CSIRO PUBLISHING Animal Production Science, 2017, 57, 2175–2187 Review http://dx.doi.org/10.1071/AN17276

Signalling from the gut lumen

John B. Furness A,B,C and Jeremy J. Cottrell A

ADepartment of Agriculture and Food, The University of Melbourne, Parkville, Vic. 3010, Australia. BFlorey Institute of Neuroscience and Mental Health, Parkville, Vic. 3010, Australia. CCorresponding author. Email: [email protected]

Abstract. The lining of the needs to be easily accessible to nutrients and, at the same time, defend against pathogens and chemical challenges. This lining is the largest and most vulnerable surface that faces the outside world. To manage the dual problems of effective nutrient conversion and defence, the gut lining has a sophisticated system for detection of individual chemical entities, pathogenic organisms and their products, and physico-chemical properties of its contents. Detection is through specific receptors that signal to the gut , the nervous system, the immune system and local tissue defence systems. These effectors, in turn, modify digestive functions and contribute to tissue defence. Receptors for nutrients include taste receptors for sweet, bitter and savoury, free fatty acid receptors, peptide and phytochemical receptors, that are primarily located on enteroendocrine cells. Hormones released by enteroendocrine cells act locally, through the circulation and via the nervous system, to optimise digestion and mucosal health. Pathogen detection is both through antigen presentation to T-cells and through pattern-recognition receptors (PRRs). Activation of PRRs triggers local tissue defence, for example, by causing release of antimicrobials from Paneth cells. Toxic chemicals, including plant toxins, are sensed and then avoided, expelled or metabolised. It continues to be a major challenge to develop a comprehensive understanding of the integrated responses of the gastrointestinal tract to its luminal contents.

Additional keywords: microbiota, mucosal , nutrient receptors.

Received 2 May 2017, accepted 3 July 2017, published online 19 July 2017

Introduction cells in other gut regions, hormones from other sources, and The gastrointestinal tract exists in a state of hypervigilance. neural signals including those from the central nervous system It contains a rich assortment of chemicals and microorganisms, (CNS). separated from the body’s internal milieu by only a single layer Roles of gastro-entero-pancreatic hormone-producing of epithelial cells for most of its length. This single layer of cells (enteroendocrine) cells in determining digestive in the small intestine is the largest vulnerable surface of the efficiency body, with a surface area of ~60 m2 (Ferraris et al. 1989). The total external (luminal) surface of the gastrointestinal tract is Optimal conversion of food to essential nutrients, including energy ~100–400 m2, compared with ~2 m2 of skin (MacDonald and substrates and structural and regulatory proteins, depends on the Monteleone 2005; Artis 2008). The lining of the gut is continually control of a range of digestive and digestion-related functions, exposed to food, drink and contaminants they may bring with many of which are influenced by the hormones released by them, to a multitude of microorganisms and their products, to enteroendocrine cells (EEC). These functions include appetite enzymatic and chemical breakdown products of complex and satiety, the rate of gastric emptying, intestinal transit, release molecules, to gastrointestinal , to potentially toxic of digestive enzymes, induction of nutrient transporters, fluid and chemicals and to food additives. electrolyte transport across the mucosa, local blood flow, pancreatic The intestine continuously monitors its contents so as to insulin , modulation of immune responses and tissue optimise nutrient conversion and defend against threats to its growth. The EEC release their hormones in response to integrity. For these purposes it possesses a range of sensory chemicals in the gut lumen, mechanical forces and the bulk receptors and mechanisms (Table 1) that activate four major properties of the luminal contents, such as pH. effector systems: the enteroendocrine system, the nervous fi system, the gut immune system and the non-immune defence Classi cation of EEC systems of the gut, including mucosal repair (Fig. 1). Enteroendocrine cells (EEC) are scattered as single cells in the This review concerns exteroception, particularly sensing of lining of the , and small and , chemicals that are in the luminal contents or arise from the which collectively form the largest endocrine organ of the body luminal contents. The digestive tract also senses messages from (Rehfeld 2004; Janssen and Depoortere 2013). Closely related the internal environment. These include hormones released by cells are found in the biliary tract and in the pancreatic islets;

Journal compilation CSIRO 2017 www.publish.csiro.au/journals/an 2176 Animal Production Science J. B. Furness and J. J. Cottrell

Table 1. Some factors that are sensed in the gut lumen and the associated receptors (italic) The table lists the major known properties of the contents and states of the gastrointestinal tract that are specifically sensed and that lead to endocrine, neural or other signals that change organ or body states. FFAR, free fatty acid receptor; GPCR, G protein-coupled receptor; Nod, nucleotide-binding oligomerisation domain; PRR, pattern-recognition receptor; TGR, Takeda G protein receptor; T1R–T2R, Tastant 1 receptor–Tastant 2 receptor; TRP, transient receptor-potential; 5-HT, 5-hydroxytryptamine

