P2X7 is adenosine triphosphate (ATP), a molecule that at high concentrations functions as a danger signal associated with tissue inflammation and damage [108], therefore constituting a damage-associated molecular pattern (DAMP). Gulbransen et al. studied the activation of enteric neuronal P2X7 receptor during inflammation in animal models and demonstrated that the myenteric neuronal density decreased during colitis, but with a pretreatment with oxidized ATP (an antagonist of P2X7 receptor) there was a protection against inflammation-induced neurons loss. On the other hand, using BzATP, an agonist of P2X7 receptor, neural packing density was reduced [109]. This phenomenon was also observed when ChAT-, calbindin-, calretinin-, anti-HuC/D-, and NOS-neurons, in which cells express P2X7 receptor, were decreased during colitis [110]. Gulbransen et al. also demonstrated that, in addition to the P2X7 receptor expression in myenteric neurons, these cells also express Panx-1, absent in EGC [109]. The P2X7 receptor expression has been associated with cell death during intestinal inflammation in human and experimental IBD [111], and the activation of P2X7 receptor-Panx-1 was proposed to contribute to neuron death by activation of a complex of caspases. In this regard, the release of ATP by Panx-1 also mediated death of EGC by phospholipase-C (PLC) pathway initiated by P2Y1 [109]. Furthermore, it has been shown that the rapid loss of myenteric neurons involves not only caspase-dependent pathway but also other multiprotein complexes, such as the inflammasome [109]. In accordance with this, blocking either P2X7 receptor or Panx-1, associated with inflammasome triggering, was shown to prevent neurons death [109]. Other purinergic receptors have been described within enteric neurons. In the myenteric and submucosal plexuses, the ChAT, calbindin, calretinin, and NOS neurons also express P2X2 receptor, which can bind to ATP and mediate synaptic transmission [112]. In the myenteric plexus, the P2X6 receptor is expressed in neurons that resemble Dogiel type II neurons [113]. The G-protein coupled purinergic receptor P2Y2 is distributed in both plexuses, in neurons and fibers. In the myenteric plexus of small intestine of guinea pig, P2Y2 receptor is associated with neuropeptide-Y and calretinin [114]. In a model of chronic inflammatory pain, this purinergic receptor is increased in peripheral cutaneous sensory neurons that innervate injured tissue [115]. Recently, it has been demonstrated that ATP liberated from the gut epithelium during cell stressful stimuli, can mediate excitation of visceral afferents through P2X receptors [116], and also stimulate mouse and human visceral nociceptors through P2Y receptors [117]. These findings appear to implicate ATP in the generation of functional GI alterations, as a neurogenic component of the inflammatory process. In addition, ATP, regarded as DAMP, triggers mechanisms downstream of P2X7 and mediates the inflammasome activation, probably contributing to the maintenance and amplification of the inflammatory response [118, 119].

7. Enteric Glial Cells

Under physiologic conditions, intestinal barrier is relatively impermeable, but during pathologic conditions, barrier disruption has been associated with the development of GI diseases, including inflammatory disorders [120]. Regarding the intestinal barrier, results of recent studies suggest an important contribution of EGC in the maintenance of normal functions. EGC are abundant in GI tract [121, 122] and are in close proximity to the intestinal epithelial border and in contact with epithelial basement membrane [121].

The genetic ablation of enteric glial cells using transgenic mice expressing herpes simplex virus thymidine kinase from the mouse glial fibrillary acidic protein (GFAP) promoter, performed by Bush et al., showed that when the animals were treated with ganciclovir, an increased inflammatory response ensued and led to death with an underlying severe jejunoileitis [123]. In another study using the same experimental model, Savidge et al. demonstrated that the involvement of EGC with the intestinal barrier function could, at least in part, be due to the release of S-nitrosoglutathione (GSNO), a small molecule that regulates the tight junctions of the epithelial cells. Of notice, GSNO was also shown to be able to restore mucosal barrier function in CD colonic mucosal specimens [124]. In another experimental study focused on EGC, Zhang et al. revealed that glial-derived neurotrophic factor (GDNF), another molecule released by EGC, could also regulate the integrity of the intestinal barrier. Using the dextran sodium sulfate- (DSS-) induced colitis model, the investigators prevented the increase in intestinal permeability and the full inflammatory response, by treating animals with GDNF [125]. Subsequently, another study proposed the existence of an EGC self-protecting mechanism, in which GDNF protects EGC from apoptosis. The authors hypothesized that alterations in such autocrine loop would lead to a defective barrier, mucosal disruption, and development and enhancement of CD inflammation [126]. It is intriguing to note that, in the noninflamed intestinal mucosa of patients with CD, the EGC network was particularly disrupted [127] corroborating the idea that the loss or decrease of EGC might contribute to the pathogenesis of IBD.

8. Involvement of Intestinal Microbiota in the ENS

Trillions of bacteria colonize the gut, with hundreds of different species unevenly distributed throughout the GI tract, basically shaped by diet and immune and genetic factors of the host [128]. In humans, chronic inflammatory disorders have been associated with abnormalities in the microbiota, which may actively modulate disease phenotypes and behavior [129, 130]. In CD, for example, it has been widely accepted that abnormalities of the gut microbiota are present, where there is either an altered composition of the microbiota or an abnormal immune response against the commensal microbiota, or both [131]. Another study in CD demonstrated an increased abundance in Bacteroidetes and Proteobacteria in contrast to a decrease in Firmicutes [132] and probably more importantly a reduction in bacterial diversity [133]. In UC, there is still limited evidence for a major pathogenic role of the microbiota, but dysbiosis has also been reported [134].

In virtue of the potential ability of microorganisms to deregulate the physiological equilibrium, innate immune system is the first to respond to microbiota antigens through the recognition of microbial associated molecular patterns (MAMPs) at the transmembrane or cytosolic receptors, known as pattern recognition receptors (PRRs) [135, 136]. These receptors comprise three distinct families: toll-like receptors (TLRs), the nucleotide oligomerization domain- (NOD-) like receptors (NLRs), and retinoic acid inducible gene I- (RIG-I-) like receptors (NLRs). The best-characterized PRR in mammals is the TLRs family [137]. The TLRs are transmembrane proteins that can be expressed in different sites of the gut [138, 139] and also in components of the nervous system [140]. They trigger the activation of nuclear factor kappa-B (NFkB) through MyD88 and other intracellular mediators, leading to the production of proinflammatory cytokines (as reviewed by Elia et al. [135]). In humans, the enhanced expression of TLR-2 and TLR-4 by crypt epithelial cells demonstrated in active IBD was hypothesized as an indication of a greater ability to respond to distinct bacterial products [141].

