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Functional gastrointestinal disorders (FGIDs)
Functional gastrointestinal disorders (FGIDs) are a group of distinct disorders of different sections of the gastrointestinal tract with the commonality that they have no clear pathophysiological cause (hence ‘functional’). FGIDs are common among the population, with Irritable Bowel Syndrome (IBS) and Functional Dyspepsia the most frequent. On a whole, FGIDs are the most commonly diagnosed disorders by gastroenterologists. Because they are difficult to define and, for the moment, have no clear pathological cause, their treatment is complicated, consisting of a variety of pharmacological, psychological, dietary, and complementary medical treatments (Whitfield and Shulman, 2009). In an ongoing effort to classify and define the FGIDs for diagnosis, the international “Rome process” has yielded the 4th edition of the Rome criteria for FGIDs in 2016 (Drossman, 2016).
Types of IBS Classification and Diagnostics
The Rome IV criteria are used to diagnose IBS and distinguish IBS from transient gut symptoms, and gut disorders with an organic origin. To qualify as IBS, symptoms must have occurred for the first time 6 months before presentation for diagnosis (Longstreth, Thompson et al., 2006), and have occurred 1 days per week during the last 3 months. Additionally, symptoms get better or worse after defecation, and stools show changes in frequency and form (Lacy, Mearin et al., 2016). The subtype is characterised as IBS-C if 25% of stools is hard or lumpy and <25% loose or watery; IBS-D if 25% of stools is loose or watery, and <25% hard or lumpy; IBS-M 25% of stools is hard or lumpy or 25% of stools is loose or watery; and IBS-U if there is insufficient abnormality to classify one of the other types (see Figure 1).
A tool often used to characterise stool form is the Bristol Stool Form Scale (Lewis and Heaton, 1997a), displayed in Table 3. According to the authors, the shape and form of stools is directly related to the transit time of stools through the gastro-intestinal tract, and is a better representation than frequency of defecation (Lewis and Heaton, 1997a). However, a later study using more modern methods to measure transit time found no correlation between whole gut transit and stool form in healthy adults, though moderate correlations with stool form were observed for constipated patients (Saad, Rao et al., 2009).
Chronic Functional Abdominal Pain
Chronic Functional Abdominal Pain (CFAP) is another FGID characterized by abdominal pain. The difference with IBS is that CFAP is not related to changes in bowel habit or stool form (Drossman, 2016). The absence of changes in bowel habit in CFAP can be taken as indication that the pain component in CFAP, but likely also in subgroups of IBS patients, is not related to motility disorders or other defects likely to influence stool transit. An altered visceral sensitivity and changes to the function of the brain-gut axis are therefore more likely causes.
Microbiota particularities/ dysbiosis
Starting with the recognition of the association of gastroenteritis, as discussed before, and the use of antibiotics as a risk factor for developing IBS (Villarreal, Aberger et al., 2012), the microbiota is increasingly recognized as a possible factor in IBS. The microbiota profiles of IBS patients tend to differ from those of healthy patients (Rajilic-Stojanovic, Biagi et al., 2011), and they can be used to predict the responsiveness of IBS patients to dietary intervention with a low-FODMAP diet (Bennet, Böhn et al., 2017; Valeur, Smastuen et al., 2018). However, because IBS is characterized by changes in bowel habits, which are very likely to influence the microbiota composition, specific differences are difficult to interpret. Additionally, it is not yet clear whether a specific microbiota pattern exists for IBS, but in general, a reduction of bacterial diversity and an increased instability have been consistently found (Collins, 2014).
It is very likely that the two-way signalling between microbiota and epithelium which can regulate secretion of mucus and other molecules involved in host-microbe interactions is involved in IBS, because IBS patients show a dysregulation of the mucus layer and β-defensin-2 peptides (Swidsinski, Loening-Baucke et al., 2008; Langhorst, Junge et al., 2009; Simren, Barbara et al., 2013). Illustrating a role for microbiota in IBS, patients show a modulated expression of certain Toll-like receptors (TLRs), which perceive pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (LPS) for the innate immune system, indicating involvement of microbiota-immune interactions in IBS. Both IBD and IBS patients show alterations in the composition of the gut microbiota (Spiller and Lam, 2011; Casen, Vebo et al., 2015), also known as ‘dysbiosis’, though it is not immediately clear whether this dysbiosis is cause, effect, or both.