Nutrients and food components The taste receptors: simple sugars, the sweet taste receptor, T1R2–T1R3; aminoacids, the umami (savoury) receptor, T1R1–T1R3; the bitter receptor family, T2Rs; the sour (acid) receptor, TRPP2. Protein breakdown products (peptones and amino acids): GPR92/93, GPRC6A, T1R1–T1R3 Free fatty acid receptors: FFARs 1–3, GPR119, GPR120 Phytochemicals (specific chemical entities of herbs and spices): TRP receptors, including TRPV1, TRPV2, TRPV5, TRPV6, TRPA1, TRPP2; the bitter receptor, T2R; olfactory receptors Mechanical distortion, stretch and tension Mechanosensitive channels of nerve endings and enteroendocrine cells Other physico-chemical attributes Temperature, osmolarity, acidity Internal secretions Bile acid receptors, TGR5 Bacteria, viruses, fungi, protozoa and helminths: their antigens and products Pattern-recognition receptors (PRRs): toll-like receptors 1–9, Nod1, 2. T-cell receptors: peptides, lipopolysaccharides, vitamin B metabolites. Toxins and emetogenic compounds Receptors for emetogenic toxins on 5-HT-containing enteroendocrine cells in the stomach and proximal small intestine. Receptors for advanced glycation end products (RAGE). Foreign compound transporters/receptors that recognise foreign compounds including pharmaceuticals: peptide transporter (PTR) family members, oligopeptide transporters and organic anion transporters (OATPs). together with EEC, these form the gastro-entero-pancreatic and that there is both diversity of 5-HT-containing EEC and a endocrine system. Until very recently, EEC were classified into wide range of roles of gut-derived 5-HT (Diwakarla et al. 2017; 12 types, each with a single letter code representing the hormone Martin et al. 2017). Although the classification of EEC needs that the cells contain and release, for example, G cells being gastrin substantial revision, the major roles of the hormones in the containing, S cells being secretin containing and I cells being control of digestive function can be identified (Table 2). cholecystokinin (CCK)-containing. The exception was L cells Nutrient receptors are mostly, but not exclusively, located on that contain both glucagon gene products (glucagon-like peptides EEC cells. A summary of the nutrient receptor types that (GLP-1, GLP-2), glicentin and oxyntomodulin) and peptide influence the release of the different hormones is included in tyrosine–tyrosine (PYY). It is now clear that the one cell–one Table 2. The patterns of co-expression of hormones, and the hormone (or hormone combination) classificationisnolonger differences along the gut, provide a much greater complexity tenable (Helander and Fändriks 2012; Gribble and Reimann than is represented in the table, and that has been covered in 2015;Fothergillet al. 2017). For example, when cells expressing depth in recent reviews (Psichas et al. 2015; Husted et al. 2017). a reporter transgene under CCK promotor control are isolated and The hormones released from the EEC can act locally on other molecularly analysed, it is found that CCK gene transcripts are cells, including immune cells, on nerve endings, or at a distance co-expressed with secretin, glucagon-like insulinotropic peptide on other organs, including the pancreatic islets and the CNS. It (GIP), GLP-1, PYY and neurotensin transcripts in subsets of is notable that the hormones do not act alone; for example, CCK EEC, and co-expression of the peptide hormones has been and 5-HT both increase the release of digestive enzymes from confirmed by mass spectrometry and immunohistochemistry the pancreas (Li et al. 2000) and CCK, GLP-1 and PYY are all (Egerod et al. 2012). Isolation of GIP-expressing and GLP- satiety factors. GLP-1 and PYY, which are commonly localised expressing EEC and correlated immunohistochemical analysis in the same cells, have synergistic effects. Infusion of GLP-1 or has confirmed overlaps in expression of GIP, GLP-1, CCK, PYY PYY, at doses that singly do not cause any reduction in energy and secretin (Habib et al. 2012). Quantitative immunohistochemical intake, in combination reduce energy intake by 30% in human analysis of colocalisation of the K cell marker, GIP, and the L cell volunteers (Schmidt et al. 2014). CCK and 5-HT also act markers, GLP-1 and PYY, in the mouse and pig gastrointestinal synergistically to suppress food intake (Voigt et al. 1995; has shown that all possible combinations of these three hormones Hayes and Covasa 2005). There is also evidence that gastric occur in EEC (Cho et al. 2015). leptin may be involved in the reduction of food intake (Picó The most numerous of the EEC, ~50% of all EEC, are those et al. 2003). that contain 5-hydroxytryptamine (serotonin; 5-HT). For many years, these were thought to be a type that is separate from EEC that contain peptide hormones. However, it is now Sweet, umami and bitter receptors clear that 5-HT and peptide hormones are co-localised in some Taste receptors are present in the intestine in addition to the oral groups of EEC, notable in EEC that contain CCK or secretin, cavity. These are G protein-coupled receptors (GPCRs) of two Gastrointestinal sensing Animal Production Science 2177

Bacterial antigen EEC Nutrient

Paneth cell Parasite Toxic chemical

Brain stem Antimicrobial

Enteric hormone Prevertebral ganglion

Vagal primary afferent neuron

Intestinofugal Spinal cord neuron

Immune signals: Spinal primary local and systemic Endocrine hormones: afferent neuron local and circulating