Using animal models, Brun et al. found that the absence of TLR2 determines changes in the architecture of the myenteric and submucosal plexuses, leading to bowel dysmotility and increased susceptibility to intestinal inflammation [142]. In another study analyzing the influence of gut microbiota on the ENS, dysbiosis induced by antibiotics led to local changes in the innate immune system including TLRs and in the expression of sensory-related systems in mice [143]. Of note, results from another experimental study have shown that the exposure of the intestinal interstitium to bacterial cell products can activate nociceptive dorsal root ganglion neurons, leading to production of inflammatory cytokines and increased excitability, directly or independent of TLR signaling [144].

The colonization of the gut by microorganisms starts early in life and is critically important in several functions of the normal GI physiology [145, 146] and also the maturation of the mucosal immune system [147]. For example, in a study with germ-free mice investigating the electrophysiological properties of neurons in the myenteric plexus of the ENS, commensal microbiota was shown to be necessary for normal excitability of gut sensory neurons [148]. Further studies on the subject confirmed that germ-free mice exhibit less excitable intrinsic primary afferent neurons [149], which can be enhanced by the exposure to polysaccharide A [150]. An additional study using germ-free mice corroborated previous findings, demonstrating that the microbiome is crucial for both intrinsic and extrinsic nerve function and gut-brain signaling [151]. Recently, Collins et al. investigated whether the microbiota could influence the postnatal development of the ENS. Investigators found that germ-free mice have a decrease in nerve density and fewer neuronal cell bodies in myenteric ganglia, while in the small bowel, an increased proportion of inhibitory nitrergic neurons was detected. These results appear to support the hypothesis that early exposure to luminal microorganisms is pivotal for the postnatal development of the ENS [152].

Another set of important receptors able to interact with the ENS and modulate neurally mediated intestinal functions is the proteinase-activated receptors (PARs), expressed in different cell types in the gut. PARs belong to a group of G protein-coupled receptors that are activated by proteolytic cleavage [153, 154]. Both PAR1 and PAR2 have been shown to be functional in ENS cells. For instance, it has been demonstrated that PAR2 activation results in depolarization and increased number of action potentials in myenteric [155] and in submucosal neurons [156]. Moreover, PAR1 and PAR2 agonists induce calcium mobilization in myenteric [157] and dorsal root ganglia [158] neurons. In addition to the association with intestinal hyperalgesia and hypersensitivity [159], PAR2 has been shown to actively participate in neurogenic inflammation in the mouse colon [160]. Regarding the potential interaction with the ENS, the release of proteases from activated mast cells, for example, was shown to cleave PAR2 on submucosal neurons, determining acute and long-term hyperexcitability [161] (Figure 2). Interestingly, a recent study has demonstrated that secreted E. faecalis proteins, namely, gelatinase, induced permeability in the colonic epithelia of mice, which was absent in PAR2-deficient animals [162]. Together, these results strongly suggest that bacterial enzymes can regulate enteric epithelial permeability and neurogenic inflammation via intestinal PAR. The main findings of PAR receptors are exposed in Table 1. These evidences indicate the existence of interactions between the gut microbiota and the host, with effects on the ENS.

Table 1: Main findings of PAR receptors.

Studies
PARs
Samples
Methods
Results

Corvera et al. (1999) [157]
PAR1 & PAR2
Small intestine of guinea pig
Primary culture
RT-PCR
PAR1 and PAR2 are expressed in myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors

Green et al. (2000) [163]
PAR2
Porcine ileum
Immunohistochemistry
Cholinergic and noncholinergic submucosal neurons

Buresi et al. (2005) [164]
PAR1
Mouse colon
RT-PCR
Immunohistochemistry
Expressed in full-thickness specimens and mucosal scraping of colon
Localized on epithelial cells and on neurons in submucosal ganglia

Ikehara et al. (2010) [165]
PAR1 & PAR2
Mouse cecum
Electrical measurements
PAR1-mediated Cl⁻ secretion might occur by activation of the receptor on the submucosal secretomotor neurons; PAR2-mediated Cl⁻ secretion might occur by activation of the receptor on the epithelial cells

Mueller et al. (2011) [166]
PAR1, PAR2, and PAR4
Human submucosal plexus
Voltage- and calcium-sensitive dye recordings
PAR1, rather than PAR2 and PAR4, activates neurons and glia

Guinea pig (comparative study)
Voltage- and calcium-sensitive dye recordings
PAR2, rather than PAR1 and PAR4, evoked strong responses in enteric neurons and glia

Finally, the accumulating evidence of multidirectional signaling involving the multiple components of the gastrointestinal system, including the bidirectional interplay by which the nervous system modulates the immune response, suggests that these neuronal circuits and neuromediators could be used for novel therapeutic strategies. In addition to gastrointestinal motility, sensitivity, and pain, such therapeutic approach should also provide the possibility of reestablishing immune tolerance and effective controlling chronic intestinal inflammation, for example, through the activation of the vagal anti-inflammatory pathway or the development of new pharmacological agents to control the afferent neuronal signaling.

9. Conclusion

The CNS interacts dynamically with the immune system to modulate inflammation through humoral and neural pathways. Neuroimmune interactions within ENS can modulate gut functions, such as motility, ion transport, and mucosal permeability, contributing to the pathophysiology of several intestinal diseases, including IBD. Intestinal inflammation has been implicated in neuroplasticity, degeneration of the ENS, and alterations in the enteric glia, with an important contribution attributed to oxidative stress. The microbiota also plays a critical role in the intestinal homeostasis and neurogenic inflammation, as it drives postnatal development of the ENS, and affects the intrinsic and extrinsic nerve function and gut-brain signaling. Further investigation of these counterregulatory mechanisms will provide additional insights into neuroimmunomodulation, potentially leading to the identification of novel therapeutic targets for the treatment of inflammatory bowel disorders.

Competing Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contributions

Claudio Bernardazzi and Beatriz Pêgo contributed equally to this work.

References

M. Costa, S. J. H. Brookes, and G. W. Hennig, “Anatomy and physiology of the enteric nervous system,” Gut, vol. 47, supplement 4, pp. iv15–iv19, 2000.

E. A. Mayer and S. M. Collins, “Evolving pathophysiologic models of functional gastrointestinal disorders,” Gastroenterology, vol. 122, no. 7, pp. 2032–2048, 2002.

S. Singh, A. Shariff, T. Roy, T. Das, and N. Rani, “Development of myenteric plexus in human foetuses: a quantitative study,” Anatomy and Cell Biology, vol. 48, no. 2, pp. 124–129, 2015.