Transit time and stool consistency directly influence microbiota composition (Vandeputte, Falony et al., 2015), and both these factors are altered in IBS and IBD, making it difficult to relate microbiota differences as a cause. Additionally, microbiota composition is affected by dietary patterns (Rajilic-Stojanovic, Jonkers et al., 2015), and patients often change their dietary habits in an effort to mitigate symptoms. Perhaps unsurprisingly, the measure of dysbiosis found in IBS patients is correlated to the gravity of their symptoms (Tap, Derrien et al., 2017), and interestingly, these differences in microbiota composition were not correlated to diet.
Theoretical background; list of FODMAPS/ diet specifics
To begin with, many symptoms of IBS, principally abdominal pain, but also bloating, are noted in response to luminal distension. Gibson and Shepherd proposed that minimizing the consumption of those dietary compounds that lead to distension of the intestine would lead to improvement of symptoms of FGIDs. A group of dietary components the same authors had previously coined ‘FODMAPs’, for Fermentable Oligo-, Di-, Mono-saccharides And Polyols (Gibson and Shepherd, 2005) have those properties that can lead to distension; they are poorly absorbed in the small intestine, osmotically active, and are rapidly fermented by the gut microbiota upon reaching the colon (Gibson and Shepherd, 2010), see Figure 8. It is generally claimed that FODMAPs themselves are not responsible for symptoms in IBS patients, but induce adverse effects due to inherent responses in these patients (Molina-Infante, Serra et al., 2016).
Metabolism; collecting the rent
Of course, a host does not always accept guests without expecting something in return. The microbiota pays us back by indispensable metabolic processes, digesting components which are indigestible to us, producing simpler molecules that we can use. Additionally, the gut microbiota is an important source of vitamins, such as riboflavin (vitamin B2), biotin (vitamin B7), folic acid (vitamin B9), cobalamin (vitamin B12), and vitamin K (Hill, 1997). In herbivore animals, cellulose and other cell wall components are processed by microbes to provide otherwise unavailable energy, particularly important in ruminants (Flint, 1997), but also in omnivores such as humans, the microbiota plays an important role in breaking down dietary carbohydrates (Cockburn and Koropatkin, 2016). Interestingly, the mucus produced by the host supports a metabolic network based on mucolytic activities of bacteria such as Akkermansia muciniphila that produces butyrate and vitamin B12 as well (Belzer, Chia et al., 2017). Butyrate produced by the microbiota is an important source of energy especially for colonocytes, who forego glucose from the blood in favour of butyrate produced by the microbiota (Roediger, 1980), other short-chain fatty acids produced are also absorbed and transported to be metabolised by rest of the body (den Besten, van Eunen et al., 2013). It is estimated that in humans, 6 to 10 per cent of energy requirements are provided by microbiota-derived SCFAs, based on the intake of a standard British diet (McNeil, 1984; Bergman, 1990), the authors point out however, that fermentable carbohydrate consumption differs greatly around the world, and therefore these energy contributions must vary substantially as well (Bergman, 1990).
The metabolites produced during microbial processing of intestinal contents are not only utilised by the host, but a complex ecology exists between different microbes as well. Many species are dependent on other species with complementary activities, and in this way, a complex metabolic network forms with mutualistic, amensalistic, or parasitic relationships between different species, where mutualism in the gut is particularly increased under anoxic conditions (Heinken and Thiele, 2015). Depending on the variety of resources they can utilize, species can be classified as either generalists or specialists. While we would normally expect generalists to be widely distributed and specialists to occur mostly in smaller niches (Cockburn and Koropatkin, 2016), in the gut, this does not necessarily hold true (Carbonero, Oakley et al., 2014), with specialists often being more widely distributed than generalist species.