Inteinsic sensory neuron

Local tissue defence signals

Fig. 1. Summary of luminal signals detected by the gut and the effector systems that they influence. A complex soup of nutrients, salts, pathogens, pathogen- derived antigens, digestive-tract secretions, desquamated , toxins and pharmaceuticals bathes the mucosa. These activate the following four major effectors: the enteroendocrine system, the nervous system, the gut immune system, and the non-immune defence system of the gut. Enteroendocrine cells (EEC, green) in the mucosal epithelium sense luminal chemicals and release hormones that act locally on nerve endings, on enteric neurons, on the epithelium and on cells of the immune system. Hormones that enter the circulation also act at remote sites. Lymphocytes are activated by antigens presented to them from the lumen. Immune cells and cells of tissue defence, such as mast cells and macrophages, also release substances that act locally within the gut wall. Some afferent neurons have cell bodies in the gut wall (intrinsic primary afferent neurons (IPANs) and intestinofugal neurons) and the cell bodies of others are in extrinsic ganglia (extrinsic primary afferent neurons). Bacterial products are detected by pattern-recognition receptors (PRRs) to activate local defence mechanisms, including secretion of antimicrobials from Paneth cells (pink). classes, namely tastant 1 (T1R) and tastant 2 (T2R; Young 2011). a T1R1–T1R3 heterodimer. The bitter-receptor family, the T2Rs, Receptors of the T1R family form the sweet taste receptor, consists of over 30 known GPCRs. The sweet, umami and a T1R2–T1R3 heterodimer, and the umami (savoury) receptor, bitter receptors are coupled to intracellular messenger systems 2178 Animal Production Science J. B. Furness and J. J. Cottrell

Table 2. Enteroendocrine cell hormones of the mammalian gastrointestinal tract The table lists the major known gut hormones, the receptors whose activation triggers their release, their major sites of storage along the gastrointestinal tract and roles that they play. This is a simplification and the text and references therein should be consulted for greater detail. See text for explanation of acronyms