W. Vermeulen, J. G. De Man, P. A. Pelckmans, and B. Y. De Winter, “Neuroanatomy of lower gastrointestinal pain disorders,” World Journal of Gastroenterology, vol. 20, no. 4, pp. 1005–1020, 2014.

C. A. Ottaway, “Neuroimmunomodulation in the intestinal mucosa,” Gastroenterology Clinics of North America, vol. 20, no. 3, pp. 511–529, 1991.

N. Arizono, S. Matsuda, T. Hattori, Y. Kojima, T. Maeda, and S. J. Galli, “Anatomical variation in mast cell nerve associations in the rat small intestine, heart, lung, and skin. Similarities of distances between neural processes and mast cells, eosinophils, or plasma cells in the jejunal lamina propria,” Laboratory Investigation, vol. 62, no. 5, pp. 626–634, 1990.

T. Yamamoto, T. Kodama, J. Lee et al., “Anti-allergic role of cholinergic neuronal pathway via α7 nicotinic ACh receptors on mucosal mast cells in a murine food allergy model,” PLoS ONE, vol. 9, no. 1, Article ID e85888, 2014.

S. Willot, C. Gauthier, N. Patey, and C. Faure, “Nerve growth factor content is increased in the rectal mucosa of children with diarrhea-predominant irritable bowel syndrome,” Neurogastroenterology and Motility, vol. 24, no. 8, pp. 734–e347, 2012.

P. T. Schmidt, L. Eriksen, M. Loftager, T. N. Rasmussen, and J. J. Holst, “Fast acting nervous regulation of immunoglobulin A secretion from isolated perfused porcine ileum,” Gut, vol. 45, no. 5, pp. 679–685, 1999.

K. A. Sharkey, “Emerging roles for enteric glia in gastrointestinal disorders,” Journal of Clinical Investigation, vol. 125, no. 3, pp. 918–925, 2015.

S. Jabari, E. C. De Oliveira, A. Brehmer, and A. B. M. Da Silveira, “Chagasic megacolon: enteric neurons and related structures,” Histochemistry and Cell Biology, vol. 142, no. 3, pp. 235–244, 2014.

T. S. Sihra, E. Bogonez, and D. G. Nicholls, “Localized Ca2+ entry preferentially effects protein dephosphorylation, phosphorylation, and glutamate release,” Journal of Biological Chemistry, vol. 267, no. 3, pp. 1983–1989, 1992.

R. Llinas, M. Sugimori, and R. B. Silver, “Microdomains of high calcium concentration in a presynaptic terminal,” Science, vol. 256, no. 5057, pp. 677–679, 1992.

K. G. Margolis and M. D. Gershon, “Neuropeptides and inflammatory bowel disease,” Current Opinion in Gastroenterology, vol. 25, no. 6, pp. 503–511, 2009.

J. Tomàs, M. M. Santafé, N. Garcia et al., “Presynaptic membrane receptors in acetylcholine release modulation in the neuromuscular synapse,” Journal of Neuroscience Research, vol. 92, no. 5, pp. 543–554, 2014.

M. Pires-Oliveira, D. Moen, and M. Akaaboune, “Dynamics of nicotinic acetylcholine receptors and receptor-associated proteins at the vertebrate neuromuscular junction,” Current Alzheimer Research, vol. 10, no. 6, pp. 631–641, 2013.

M. Israël and Y. Dunant, “A unifying hypothesis for acetylcholine release,” Neurochemistry International, vol. 28, no. 1, pp. 1–9, 1996.

T. S. Hnasko and R. H. Edwards, “Neurotransmitter corelease: mechanism and physiological role,” Annual Review of Physiology, vol. 74, pp. 225–243, 2012.

D.-Y. Kim and M. Camilleri, “Serotonin: a mediator of the brain-gut connection,” The American Journal of Gastroenterology, vol. 95, no. 10, pp. 2698–2709, 2000.

G. Flemström and M. Sjöblom, “Epithelial cells and their neighbors. II. New perspectives on efferent signaling between brain, neuroendocrine cells, and gut epithelial cells,” American Journal of Physiology—Gastrointestinal and Liver Physiology, vol. 289, no. 3, pp. G377–G380, 2005.

J. Wheatcroft, D. Wakelin, A. Smith, C. R. Mahoney, G. Mawe, and R. Spiller, “Enterochromaffin cell hyperplasia and decreased serotonin transporter in a mouse model of postinfectious bowel dysfunction,” Neurogastroenterology and Motility, vol. 17, no. 6, pp. 863–870, 2005.

M. B. Hansen and A.-B. Witte, “The role of serotonin in intestinal luminal sensing and secretion,” Acta Physiologica, vol. 193, no. 4, pp. 311–323, 2008.

M. Kidd, B. I. Gustafsson, I. Drozdov, and I. M. Modlin, “IL1β- and LPS-induced serotonin secretion is increased in EC cells derived from Crohn's disease,” Neurogastroenterology and Motility, vol. 21, no. 4, pp. 439–450, 2009.

M. Bogunovic, S. H. Davé, J. S. Tilstra et al., “Enteroendocrine cells express functional Toll-like receptors,” American Journal of Physiology—Gastrointestinal and Liver Physiology, vol. 292, no. 6, pp. G1770–G1783, 2007.

P. Holzer and U. Holzer-Petsche, “Tachykinins in the gut. Part I. Expression, release and motor function,” Pharmacology and Therapeutics, vol. 73, no. 3, pp. 173–217, 1997.

P. Holzer and U. Holzer-Petsche, “Tachykinins in the gut. Part II. Roles in neural excitation, secretion and inflammation,” Pharmacology and Therapeutics, vol. 73, no. 3, pp. 219–263, 1997.

C. Pothoulakis, I. Castagliuolo, J. T. Lamont et al., “CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 3, pp. 947–951, 1994.

I. Marriott and K. L. Bost, “Substance P receptor mediated macrophage responses,” Advances in Experimental Medicine and Biology, vol. 493, pp. 247–254, 2001.

M. Riegler, I. Castagliuolo, P. T. C. So et al., “Effects of substance P on human colonic mucosa in vitro,” American Journal of Physiology—Gastrointestinal and Liver Physiology, vol. 276, no. 6, part 1, pp. G1473–G1483, 1999.

H.-W. Koon, D. Zhao, X. Na, M. P. Moyer, and C. Pothoulakis, “Metalloproteinases and transforming growth factor-α mediate substance p-induced mitogen-activated protein kinase activation and proliferation in human colonocytes,” Journal of Biological Chemistry, vol. 279, no. 44, pp. 45519–45527, 2004.