House rules; reciprocal control
In the relation between the host and the intestinal microbiome, both actors influence each other and exert a modicum of control. Under healthy conditions, the host enforces sterility where it is necessary, and supports the microbiota where desired. The microbiota in its turn, communicates with the host to support immune tolerance and optimal conditions to its prosperity.
The microbiota, through their gene expression and products, but also metabolites such as SCFAs, influences the host immune cells and cytokine production (Geuking, Koller et al., 2014), while it itself is subject to change by host-produced antimicrobial peptides and IgA secretions, which have regulatory functions, which can be, but are not always, microbicidal (Neish, 2009). The innate and adaptive immunity both play a significant role in monitoring and regulating the microbiota. For the innate immune system, some of these are produced independently from circumstances, such as α- and β-defensins (Pütsep, Axelsson et al., 2000), and Crohn’s disease patients exhibit a defect in α-defensin production by Paneth cells, underlining its important function (Wehkamp, Salzman et al., 2005),the abundance of other secretions, such as C-type-lectin Reg3γ, is regulated by the activity of pattern recognition receptors (PRRs) (Gong, Xu et al., 2010), important regulators of innate immunity. Reg3γ is poorly expressed in germ-free mice, and introduction of a mixed mouse microbiota increases this expression 20-fold, related to the ability of the bacteria to reach the epithelium (Cash, Whitham et al., 2006), indicating the involvement of epithelial sensing, most likely through PRRs. Many of these PRRs belong to the toll-like receptor family (TLRs), but Nod1 and Nod2, as well as several other types of receptors are important PRRs too. Many PRRs are activated by microbe-associated molecular patterns (MAMPs), which are represented among others by lipopeptides, lipoproteins, flagellins, and lipopolysaccharides; i.e., molecules that indicate the presence of microbes (Medzhitov and Janeway, 2002). Upon recognition of such a molecule, the PRR is activated and, depending on which PRR it is, activates regulatory pathways such as the mitogen-activated protein kinase (MAPK), nuclear factor B (NF-B)/Rel pathways, or the inflammasome (Neish, 2009). PRRs can be present on the cell membrane, on endosomes, or in the cytoplasm (Neish, 2009). In most tissues, PRRs activate inflammatory pathways, but because of the high microbial presence in the gut, PRRs there are also used to retain tolerance. For example, while activation of basolaterally positioned TLR9 leads to NF-B activation by degradation of IBα, apical activation leads to stabilisation of IBα and p105, preventing activation of the NF-B pathway (Lee, Mo et al., 2006). TLR9-/- mice have a lower NF-B activation threshold, and consequently are more susceptible to experimental colitis induced by DSS (Lee, Mo et al., 2006), further indicating the vital role of apical PRRs in maintaining immune tolerance in the gut. Similarly, flagellin-sensitive TLR5 is located only basolaterally to be activated when bacteria are present on the ‘wrong’ side of the epithelium, inducing inflammatory processes upon activation by bacteria penetrating the epithelium (Gewirtz, Navas et al., 2001).
A sticky history of organisation
Much has been written about the organisation, and even the existence, of the gastrointestinal mucus barrier. The transparent mucus of the internal, difficult to access, mucosae is prone to dry out and notoriously hard to study. It has been debated extensively whether a permanent barrier even covers the internal mucosae or not.