Hormone Luminal receptors Primary locations Principal functions influenced Ghrelin T1R1/T1R3; T2Rs Stomach, small intestine Appetite increase, growth hormone release Histamine Closed cell Stomach Stimulation of gastric acid secretion Gastrin GPR92/93; GPRC6A Stomach Stimulation of gastric acid secretion Somatostatin GPR92/93;GPRC6A Stomach, small intestine Inhibition of gastrin release (stomach); modulation of insulin (and pancreas) release (pancreas) 5-HT FFARs 2, 3, TRPA1; toxin Stomach, small and large Facilitation of intestinal motility reflexes and secretion. receptors; TLRs, intestine Triggering of emesis and nausea in response to toxins. mechanoreceptors Decreased appetite. Enhancement of inflammation. Also affects bone formation CCK T2Rs; FFA1; GPR120; GPR93; Proximal small intestine Activation of gallbladder contraction and stimulation of CaSR; TRPA1; TLRs pancreatic enzyme secretion, suppression of appetite. GIP GPRs 119120; FFAR1 Proximal small intestine Stimulation of insulin release GLP-1 T2Rs;T1R2–T1R3;FFARs1, 2,3; Distal small intestine, colon Enhancement of carbohydrate uptake, slowing of intestinal GPRs119, 92/93, 120; CaSR transit, appetite regulation, enhancement of insulin release GLP-2 T2Rs;T1R2–T1R3;FFARs1, 2,3; Distal small intestine, colon Promotion of epithelial growth and repair GPRs119, 92/93, 120; CaSR PYY T2Rs;T1R2–T1R3;FFARs1, 2,3; Distal small intestine, colon; Suppression of appetite, slowing gastric emptying, slowing of GPRs119, 92/93, 120; CaSR proximal small intestine transit and stomach in pigs Oxyntomodulin FFARs 1, 2, 3; GPRs119, 92/93, Distal small intestine Induces satiety 120 Leptin Receptors for protein fragments Stomach, chief cells Reduces food intake Motilin Bile receptors Small intestine Initiation of migrating myoelectric complex in pig, dog and human. Not present in rodents. Neurotensin FFARs Small and large intestine Inhibition of intestinal contractions Secretin Acid receptor Proximal small intestine Reduction of acidity in upper small intestine by stimulation of bicarbonate release. Insulin-like peptide 5 Not known Colon Hypothesised to control colonic motility through the G-protein, a-gustducin. The sour receptor, TRPP2, is Iwatsuki et al. 2012) CCK-releasing cells of the proximal a member of the transient receptor-potential (TRP) family of intestine also express umami receptors (Daly et al. 2013). ligand-gated ion channels (Huang et al. 2006). Ghrelin cells express bitter taste receptors (Janssen et al. 2011). Sweet taste receptors detect glucose and other simple sugars, and are also activated by artificial sweeteners, such as saccharin, Free fatty acid receptors sucralose and acesulfame. In the proximal intestine they are in Fats initiate the release of bile salts to emulsify the fats, the release cells that express glucagon-like insulinotropic peptide (GIP) and of lipases to break them down, the slowing of intestinal transit in the distal small intestine, they are in cells containing GLP-1 and to allow time for their digestion, and satiety. They do this by PYY. Sweet taste receptors do not appear to be expressed activating GPCRs, including free fatty acid receptor 1 (FFAR1, in the stomach at all (Iwatsuki and Uneyama 2012). GIP and also known as GPR40), FFAR2 (GPR43), FFAR3 (GPR41), GLP-1 are incretins, which increase the sensitivity of pancreatic GPR119 (which binds free fatty acid metabolites, including fatty b cells to glucose, thus enhancing insulin secretion and, therefore, acyl ethanolamides) and GPR120. FFARs respond to fatty acids glucose storage (Gerspach et al. 2011). These hormones are thus that are produced by bacteria, especially in the colon and in the anti-diabetogenic (Irwin and Flatt 2015). GLP-2, also released in ruminant stomach. response tocarbohydrate ingestion, indirectly induces the glucose transporter sodium-glucose linked transporter 1 (also known as sodium-dependent glucose cotransporter 1) in enterocytes and Fats and digestive enzyme release enhances glucose absorption (Shirazi-Beechey et al. 2011). The major hormone that triggers the release of digestive Induction of glucose transport and augmentation of glucose enzymes is CCK, released from cells in the upper small intestine sensitivity of pancreatic b cells occurs rapidly and the addition that express FFAR1, acted on by short and medium chain fatty acids of synthetic sweet taste receptor stimulants has been suggested to (Edfalk et al. 2008). Like many gut hormones, CCK excites the enhance carbohydrate uptake in production animals (Moran et al. mucosal endings of vagal afferent neurons (Strader and Woods 2010, 2014). 2005). The vagal afferent signals are conveyed to the nucleus Umami receptors in the stomach are expressed by ghrelin- tractus solitarius in the lower brain stem. The output, modified containing EEC and also by specialised epithelial cells that are by other sensory information reaching the lower brain stem, is proposed to have a sensory role, the brush cells (Hass et al. 2010; activation of vagal efferent pathways that innervate the gallbladder Gastrointestinal sensing Animal Production Science 2179 and the pancreas. Circulating CCK can also contribute to Bitter taste receptors gallbladder contraction, by enhancing transmission from vagal Bitter taste receptors belong to the T2R family, in which there are efferent nerve endings (Mawe 1998). Greater, possibly supra- over 30 types that detect compounds which, in the oral cavity, physiological, amounts of CCK are required to cause enzyme taste bitter. They are found throughout the gastrointestinal tract release by a direct action on the pancreas (Furness 2006). (Wu et al. 2002; Avau and Depoortere 2016). It is thought that a Although CCK is released in response to fats, CCK triggers major role of bitter taste receptors in the intestine is to recognise the release of amylases and proteases as well as lipases from the potentially injurious or poisonous substances that may be eaten pancreas. Thus, lipid in the small intestine promotes digestion and to induce reactions to expel the potential toxin (see section of all major food components. It is notable that CCK cells also Sensing and reacting to noxious chemicals and pharmaceuticals express umami