A. F. Stucchi, S. Shofer, S. Leeman et al., “NK-1 antagonist reduces colonic inflammation and oxidative stress in dextran sulfate-induced colitis in rats,” American Journal of Physiology—Gastrointestinal and Liver Physiology, vol. 279, no. 6, pp. G1298–G1306, 2000.

C. R. Mantyh, T. S. Gates, R. P. Zimmerman et al., “Receptor binding sites for substance P, but not substance K or neuromedin K, are expressed in high concentrations by arterioles, venules, and lymph nodules in surgical specimens obtained from patients with ulcerative colitis and Crohn disease,” Proceedings of the National Academy of Sciences of the United States of America, vol. 85, no. 9, pp. 3235–3239, 1988.

T. Goode, J. O'Connell, P. Anton et al., “Neurokinin-1 receptor expression in inflammatory bowel disease: molecular quantitation and localization,” Gut, vol. 47, no. 3, pp. 387–396, 2000.

K. Lieb, B. L. Fiebich, M. Berger, J. Bauer, and K. Schulze-Osthoff, “The neuropeptide substance P activates transcription factor NF-κB and κB-dependent gene expression in human astrocytoma cells,” Journal of Immunology, vol. 159, no. 10, pp. 4952–4958, 1997.

A. Azzolina, A. Bongiovanni, and N. Lampiasi, “Substance P induces TNF-α and IL-6 production through NFκB in peritoneal mast cells,” Biochimica et Biophysica Acta (BBA)—Molecular Cell Research, vol. 1643, no. 1–3, pp. 75–83, 2003.

B. L. Fiebich, S. Schleicher, R. D. Butcher, A. Craig, and K. Lieb, “The neuropeptide substance P activates p38 mitogen-activated protein kinase resulting in IL-6 expression independently from NF-κB,” The Journal of Immunology, vol. 165, no. 10, pp. 5606–5611, 2000.

S. Simeonidis, I. Castagliuolo, A. Pan et al., “Regulation of the NK-1 receptor gene expression in human macrophage cells via an NF-κB site on its promoter,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 5, pp. 2957–2962, 2003.

B. N. Lambrecht, P. R. Germonpré, E. G. Everaert et al., “Endogenously produced substance P contributes to lymphocyte proliferation induced by dendritic cells and direct TCR ligation,” European Journal of Immunology, vol. 29, no. 12, pp. 3815–3825, 1999.

W.-Z. Ho, J.-P. Lai, X.-H. Zhu, M. Uvaydova, and S. D. Douglas, “Human monocytes and macrophages express substance P and neurokinin-1 receptor,” Journal of Immunology, vol. 159, no. 11, pp. 5654–5660, 1997.

J.-P. Lai, S. D. Douglas, and W.-Z. Ho, “Human lymphocytes express substance P and its receptor,” Journal of Neuroimmunology, vol. 86, no. 1, pp. 80–86, 1998.

J.-P. Lai, S. D. Douglas, E. Rappaport, J. M. Wu, and W.-Z. Ho, “Identification of a δ isoform of preprotachykinin mRNA in human mononuclear phagocytes and lymphocytes,” Journal of Neuroimmunology, vol. 91, no. 1-2, pp. 121–128, 1998.

D. G. Payan, D. R. Brewster, and E. J. Goetzl, “Specific stimulation of human T lymphocytes by substance P,” Journal of Immunology, vol. 131, no. 4, pp. 1613–1615, 1983.

A. M. Stanisz, R. Scicchitano, P. Dazin, J. Bienenstock, and D. G. Payan, “Distribution of substance P receptors on murine spleen and Peyer's patch T and B cells,” Journal of Immunology, vol. 139, no. 3, pp. 749–754, 1987.

A. Wozniak, G. Mclennan, W. H. Betts, G. A. Murphy, and R. Scicchitano, “Activation of human neutrophils by substance P: effect on FMLP-stimulated oxidative and arachidonic acid metabolism and on antibody-dependent cell-mediated cytotoxicity,” Immunology, vol. 68, no. 3, pp. 359–364, 1989.

C. Feistritzer, J. Clausen, D. H. Sturn et al., “Natural killer cell functions mediated by the neuropeptide substance P,” Regulatory Peptides, vol. 116, no. 1–3, pp. 119–126, 2003.

H. W. Koon, D. Q. Shih, T. C. Hing et al., “Substance P induces CCN1 expression via histone deacetylase activity in human colonic epithelial cells,” The American Journal of Pathology, vol. 179, no. 5, pp. 2315–2326, 2011.

H.-W. Koon, D. Zhao, H. Xu et al., “Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis,” American Journal of Pathology, vol. 173, no. 2, pp. 400–410, 2008.

A. Lanas, “Role of nitric oxide in the gastrointestinal tract,” Arthritis Research and Therapy, vol. 10, supplement 2, p. S4, 2008.

C. H. Cho, “Current roles of nitric oxide in gastrointestinal disorders,” Journal of Physiology Paris, vol. 95, no. 1–6, pp. 253–256, 2001.

J. L. Wallace and M. J. S. Miller, “Nitric oxide in mucosal defense: a little goes a long way,” Gastroenterology, vol. 119, no. 2, pp. 512–520, 2000.

M. J. Martín, M. D. Jiménez, and V. Motilva, “New issues about nitric oxide and its effects on the gastrointestinal tract,” Current Pharmaceutical Design, vol. 7, no. 10, pp. 881–908, 2001.

C. Cirillo, G. Sarnelli, G. Esposito et al., “Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived S100B protein,” Neurogastroenterology and Motility, vol. 21, no. 11, pp. 1209–e112, 2009.

W. Sigge, T. Wedel, W. Kühnel, and H.-J. Krammer, “Morphologic alterations of the enteric nervous system and deficiency of non-adrenergic non-cholinergic inhibitory innervation in neonatal necrotizing enterocolitis,” European Journal of Pediatric Surgery, vol. 8, no. 2, pp. 87–94, 1998.

A. J. Porter, D. A. Wattchow, S. J. H. Brookes, and M. Costa, “Projections of nitric oxide synthase and vasoactive intestinal polypeptide-reactive submucosal neurons in the human colon,” Journal of Gastroenterology and Hepatology, vol. 14, no. 12, pp. 1180–1187, 1999.

A. Fischer, M. Gluth, U.-F. Pape, B. Wiedenmann, F. Theuring, and D. C. Baumgart, “Adalimumab prevents barrier dysfunction and antagonizes distinct effects of TNF-α on tight junction proteins and signaling pathways in intestinal epithelial cells,” American Journal of Physiology—Gastrointestinal and Liver Physiology, vol. 304, no. 11, pp. G970–G979, 2013.