In 1955, in the Croonian Lecture, Sir Howard Florey laid out the current understanding of various mucus secretions of mammals at that time. For intestinal mucus, he presented mainly a lubricative and protective function. Mucus was thought able to entrap food particles and bacteria touching the epithelium “…wrapping them up as it were in an envelope and expediting their forward movement…”, but without impeding diffusion of smaller molecules and not impeding absorption. Rationale behind this understanding is the higher concentration of goblet cells in the large intestine compared to the small intestine, together with the liquid chyme in the small intestine and the solid stools in the large intestine, where lubrication is thus of higher importance (Florey, 1955). Additionally, questions were raised about whether mucus release in response to irritants such as mustard oil is mediated by the central nervous system or autonomously by the tissue. Some years later, starting from 1965, a ‘surface coat’ in the intestine covering epithelial cells and the microvilli was described, separately from the mucus produced by goblet cells, observed using electron microscopy (Ito, 1965; Rifaat, Iseri et al., 1965; Mukherjee and Williams, 1967; Monis, Candiotti et al., 1969). This surface coat is produced by each cell individually and can vary quite abruptly from cell to the next (see Figure 21). This layer is known as the glycocalyx, a term proposed by Stanley Bennett in 1962 (published in 1963) meaning ‘sweet husk’, from Greek, in reference to the polysaccharides present in this layer (Bennett, 1963) in many species across the biological Kingdoms.
Visceral sensitivity: EMG, von Frey
Since a central IBS symptom relieved with the low-FODMAP diet is abdominal pain/discomfort, we set out to measure the possible induction of increased sensitivity by FODMAP ingestion. The reference method of recording of electromyographic response to colorectal balloon distension, as well as an alternative method of mechanical behavioural testing using von Frey filaments were performed. This alternative was necessary as FOS-treatment lead to increased amounts of intestinal content that caused the colorectal cavity to remain full even after fasting and habituation periods, which would impede reliable data gathering.
Electrodes are implanted into the abdominal muscles and colorectal distensions are used as noxious stimuli to evaluate visceral hyperalgesia by electromyographic (EMG) recording. Striated muscle’s EMG activity was recorded and analysed according to (Larsson, Arvidsson et al., 2003). Basal EMG activity was subtracted from the EMG activity registered during the periods of distension.
This procedure is considered the gold standard in visceral sensitivity analysis, measuring the abdominal contractions in response to a painful stimulus in a quantifiable way. Visceral hypersensitivity is observable through an increased response to the colorectal distension stimulus, both by a response to stimuli that do not provoke a reaction in normosensitive subjects (allodynia), as well as an increased response to painful stimuli (hyperalgesia).
Table of contents :
1 General Introduction
1.1 A fresh look at IBS – opportunities for systems medicine approaches
1.2 IBS, FODMAPs
1.2.1 Functional gastrointestinal disorders (FGIDs)
1.2.2 Aetiology IBS and physiopathology
1.2.3 Clinical practice
1.2.4 FODMAP diet
1.3 Intestinal microbiota
1.3.1 Function
1.3.2 Toxic metabolite hypothesis
1.4 Bacterial Metabolites; aldehydes/ methylglyoxal
1.4.1 Chemical properties of methylglyoxal
1.4.2 Biological properties of methylglyoxal
1.4.3 Deleterious effects of dicarbonyl stress
1.4.4 Detoxification – glyoxalase system
1.5 Intestinal barrier function
1.5.1 Epithelial barrier
1.5.2 Permeability
1.5.3 Mucus barrier
1.6 Aims and Outline of Thesis
1.6.1 Hypotheses
1.6.2 Approach
1.6.3 Experimental procedures
2 Project Results
2.1 Mucus organisation is shaped by colonic content; a new view
2.2 FODMAPs increase visceral sensitivity in mice through glycation processes, increasing mast cell counts in colonic mucosae
2.3 Increased FODMAP intake alters colonic mucus barrier function through glycation processes and increased mastocyte counts
3 General Discussion
3.1 Fermentable carbohydrates and IBS symptoms
3.2 Immune activation
3.3 Permeability
3.4 Role of glycation in efficacy of low-FODMAP diet
3.5 Mucus barrier
3.6 Concluding remarks
4 Acknowledgments
5 Publications, Dissemination and Training Activities
5.1 Publications part of this thesis
5.2 Other Publications
5.3 Oral presentations
5.4 Poster presentations
5.5 Other training and dissemination activities
6 References