receptors (Daly et al. 2013) and the aromatic below). amino acid-responsive calcium-sensitive receptor (CaSR; Wang et al. 2011). Free fatty acids also cause the release of the insulinotropic hormones GIP and GLP1 (Xiong et al. 2013). EEC responses to physico-chemical states In addition to their activation through specific receptors, EEC react Fats and satiety to more generalised cues. For example, 5-HT cells are activated by Lipids are the most energy dense (~9 Cal/g or 38 kJ/g) of the mechanical distortion and the released 5-HT initiates or augments fl major food types, and the most satiating. CCK, 5-HT and three propulsive re exes in the intestine (Bülbring and Crema 1958; products of L cells, GLP1, oxyntomodulin and PYY, are satiety Keating and Spencer 2010;Herediaet al. 2013; Smith and Gershon factors (Chaudhri et al. 2008). The major effects of CCK, GLP1 2015; Spencer et al. 2015). Mechanical stimuli, luminal glucose, and oxyntomodulin on satiety are mediated through the vagus fatty acids, odorants and toxins have all been shown to stimulate fi nerves, although direct effects on the hypothalamus can be enterochromaf n cell (EC)5-HTrelease(Braunet al.2007).ThepH fl demonstrated (Abbott et al. 2005; Strader and Woods 2005; intheantralpartofthestomachinuences both gastrin and Ogawa et al. 2012). PYY acts directly on the hypothalamus, somatostatin release. If the food neutralises gastric acid, and the through Y2 receptors, and also causes CCK release that chyme reaching the antrum is thus less acidic, gastrin is released contributes to the satiety effect of PYY through CCK activation to promote acid release from parietal cells. Conversely, if the of the vagus (Strader and Woods 2005; Raybould 2007). antrum becomes more acidic, somatostatin release increases and somatostatin acts on gastric EEC to restrain gastrin release. Acidic Fats and intestinal transit: the ileal brake conditions in the duodenum stimulate secretin release, which acts to increase bicarbonate entry into the lumen and neutralise the Fats trigger the release of PYY and GLP-1 from EEC in the acid. distal small intestine. These cells express a complete range of fatty acid receptors, including GPR119, FFAR1-3 and FFAR4 (GPR120; Cox 2016). A major effect of PYY is to activate a Sensory neurons that react to physico-chemical neural circuit that inhibits propulsive activity in the more properties of the luminal content and to states proximal intestine (duodenum and jejunum; Lin et al. 1996; of tissues Furness 2006). Thus, PYY slows transit in the small intestine Extrinsic sensory neurons to allow time for fat digestion. There is evidence that GLP-1 also Both extrinsic and intrinsic neural pathways carry gastrointestinal contributes to the ileal brake and that both PYY and GLP-1 sensory (primary afferent) information. The extrinsic sensory contribute to reducing the rate of gastric emptying. pathways are the vagal (with first-order cell bodies in nodose and jugular ganglia), thoracolumbar (first-order cell bodies in Receptors for proteins and protein fragments, including fi umami receptors thoracolumbar dorsal root ganglia, DRGs), lumbosacral ( rst- order cell bodies in lumbosacral DRGs) and the viscerofugal Proteins are broken down by acid hydrolysis and proteases in the pathways, with first-order neurons in the gut wall (Furness et al. stomach, to produce denatured protein fragments (peptones), 2014). Vagal afferents carry information of the physiological peptides and amino acids. GLP-1 is released by protein digests, state of the digestive tract and probably do not carry any pain but not by undigested proteins, suggesting that the L cells express signals. However, cutting the thoracolumbar afferents (which peptone receptors (Cordier-Bussat et al. 1998). Peptone receptors follow the sympathetic pathways, prevents abdominal visceral also occur in the stomach, where the peptone receptor GPR92/93 pain, implying that most pain and discomfort of gastrointestinal has been located in gastrin (G) and somatostatin (D) cells (Haid origin is conveyed by these afferent neurons (Ray and Neill 1947; et al. 2012). G cells, and a small proportion of D cells, also express Bingham et al. 1950). GPRC6A, which responds to basic amino acids, including arginine, lysine and ornithine (Haid et al. 2011). The umami receptor (T1R1–T1R3 heterodimer), which is activated by Vagal, thoracolumbar and lumbosacral sensory neurons glutamate, is expressed by ghrelin cells in the stomach and by These give rise to endings in the gut wall that respond to both CCK cells in the small intestine. In the oral cavity, the umami chemical and mechanical stimuli (Kentish et al. 2012; Brookes receptor signals a savoury taste, typical of meats (Iwatsuki et al. et al. 2013). 2012). Recordings of gastric sensory nerve activity have indicated Sensory endings in the mucosa (mucosal afferents) do not that umami receptors are substantially more sensitive to glutamate penetrate the epithelium, and they sense nutrients indirectly via than to any other amino acid (Iwatsuki et al. 2012). signals that are released from the epithelium, primarily from EEC 2180 Animal Production Science J. B. Furness and J. J. Cottrell cells (Berthoud et al. 1995). Vagal sensory nerve endings have Intestinofugal neurons receptors for EEC hormones, including CCK (Raybould 2007), Intestinofugal neurons are an unusual type of neuron, peculiar GLP-1 and GLP-2 (Bucinskaite et al. 2009), 5-HT (Andrews to the gastrointestinal tract, that have cell bodies in the gut wall et al. 1990), ghrelin (Date et al. 2002) and PYY (Abbott et al. and send their processes to prevertebral ganglia (where they 2005). The effect of PYY on the vagus is partly indirect, through form synapses with post-ganglionic sympathetic neurons), to the release of CCK (Strader and Woods 2005; Raybould 2007). other digestive organs, and to the CNS (Furness et al. 2014). Vagal mucosal afferents are not sensitive to distension, but can Intestinofugal neurons that project to sympathetic ganglia are be activated by light stroking of the mucosa (Brookes et al. within the afferent limbs of nerve pathways of entero-enteric 2013). reflexes, that is, reflexes that are initiated from the intestine, and Bacterial