Y. Merga, B. J. Campbell, and J. M. Rhodes, “Mucosal barrier, bacteria and inflammatory bowel disease: possibilities for therapy,” Digestive Diseases, vol. 32, no. 4, pp. 475–483, 2014.

M. Vivinus-Nébot, G. Frin-Mathy, H. Bzioueche et al., “Functional bowel symptoms in quiescent inflammatory bowel diseases: role of epithelial barrier disruption and low-grade inflammation,” Gut, vol. 63, no. 5, pp. 744–752, 2014.

K. Geboes and S. Collins, “Structural abnormalities of the nervous system in Crohn's disease and ulcerative colitis,” Neurogastroenterology and Motility, vol. 10, no. 3, pp. 189–202, 1998.

J. I. Antonius, F. E. Gump, R. Lattes, and M. Lepore, “A study of certain microscopic features in regional enteritis, and their possible prognostic significance,” Gastroenterology, vol. 38, pp. 889–905, 1960.

R. S. Strobach, A. H. Ross, R. S. Markin, R. K. Zetterman, and J. Linder, “Neural patterns in inflammatory bowel disease: an immunohistochemical survey,” Modern Pathology, vol. 3, no. 4, pp. 488–493, 1990.

K. Geboes, “Immunopathological studies of the small intestinal intramural nervous system and of intramural vessels in Crohn's disease,” Verhandelingen-Koninklijke Academie voor Geneeskunde van Belgie, vol. 55, no. 4, pp. 267–303, 1993.

A. M. Dvorak and W. Silen, “Differentiation between Crohn's disease and other inflammatory conditions by electron microscopy,” Annals of Surgery, vol. 201, no. 1, pp. 53–63, 1985.

V. Villanacci, G. Bassotti, R. Nascimbeni et al., “Enteric nervous system abnormalities in inflammatory bowel diseases,” Neurogastroenterology and Motility, vol. 20, no. 9, pp. 1009–1016, 2008.

B. Ohlsson, B. Veress, S. Lindgren, and G. Sundkvist, “Enteric ganglioneuritis and abnormal interstitial cells of Cajal: features of inflammatory bowel disease,” Inflammatory Bowel Diseases, vol. 13, no. 6, pp. 721–726, 2007.

J. Schneider, E. C. Jehle, M. J. Starlinger et al., “Neurotransmitter coding of enteric neurones in the submucous plexus is changed in non-inflamed rectum of patients with Crohn's disease,” Neurogastroenterology and Motility, vol. 13, no. 3, pp. 255–264, 2001.

D. R. Linden, J. M. Couvrette, A. Ciolino et al., “Indiscriminate loss of myenteric neurones in the TNBS-inflamed guinea-pig distal colon,” Neurogastroenterology and Motility, vol. 17, no. 5, pp. 751–760, 2005.

L. Boyer, M. Ghoreishi, V. Templeman et al., “Myenteric plexus injury and apoptosis in experimental colitis,” Autonomic Neuroscience: Basic and Clinical, vol. 117, no. 1, pp. 41–53, 2005.

K. M. Murphy and B. Stockinger, “Effector T cell plasticity: flexibility in the face of changing circumstances,” Nature Immunology, vol. 11, no. 8, pp. 674–680, 2010.

W. O'Connor, L. A. Zenewicz, and R. A. Flavell, “The dual nature of TH17 cells: shifting the focus to function,” Nature Immunology, vol. 11, no. 6, pp. 471–476, 2010.

F. Annunziato, L. Cosmi, V. Santarlasci et al., “Phenotypic and functional features of human Th17 cells,” Journal of Experimental Medicine, vol. 204, no. 8, pp. 1849–1861, 2007.

K. Gerlach, Y. Hwang, A. Nikolaev et al., “ cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells,” Nature Immunology, vol. 15, no. 7, pp. 676–686, 2014.

S. Fujino, A. Andoh, S. Bamba et al., “Increased expression of interleukin 17 in inflammatory bowel disease,” Gut, vol. 52, no. 1, pp. 65–70, 2003.

E. A. Mayer, K. Tillisch, and A. Gupta, “Gut/brain axis and the microbiota,” Journal of Clinical Investigation, vol. 125, no. 3, pp. 926–938, 2015.

L. V. Borovikova, S. Ivanova, M. Zhang et al., “Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin,” Nature, vol. 405, no. 6785, pp. 458–462, 2000.

K. J. Tracey, “The inflammatory reflex,” Nature, vol. 420, no. 6917, pp. 853–859, 2002.

M. Rosas-Ballina, P. S. Olofsson, M. Ochani et al., “Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit,” Science, vol. 334, no. 6052, pp. 98–101, 2011.

F. A. Sayani, C. M. Keenan, M. D. Van Sickle et al., “The expression and role of Fas ligand in intestinal inflammation,” Neurogastroenterology and Motility, vol. 16, no. 1, pp. 61–74, 2004.

Y. Kawahito, H. Sano, S. Mukai et al., “Corticotropin releasing hormone in colonic mucosa in patients with ulcerative colitis,” Gut, vol. 37, no. 4, pp. 544–551, 1995.

G. Matteoli and G. E. Boeckxstaens, “The vagal innervation of the gut and immune homeostasis,” Gut, vol. 62, no. 8, pp. 1214–1222, 2013.

S. M. Collins, “The immunomodulation of enteric neuromuscular function: implications for motility and inflammatory disorders,” Gastroenterology, vol. 111, no. 6, pp. 1683–1699, 1996.

X. Sagaert, T. Tousseyn, G. De Hertogh, and K. Geboes, “Macrophage-related diseases of the gut: a pathologist's perspective,” Virchows Archiv, vol. 460, no. 6, pp. 555–567, 2012.

B. Koscsó, K. Gowda, T. D. Schell, and M. Bogunovic, “Purification of dendritic cell and macrophage subsets from the normal mouse small intestine,” Journal of Immunological Methods, vol. 421, pp. 1–13, 2015.

G. Peña, B. Cai, L. Ramos, G. Vida, E. A. Deitch, and L. Ulloa, “Cholinergic regulatory lymphocytes re-establish neuromodulation of innate immune responses in sepsis,” Journal of Immunology, vol. 187, no. 2, pp. 718–725, 2011.

W. J. de Jonge, E. P. van der Zanden, F. O. The et al., “Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway,” Nature Immunology, vol. 6, no. 8, pp. 844–851, 2005.