products activate vagal afferents; the probiotic then act back on the stomach or intestine (Szurszewski and bacterium Lactobacillus rhamnosus increased spontaneous Miller 1994; Furness 2006). The sympathetic neurons that are fi vagal afferent ring and augmented vagal afferent discharge in innervated by intestinofugal neurons inhibit motility and fluid response to intraluminal distension (Perez-Burgos et al. 2013). secretion. The effect of inhibiting motility in the stomach and Bacteria or bacterial products could interact directly with toll- proximal small intestine is to slow transit of contents; this like receptors (TLR) expressed by the afferent neurons (Hosoi slowing is commonly referred to as the ileal brake, which is et al. 2005) and the afferents could be activated indirectly contributed to by the EEC hormone, PYY (see above). through stimulation of EEC cells (e.g. 5-HT-containing EC cells) by bacterial products. Intrinsic sensory (intrinsic primary afferent) neurons Mechanoreceptor endings are found in the external layers of the Although it was recognised more than 100 years ago that neural gastrointestinal tract. One type, intraganglionic laminar endings fl fl (IGLEs), is located in close proximity to enteric ganglia, between re exes in uencing movement and secretion occur in the isolated intestine, it was not until the 1990s that intrinsic sensory the circular and longitudinal muscle layers of the oesophagus, fi stomach, and small and large intestines (Berthoud et al. 1997; neurons were identi ed. Intrinsic sensory neurons, commonly Castelucci et al. 2003; Wang and Powley 2007). They have low referred to as intrinsic primary afferent neurons (IPANs), are mechanical thresholds for activation and are sensitive to distension large multipolar neurons with characteristic electrophysiological properties, notably substantial after-hyperpolarising potentials or direct application of pressure to the endings (Zagorodnyuk et al. fi 2001;Lynnet al. 2003). IGLEs of vagal origin are mainly found that follow their action potentials and limit their ring rates in the oesophagus and the stomach, whereas IGLEs of lumbosacral (Gershon and Kirchgessner 1991; Furness et al. 2004). IPANs origin are found in the colon and rectum. Another type of detect distortion of the mucosa, chemical changes in the lumen, mechanosensor, the intramuscular array, is located within the tension changes in the muscle and compressive forces on enteric circular smooth muscle layers and forms synapse-like complexes ganglia. They respond to some gut hormones, including 5-HT and with interstitial cells of Cajal (Powley and Phillips 2011). Powley CCK (Ellis et al. 2013; Gwynne et al. 2014). Their activation initiates mixing and propulsive reflexes and local secretomotor and Phillips (2011) postulated that IMAs, interstitial cells of fl Cajal and smooth muscle work cooperatively or synergistically and vasodilator re exes (Furness 2006). They are present in the to transduce specific stretch or muscle length information. small and large intestine, but are absent from enteric ganglia of Muscular–mucosal afferents respond to both stretch and mucosal the oesophagus and are very rare in the stomach. This is consistent with the oesophagus and stomach being controlled though vago- stroking (Hughes et al. 2009). fl throughout the body have a dense sensory innervation vagal re exes and signals that originate in the CNS (Furness et al. from unmyelinated C fibre afferents, with particularly high 2014). densities observed in vessels that supply the gut, including small Immune defence arteries within the gut wall (Furness et al. 1982). The fibres arise from DRGs and their sensory endings around the mesenteric The gut immune system is always in a state of activity, because arteries close to the gut are mechanosensitive (Brookes et al. there is always luminal exposure to foreign substances, bacteria, 2013). Neurogenic inflammation, which appears to be mediated other microorganisms and microbial products. Thus, there is by the peptide calcitonin gene-related peptide released from the ongoing activity of both the innate and adaptive immune vascular afferents, has an important mucosal protective role in the systems and cytokines are present in the healthy intestine. gastrointestinal tract, especially in the stomach (Holzer 2007). Many extrinsic afferent neurons that innervate the gut, Bacteria: good and bad termed silent nociceptors, are mechanically insensitive in The mammalian gut, and the intestines of other vertebrates conditions of normal health, but become sensitive to and invertebrates, contain vast numbers of microbiota, namely, mechanical forces in the physiological range when the tissue is bacteria, fungi and viruses (Fagarasan et al. 2010). Bacteria are inflamed (Ness and Gebhart 1990). These afferents can remain essential to normal life, and, in health, many species exist in a excitable when a severe inflammation has subsided. There is symbiosis with the host. Bacterial symbionts benefit the host evidence that muscular–mucosal afferents and vascular afferents, by metabolising food components such as plant carbohydrates, but not muscle afferents, become sensitised and acquire including pectins and celluloses, that would otherwise not provide mechanosensitivity following inflammation (Feng et al. 2012; host nutrition. It should be noted that all mammals lack cellulases Brookes et al. 2013). Persistent hypersensitivity that remains andsodependonbacteriafortheirconversion.Symbioticbacteria after inflammation has subsided may contribute to the irritable also aid in maintaining epithelial integrity, defending against bowel syndrome. pathogenic bacteria and promoting development and maturation Gastrointestinal sensing Animal Production Science 2181 of the mucosa (Backhed et al. 2005;Artis2008; Cerf-Bensussan Away from M cells, the epithelium is not completely impervious and Gaboriau-Routhiau 2010). In ruminants and in hindgut to bacteria; some cross it and can be found in the wall of the fermenters, such as the horse, bacteria are essential to break down intestine, in the liver and in mesenteric lymph nodes. If there is plant material and produce short-chain fatty acids (SCFAs), mucosal damage or leakiness, there can be greater passage of whereas in humans a very small proportion of energy supply is microbiota that contributes to liver disease (Leung et al. 2016b). obtained from SCFAs produced by bacteria (Furness and Bravo Bacterial antigens also cross the mucosal epithelium. Here, away 2015). In return, the symbiotic bacteria benefit from the moist, from Peyer’s patches, they can be sampled by antigen-presenting warm, nutrient-rich environment. The gut both senses its bacterial cells and exhibited to T-cells. A small proportion of dendritic cells load and interacts with the microbiome to shape its own ecosystem extend processes between enterocytes and probably sample the (Garrett et al. 2010; Blanton et al. 2016; Fischbach and Segre luminal contents directly (Artis 2008). 2016). The bacterial populations are modulated by diet in all Numerous among mucosal T-cells are conventional CD4+ mammals, including production animals (Burrough et al. 2015). and CD8+ cells whose surface repertoire of antigen receptors The important influence of the resident microbiota on gut health, bind to peptide fragments from proteins. Also in the mucosa are overall animal health, animal maturation and productivity is natural killer T-cells that recognise lipid molecules presented by apparent in production animals (Yeoman and White 2014). The dendritic cells. microbiota of farm animals can be disturbed by antibiotic treatment and diet. Recognition of the important influences of the microbiota Local defence against microorganisms has led to the development of probiotic and prebiotic additives Bacterial antigens are recognised by PRRs, primarily the TLRs intended to ensure gut and, hence, whole-animal health (Yeoman and nucleotide-binding oligomerisation domain (Nod) receptors and White 2014). that bind conserved structures of bacteria, viruses, fungi and Roles of bacteria in maturation of the gut are emphasised by certain parasites (Akira et al. 2006). TLRs 1–9 and Nods 1 and 2 studies of mice reared under gnotobiotic or germ-free conditions. are expressed by intestinal epithelial cells (MacDonald and In germ-free mice, the Peyer’s patches lack germinal centre Monteleone 2005). formation, isolated lymphoid follicles do not form, the composition In response to TLR and Nod activation, the release of anti- of CD4+ T-cells and IgA-producing B-cells in the lamina propria is microbial defence molecules (ADMs), which includes anti- altered and mucosa-associated invariant T-cells are deficient, microbial peptides and lectins, provides an initial defence against compared with those in conventionally housed animals (Treiner pathogenic bacteria (Dann and Eckmann 2007; Ouellette 2010). et al. 2003; Artis 2008). Provision of bacteria, or bacterial products, The major source of ADMs in the small intestine is Paneth cells, to germ-free mice restores the mucosal immune system (Klaasen but ADMs are also released from intestinal epithelial cells and et al. 1993;Mazmanianet al. 2005). It is notable that germ-free they are secreted by neutrophils in the intestine, as in other organs. mice to which normal gut microbiota are introduced in adulthood Paneth cells express several TLRs, including TLR2, 4, 5, and 9, have increased carbohydrate uptake and 60% more fat deposition, and Nod2 (Vaishnava et al. 2008). The major classes of ADMs in compared with animals that remain germ free, despite the mice the intestine are as follows: defensins,small(2–6 kDa) cationic with introduced microbiota consuming less food (Bäckhed et al. peptides that kill bacteria primarily by disruption of cell walls; 2004). Gut microbes stimulate EC-cell 5-HT production via lysozyme C, an enzyme that hydrolyses bacterial peptidoglycans, SCFAs (Reigstad et al. 2015). The physiological relevance of thus restricting their pro-inflammatory effects; phospolipases, this effect is not known with certainty, but it is recognised including phospholipase A2, some of which have preferential that bacterial overload or the presence of bacterial pathogens activity at bacterial membranes; bacteriocidal lectins,themain increases secretion of fluid into the colon and the propulsion of lectins of human Paneth cells being regenerating islet-derived 3- its contents, both effects that can be mediated by 5-HT. These alpha and g;andhost defence-related ribonucleases (Bevins and responses are involved in the expulsion of pathogens from the Salzman 2011). Some defensins also attack funguses, viruses and large bowel (see section Sensing and reacting to noxious chemicals protozoa. The adaptor molecule, myeloid differentiation primary and pharmaceuticals below). response protein 88, is downstream from Nod2, and manipulation of myeloid differentiation primary response protein 88 has shown Immune cell detection of bacterial antigens that Paneth-cell ADMs limit bacterial numbers in the mucus at the mucosal surface, and, consequently, reduce the movement of Gastrointestinal mucosal T-cells have the following three bacteria into the body (Vaishnava et al. 2008). Paneth cells also locations: (1) lymphoid aggregations: the Peyer’s patches and produce a range of inflammatory mediators, which include numerous small lymphoid follicles; (2) as individual cells interleukin-17A, tumour necrosis factor-a, interleukin-1b and scattered in the connective tissue beneath the mucosal lipokines (Ouellette 2010). epithelium; and (3) as intraepithelial lymphocytes, which are mostly CD8+ T-cells, within the epithelial layer (Ganusov and De Boer 2007). Presentation of antigen to Peyer’s patches is Involvement of EEC cell hormones in immune and tissue mediated by microfold (M) cells (Neutra et al. 2001) that defence roles sample the lumen and transfer antigen to dendritic cells. M cells A major function of GLP-2 is augmentation of intestinal probably do not process antigens themselves; they do not absorptive capacity, which it does by enhancing mucosal growth express MHC Class II molecules (Mowat 2003), although they (Engelstoft et al. 2008). It also modulates mucosal defence, in do express pattern-recognition receptors (PRRs; Kyd and Cripps particular by anti-inflammatory effects that are mediated indirectly 2008). through the stimulation of submucosal