C. Cailotto, P. J. Gomez-Pinilla, L. M. Costes et al., “Neuro-anatomical evidence indicating indirect modulation of macrophages by vagal efferents in the intestine but not in the spleen,” PLoS ONE, vol. 9, no. 1, Article ID e87785, 2014.

H. S. P. de Souza and C. Fiocchi, “Immunopathogenesis of IBD: current state of the art,” Nature Reviews Gastroenterology and Hepatology, vol. 13, no. 1, pp. 13–27, 2016.

H. Wang, M. Yu, M. Ochani et al., “Nicotinic acetylcholine receptor α7 subunit is an essential regulator of inflammation,” Nature, vol. 421, no. 6921, pp. 384–388, 2003.

G. Matteoli, P. J. Gomez-Pinilla, A. Nemethova et al., “A distinct vagal anti-inflammatory pathway modulates intestinal muscularis resident macrophages independent of the spleen,” Gut, vol. 63, no. 6, pp. 938–948, 2014.

D. Foell, T. Kucharzik, M. Kraft et al., “Neutrophil derived human S100A12 (EN-RAGE) is strongly expressed during chronic active inflammatory bowel disease,” Gut, vol. 52, no. 6, pp. 847–853, 2003.

S. Sanovic, D. P. Lamb, and M. G. Blennerhassett, “Damage to the enteric nervous system in experimental colitis,” American Journal of Pathology, vol. 155, no. 4, pp. 1051–1057, 1999.

A. M. Dvorak, “Ultrastructural evidence for release of major basic protein-containing crystalline cores of eosinophil granules in vivo: cytotoxic potential in Crohn's disease,” Journal of Immunology, vol. 125, no. 1, pp. 460–462, 1980.

O. Saitoh, K. Kojima, K. Sugi et al., “Fecal eosinophil granule-derived proteins reflect disease activity in inflammatory bowel disease,” The American Journal of Gastroenterology, vol. 94, no. 12, pp. 3513–3520, 1999.

M. E. Rothenberg, “Gastrointestinal eosinophils,” Allergy, vol. 56, supplement 67, pp. 21–22, 2001.

S. A. Woodruff, J. C. Masterson, S. Fillon, Z. D. Robinson, and G. T. Furuta, “Role of eosinophils in inflammatory bowel and gastrointestinal diseases,” Journal of Pediatric Gastroenterology and Nutrition, vol. 52, no. 6, pp. 650–661, 2011.

C. M. Smyth, N. Akasheh, S. Woods et al., “Activated eosinophils in association with enteric nerves in inflammatory bowel disease,” PLoS ONE, vol. 8, no. 5, Article ID e64216, 2013.

S. P. Hogan, A. Mishra, E. B. Brandt et al., “A pathological function for eotaxin and eosinophils in eosinophilic gastrointestinal inflammation,” Nature Immunology, vol. 2, no. 4, pp. 353–360, 2001.

M. Jönsson, Ö. Norrgård, and S. Forsgren, “Substance P and the neurokinin-1 receptor in relation to eosinophilia in ulcerative colitis,” Peptides, vol. 26, no. 5, pp. 799–814, 2005.

I. M. McGowan, R. M. Fairhurst, F. Shanahan, and P. A. Anton, “Mucosal substance P receptor expression in HIV infection and inflammatory bowel disease,” NeuroImmunoModulation, vol. 4, no. 2, pp. 70–76, 1997.

S. J. Galli, M. Tsai, J. R. Gordon, E. N. Geissler, and B. K. Wershil, “Analyzing mast cell development and function using mice carrying mutations at W/c-kit or Sl/MGF (SCF) loci,” Annals of the New York Academy of Sciences, vol. 664, pp. 69–88, 1992.

P. Rømert and H. B. Mikkelsen, “c-kit immunoreactive interstitial cells of Cajal in the human small and large intestine,” Histochemistry and Cell Biology, vol. 109, no. 3, pp. 195–202, 1998.

S. N. Abraham and A. L. St John, “Mast cell-orchestrated immunity to pathogens,” Nature Reviews Immunology, vol. 10, no. 6, pp. 440–452, 2010.

M. Gué, C. Del Rio-Lacheze, H. Eutamene, V. Théodorou, J. Fioramonti, and L. Buéno, “Stress-induced visceral hypersensitivity to rectal distension in rats: role of CRF and mast cells,” Neurogastroenterology and Motility, vol. 9, no. 4, pp. 271–279, 1997.

J. Lee, T. Yamamoto, S. Hayashi, H. Kuramoto, and M. Kadowaki, “Enhancement of CGRP sensory afferent innervation in the gut during the development of food allergy in an experimental murine model,” Biochemical and Biophysical Research Communications, vol. 430, no. 3, pp. 895–900, 2013.

G. Barbara, V. Stanghellini, R. De Giorgio et al., “Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome,” Gastroenterology, vol. 126, no. 3, pp. 693–702, 2004.

G. Barbara, C. Cremon, and V. Stanghellini, “Inflammatory bowel disease and irritable bowel syndrome: similarities and differences,” Current Opinion in Gastroenterology, vol. 30, no. 4, pp. 352–358, 2014.

G. Barbara, B. Wang, V. Stanghellini et al., “Mast cell-dependent excitation of visceral-nociceptive sensory neurons in irritable bowel syndrome,” Gastroenterology, vol. 132, no. 1, pp. 26–37, 2007.

X.-Y. Wang, N. Zarate, J. D. Soderholm, J. M. Bourgeois, L. W. C. Liu, and J. D. Huizinga, “Ultrastructural injury to interstitial cells of Cajal and communication with mast cells in Crohn's disease,” Neurogastroenterology and Motility, vol. 19, no. 5, pp. 349–364, 2007.

N. Mehta, M. Kaur, M. Singh et al., “Purinergic receptor P2X₇: a novel target for anti-inflammatory therapy,” Bioorganic and Medicinal Chemistry, vol. 22, no. 1, pp. 54–88, 2014.

B. D. Gulbransen, M. Bashashati, S. A. Hirota et al., “Activation of neuronal P2X7 receptor-pannexin-1 mediates death of enteric neurons during colitis,” Nature Medicine, vol. 18, no. 4, pp. 600–604, 2012.

M. V. da Silva, A. R. Marosti, C. E. Mendes, K. Palombit, and P. Castelucci, “Differential effects of experimental ulcerative colitis on P2X7 receptor expression in enteric neurons,” Histochemistry and Cell Biology, vol. 143, no. 2, pp. 171–184, 2014.

A. R. Neves, M. T. L. Castelo-Branco, V. R. Figliuolo et al., “Overexpression of ATP-activated P2X7 receptors in the intestinal mucosa is implicated in the pathogenesis of Crohn's disease,” Inflammatory Bowel Diseases, vol. 20, no. 3, pp. 444–457, 2014.