neurons and the release of 2182 Animal Production Science J. B. Furness and J. J. Cottrell the tissue-repair factors, keratinocyte growth factor and insulin- groups of proteins through a series of intermediary reactions like growth factor (Sigalet et al. 2007; Rowland and Brubaker (the browning reaction), a reaction accelerated by heating. Thus, 2011;Yustaet al. 2012). Deletion of the GLP-2 receptor increases substantial amounts of AGEs occur when foods are overheated. the severity of indomethacin-induced enteritis and the extent of They can also be formed endogenously in hyperglycemic bacterial translocation in the small intestine (Lee et al. 2012). conditions. There is good evidence that these reactive molecules This was accompanied by decreased expression of genes for contribute to gastrointestinal complications in diabetic patients anti-microbial products in Paneth cells and reduced mucosal (Birlouez-Aragon et al. 2010) and to liver disease (Leung et al. anti-bacterial capacity, although Paneth cell numbers were not 2016a). Receptors for AGEs (RAGEs) are present in the epithelial changed (Lee et al. 2012). GLP-2 increases intestinal blood lining of the small intestine, in the villi and the crypts, and on enteric flow, which could facilitate both digestion and absorption of neurons (Chen et al. 2012). These receptors bind AGEs and a range nutrients and tissue repair (Bremholm et al. 2009). of other ligands, to initiate pro-inflammatory cascades (Zong et al. Another gut hormone, 5-HT, has pro-inflammatory effects. 2010). RAGE-dependent inflammation may contribute to the For instance, the severity of experimental colitis is significantly AGE-induced damage to neurons in the intestine (Jeyabal et al. reduced when its synthesis is disrupted by knocking out tryptophan 2008;Riveraet al. 2014). hydroxylase 1 and restoration of 5-HT enhances colitis (Ghia et al. 2009). 5-HT drives pro-inflammatory cytokine production Eliminating toxins: nausea, emesis and diarrhoea from macrophages and enhances T-cell activation (Li et al. 2011). Emesis is initiated by toxins or irritants in the gastrointestinal tract, by toxins that cross into the circulation and reach the Sensing and reacting to noxious chemicals vomiting centres in the lower brain stem (area postrema), by and pharmaceuticals nausea induced by smell and by vestibular disturbance (motion The gut detects noxious chemicals, for example, plant alkaloids, sickness). The gastrointestinal component is dependent on the toxic products of bacteria, and products of putrefaction. The smell, release of 5-HT from EEC cells, which acts on vagal nerve taste and association of potentially toxic substances with nausea endings in the gut wall to carry the signal to vomiting centres elicit aversive behavioural changes, so that the substances are in the brain (Andrews et al. 1990). Antagonists of 5-HT3 receptors avoided. Foreign chemicals initiate expulsion, through vomiting are effective anti-emetics. It has been suggested that the reason and diarrhoea, and chemicals are detoxified through recognition for the presence of large amounts of 5-HT in the EEC cells of the by transporters and metabolism by detoxifying enzymes. stomach and duodenum is to provide a defence against toxic Pharmaceutical compounds, although not present in the period of substances (Sanger and Andrews 2006). Thus 5-HT-containing evolution of toxin sensing, are detoxified through the same EEC cells in the stomach and proximal small intestine are pathways. The initial sensing of toxins is through olfaction and detectors of emetogenic toxins and of hypertonic salt solutions taste, notably odorant receptors and bitter taste receptors in the that are effective initiators of vomiting. It is notable that 5-HT- oral cavity. Recognition continues after food is swallowed; for containing EEC cells express TLRs (Bogunovic et al. 2007), and example, administration of bitter compounds into the stomach are thus predicted to be activated by bacterial antigens. activates second-order neurons in the brain stem (Hao et al. The initiation of strong, rapidly moving, nerve-mediated 2008), through a signalling pathway that may be related to emesis. propulsive contractions, in both the small and large intestines, assists in expelling undesired contents from the gut. These Recognition and detoxification of luminal chemicals propulsive contractions are evoked by pathogens and their Foreign compounds in the intestine are recognised by products, including parasitic nematodes, the bacterial pathogen, specific foreign compound transporters, including the peptide- Vibrio cholera, and bacterial toxins, as well as by irritants, such as transporter (PTR) family (Rubio-Aliaga and Daniel 2008), ricinoleic acid, the active component of castor oil (Furness 2006). oligopeptide transporters (Smith et al. 2013) and organic Pathogens and their products, including cholera toxin, anion transporters (Hagenbuch and Gui 2008). After they are enterotoxins and rotavirus, are sensed by enteric neurons in transported across the epithelium, the compounds are exposed to secretomotor reflex pathways (Lundgren et al. 2000; Furness a range of detoxifying enzymes in the wall of the intestine, some 2006). This causes copious watery diarrhoea, which, along with of the enzymes being expressed by the enterocytes themselves the stimulation of propulsion, expels toxins and pathogens. (Thelen and Dressman 2009). Detoxifying enzymes in the The pathways can be overstimulated, and infectious diarrheas, intestine include cytochromes P450, uridine diphosphate if unchecked, can cause death through water and electrolyte loss glucuronosyltransferases, sulfotransferases, acetyl transferases, (Field 2003). glutathione S-transferases, esterases, epoxide hydrolase and alcohol dehydrogenase (Thelen and Dressman 2009). This is The integrated response to a meal fi the rst line of defence; those chemicals that escape into the The sight, smell or even the thought of food prepares the portal venous drainage are exposed to a similar range of digestive tract, by increasing salivary and gastric acid secretion detoxifying enzymes in the liver. and relaxing the stomach wall. 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