P. A. Girotti, R. Misawa, K. Palombit, C. E. Mendes, J. C. Bittencourt, and P. Castelucci, “Differential effects of undernourishment on the differentiation and maturation of rat enteric neurons,” Cell and Tissue Research, vol. 353, no. 3, pp. 367–380, 2013.

Q. Yu, Z. Zhao, J. Sun et al., “Expression of P₂X₆ receptors in the enteric nervous system of the rat gastrointestinal tract,” Histochemistry and Cell Biology, vol. 133, no. 2, pp. 177–188, 2010.

Z. Xiang and G. Burnstock, “Distribution of P2Y2 receptors in the guinea pig enteric nervous system and its coexistence with P2X2 and P2X3 receptors, neuropeptide Y, nitric oxide synthase and calretinin,” Histochemistry and Cell Biology, vol. 124, no. 5, pp. 379–390, 2005.

H. Zhu, Y. Yu, L. Zheng et al., “Chronic inflammatory pain upregulates expression of P₂Y₂ receptor in small-diameter sensory neurons,” Metabolic Brain Disease, vol. 30, no. 6, pp. 1349–1358, 2015.

M. Shinoda, B. Feng, and G. F. Gebhart, “Peripheral and central P2X₃ receptor contributions to colon mechanosensitivity and hypersensitivity in the mouse,” Gastroenterology, vol. 137, no. 6, pp. 2096–2104, 2009.

J. R. Hockley, M. M. Tranter, C. McGuire et al., “P2Y receptors sensitize mouse and human colonic nociceptors,” Journal of Neuroscience, vol. 36, no. 8, pp. 2364–2376, 2016.

T.-D. Kanneganti, M. Lamkanfi, and G. Núñez, “Intracellular NOD-like receptors in host defense and disease,” Immunity, vol. 27, no. 4, pp. 549–559, 2007.

T. Nunes and H. S. de Souza, “Inflammasome in intestinal inflammation and cancer,” Mediators of Inflammation, vol. 2013, Article ID 654963, 8 pages, 2013.

D. Hollander, “Inflammatory bowel diseases and brain-gut axis,” Journal of Physiology and Pharmacology, vol. 54, supplement 4, pp. 183–190, 2003.

J. Cabarrocas, T. C. Savidge, and R. S. Liblau, “Role of enteric glial cells in inflammatory bowel disease,” Glia, vol. 41, no. 1, pp. 81–93, 2003.

M. Neunlist, P. Aubert, S. Bonnaud et al., “Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGF-β1-dependent pathway,” American Journal of Physiology, vol. 292, no. 1, pp. G231–G241, 2007.

T. G. Bush, T. C. Savidge, T. C. Freeman et al., “Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice,” Cell, vol. 93, no. 2, pp. 189–201, 1998.

T. C. Savidge, P. Newman, C. Pothoulakis et al., “Enteric glia regulate intestinal barrier function and inflammation via release of S-nitrosoglutathione,” Gastroenterology, vol. 132, no. 4, pp. 1344–1358, 2007.

D. K. Zhang, F. Q. He, T. K. Li et al., “Glial-derived neurotrophic factor regulates intestinal epithelial barrier function and inflammation and is therapeutic for murine colitis,” Journal of Pathology, vol. 222, no. 2, pp. 213–222, 2010.

M. Steinkamp, H. Gundel, N. Schulte et al., “GDNF protects enteric glia from apoptosis: evidence for an autocrine loop,” BMC Gastroenterology, vol. 12, article 6, 2012.

A. Cornet, T. C. Savidge, J. Cabarrocas et al., “Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn's disease?” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 23, pp. 13306–13311, 2001.

F. E. Dewhirst, C.-C. Chien, B. J. Paster et al., “Phylogeny of the defined murine microbiota: altered Schaedler flora,” Applied and Environmental Microbiology, vol. 65, no. 8, pp. 3287–3292, 1999.

M.-H. Wang and J.-P. Achkar, “Gene-environment interactions in inflammatory bowel disease pathogenesis,” Current Opinion in Gastroenterology, vol. 31, no. 4, pp. 277–282, 2015.

M. Friswell, B. Campbell, and J. Rhodes, “The role of bacteria in the pathogenesis of inflammatory bowel disease,” Gut and Liver, vol. 4, no. 3, pp. 295–306, 2010.

R. B. Sartor, “Microbial influences in inflammatory bowel diseases,” Gastroenterology, vol. 134, no. 2, pp. 577–594, 2008.

S. M. Man, N. O. Kaakoush, and H. M. Mitchell, “The role of bacteria and pattern-recognition receptors in Crohn's disease,” Nature Reviews Gastroenterology and Hepatology, vol. 8, no. 3, pp. 152–168, 2011.

R. Hansen, R. K. Russell, C. Reiff et al., “Microbiota of de-novo pediatric IBD: increased faecalibacterium prausnitzii and reduced bacterial diversity in Crohn's but not in ulcerative colitis,” American Journal of Gastroenterology, vol. 107, no. 12, pp. 1913–1922, 2012.

A. Andoh, H. Imaeda, T. Aomatsu et al., “Comparison of the fecal microbiota profiles between ulcerative colitis and Crohn's disease using terminal restriction fragment length polymorphism analysis,” Journal of Gastroenterology, vol. 46, no. 4, pp. 479–486, 2011.

P. P. Elia, Y. F. M. Tolentino, C. Bernardazzi, and H. S. P. De Souza, “The role of innate immunity receptors in the pathogenesis of inflammatory bowel disease,” Mediators of Inflammation, vol. 2015, Article ID 936193, 10 pages, 2015.

C. A. Janeway Jr. and R. Medzhitov, “Innate immune recognition,” Annual Review of Immunology, vol. 20, pp. 197–216, 2002.

S. Akira, S. Uematsu, and O. Takeuchi, “Pathogen recognition and innate immunity,” Cell, vol. 124, no. 4, pp. 783–801, 2006.

J. M. Wells, L. M. P. Loonen, and J. M. Karczewski, “The role of innate signaling in the homeostasis of tolerance and immunity in the intestine,” International Journal of Medical Microbiology, vol. 300, no. 1, pp. 41–48, 2010.

J. M. Wells, O. Rossia, M. Meijerink, and P. Van Baarlen, “Epithelial crosstalk at the microbiota-mucosal interface,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, supplement 1, pp. 4607–4614, 2011.

T. Kawai and S. Akira, “The role of pattern-recognition receptors in innate immunity: update on toll-like receptors,” Nature Immunology, vol. 11, no. 5, pp. 373–384, 2010.

M. Brown, K. R. Hughes, S. Moossavi, A. Robins, and Y. R. Mahida, “Toll-like receptor expression in crypt epithelial cells, putative stem cells and intestinal myofibroblasts isolated from controls and patients with inflammatory bowel disease,” Clinical and Experimental Immunology, vol. 178, no. 1, pp. 28–39, 2014.

P. Brun, M. C. Giron, M. Qesari et al., “Toll-like receptor 2 regulates intestinal inflammation by controlling integrity of the enteric nervous system,” Gastroenterology, vol. 145, no. 6, pp. 1323–1333, 2013.

M. Aguilera, P. Vergara, and V. Martínez, “Environment-related adaptive changes of gut commensal microbiota do not alter colonic toll-like receptors but modulate the local expression of sensory-related systems in rats,” Microbial Ecology, vol. 66, no. 1, pp. 232–243, 2013.

F. Ochoa-Cortes, T. Ramos-Lomas, M. Miranda-Morales et al., “Bacterial cell products signal to mouse colonic nociceptive dorsal root ganglia neurons,” American Journal of Physiology—Gastrointestinal and Liver Physiology, vol. 299, no. 3, pp. G723–G732, 2010.

B. S. Wostmann, “The germfree animal in nutritional studies,” Annual Review of Nutrition, vol. 1, pp. 257–279, 1981.

L. V. Hooper and J. I. Gordon, “Commensal host-bacterial relationships in the gut,” Science, vol. 292, no. 5519, pp. 1115–1118, 2001.

K. E. Shroff, K. Meslin, and J. J. Cebra, “Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut,” Infection and Immunity, vol. 63, no. 10, pp. 3904–3913, 1995.

K.-A. Mcvey Neufeld, Y. K. Mao, J. Bienenstock, J. A. Foster, and W. A. Kunze, “The microbiome is essential for normal gut intrinsic primary afferent neuron excitability in the mouse,” Neurogastroenterology and Motility, vol. 25, no. 2, pp. 183–e88, 2013.

J. B. Furness, W. A. A. Kunze, P. P. Bertrand, N. Clerc, and J. C. Bornstein, “Intrinsic primary afferent neurons of the intestine,” Progress in Neurobiology, vol. 54, no. 1, pp. 1–18, 1998.

Y.-K. Mao, D. L. Kasper, B. Wang, P. Forsythe, J. Bienenstock, and W. A. Kunze, “Bacteroides fragilis polysaccharide A is necessary and sufficient for acute activation of intestinal sensory neurons,” Nature Communications, vol. 4, article 1465, 2013.

K. A. Mcvey Neufeld, A. Perez-Burgos, Y. K. Mao, J. Bienenstock, and W. A. Kunze, “The gut microbiome restores intrinsic and extrinsic nerve function in germ-free mice accompanied by changes in calbindin,” Neurogastroenterology and Motility, vol. 27, no. 5, pp. 627–636, 2015.

J. Collins, R. Borojevic, E. F. Verdu, J. D. Huizinga, and E. M. Ratcliffe, “Intestinal microbiota influence the early postnatal development of the enteric nervous system,” Neurogastroenterology and Motility, vol. 26, no. 1, pp. 98–107, 2014.

N. Vergnolle, “Protease-activated receptors as drug targets in inflammation and pain,” Pharmacology and Therapeutics, vol. 123, no. 3, pp. 292–309, 2009.

S. R. Coughlin and E. Camerer, “PARticipation in inflammation,” The Journal of Clinical Investigation, vol. 111, no. 1, pp. 25–27, 2003.

D. R. Linden, B. P. Manning, N. W. Bunnett, and G. M. Mawe, “Agonists of proteinase-activated receptor 2 excite guinea pig ileal myenteric neurons,” European Journal of Pharmacology, vol. 431, no. 3, pp. 311–314, 2001.

D. E. Reed, C. Barajas-Lopez, G. Cottrell et al., “Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons,” Journal of Physiology, vol. 547, no. 2, pp. 531–542, 2003.

C. U. Corvera, O. Déry, K. McConalogue et al., “Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2,” Journal of Physiology, vol. 517, part 3, pp. 741–756, 1999.

M. Steinhoff, N. Vergnolle, S. H. Young et al., “Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism,” Nature Medicine, vol. 6, no. 2, pp. 151–158, 2000.

A. Coelho, N. Vergnolle, B. Guiard, J. Fioramonti, and L. Bueno, “Proteinases and proteinase-activated receptor 2: a possible role to promote visceral hyperalgesia in rats,” Gastroenterology, vol. 122, no. 4, pp. 1035–1047, 2002.

C. Nguyen, A.-M. Coelho, E. Grady et al., “Colitis induced by proteinase-activated receptor-2 agonists is mediated by a neurogenic mechanism,” Canadian Journal of Physiology and Pharmacology, vol. 81, no. 9, pp. 920–927, 2003.

N. Vergnolle, “The enteric nervous system in inflammation and pain: the role of proteinase-activated receptors,” Canadian Journal of Gastroenterology, vol. 17, no. 10, pp. 589–592, 2003.

N. Maharshak, E. Y. Huh, C. Paiboonrungruang et al., “Enterococcus faecalis gelatinase mediates intestinal permeability via protease-activated receptor 2,” Infection and Immunity, vol. 83, no. 7, pp. 2762–2770, 2015.

B. T. Green, N. W. Bunnett, A. Kulkarni-Narla, M. Steinhoff, and D. R. Brown, “Intestinal type 2 proteinase-activated receptors: expression in opioid-sensitive secretomotor neural circuits that mediate epithelial ion transport,” Journal of Pharmacology and Experimental Therapeutics, vol. 295, no. 1, pp. 410–416, 2000.

M. C. Buresi, N. Vergnolle, K. A. Sharkey et al., “Activation of proteinase-activated receptor-1 inhibits neurally evoked chloride secretion in the mouse colon in vitro,” American Journal of Physiology-Gastrointestinal and Liver Physiology, vol. 288, no. 2, pp. G337–G345, 2005.

O. Ikehara, H. Hayashi, Y. Watanabe et al., “Proteinase-activated receptors-1 and 2 induce electrogenic Cl- secretion in the mouse cecum by distinct mechanisms,” American Journal of Physiology—Gastrointestinal and Liver Physiology, vol. 299, no. 1, pp. G115–G125, 2010.

K. Mueller, K. Michel, D. Krueger et al., “Activity of protease-activated receptors in the human submucous plexus,” Gastroenterology, vol. 141, no. 6, pp. 2088–2097.e1, 2011.