Intestinal epithelium plays a pivotal role as a barrier that selectively regulates paracellular permeability and transport, and therefore prevents access by potentially harmful pathogens and their toxins from the intestinal lumen. One of the major components of the intestinal mucosal barrier are the intracellular tight junctions (TJs), which—along with occludin and claudins—comprise a complex system that, by interacting with specific zonula occludens (ZO)-1, ZO-2 and ZO-3 proteins regulates permeability to small molecules and electrolytes (Fig. 1.2). An abnormal TJs structure or function alters intestinal permeability and may contribute to worsening of IBS symptoms [2]. In line, in vivo studies as well as assays on mucosal biopsies of the small and large intestines from diarrhea-predominant-IBS (IBS-D) and constipation-predominant-IBS (IBS-C) patients revealed an augmented intestinal permeability and decreased mRNA expression of ZO-1 and occludin, compared with healthy controls [3]. In contrast, Camilleri et al. [4] reported an increased expression of occludin in the intestinal mucosa in patients with IBS-C; hence, to date it is still unclear whether the expression of occludin is more likely associated with diarrhea or constipation IBS. Several in vivo studies in mice also showed increased intestinal permeability upon administration of colonic supernatants from all IBS subtypes and fecal supernatants from IBS-D patients, which was positively correlated with somatic and visceral pain.

Alterations within the intestinal epithelium affect sensineural processing and change both motor and sensory activities of the gut, contributing to symptom progression in IBS patients. However, extra-epithelial factors may also contribute to visceral hypersensitivity and abdominal pain.

MCs serve as a contributor in various intestinal disorders, including IBS, inflammatory bowel disease (IBD) or food allergy. The density of MCs in both small and large intestine is increased in IBS patients, compared to healthy individuals, but their distribution within the lower GI tract varies—some reports demonstrate higher accumulation in the small rather than large intestine, while others show the opposite. Deviations from the number of MCs appear independently of gender and the subtype of IBS; nonetheless, the presence of MCs per se does not always imply a pathogenic importance, unless they are activated.

Due to close proximity of MCs to enteric nerves and varicosities, MCs activation increases enteric neuron excitability and enhances potential firing of extrinsic sensory neurons in a MC-derived, mediator dependent manner. Multiple factors such as bacteria, viruses, parasites, toxins or endogenous peptides may activate MCs, prompting their degranulation and subsequent release of various mediators from cytoplasmic granules, particularly histamine, chymase, tryptase and cytokines, or induce synthesis of leukotriene C₄, platelet activating factor or prostaglandins (PGs). There is a growing appreciation for the hypothesis that MCs activation positively correlates with intestinal symptoms, such as abdominal pain, discomfort, bloating, changes in bowel habits, and psychological symptoms including depression and fatigue (for review see [11]).

Several studies reported a high concentration of histamine, proteases and tryptase in IBS mucosa, which excite and sensitize sensory nerves. Interestingly, histamine directly increases sensory response through H₁ receptor-mediated mechanism. The blockade of H₁ receptor results in a decreased response of mesenteric afferents, and inactivation of proteases in the mucosal supernatants of IBS patients [12]. Tryptase, in turn, is abundantly expressed in MC granules and is considered as a marker for their activation. It possibly activates spinal afferent terminals and enhances intestinal permeability through the protease-activated receptor-2 (PAR-2) on enterocytes, which subsequently causes long-lasting neuronal hyperexcitability and redistribution of TJs allowing the intraepithelial passage of macromolecules [13]. Interestingly, infusion of mucosal supernatants from IBS patients into the colon of rodents i.e. mice or rats, induces visceral hyperalgesia and allodynia. Both effects are reversed by PAR-2 antagonist and serine protease inhibitors. No hyperalgesia was documented in PAR-2—deficient mice [14], what supports its role in sensory activation.

Serum samples from IBS sufferers contain elevated levels of proinflammatory IL-1β, IL-6, IL-8 and TNF-α, which directly affect neuronal activity and alter intestinal contractility, absorption and/or secretion. Notably, breakdown of the mucosal barrier by proinflammatory cytokines allows foreign particles to invade the intestinal barrier and induce the immune response in the submucosal and myenteric neuronal plexi (for review see: [15]). Interestingly, IBS-D patients have higher accumulation of MCs within the small intestine, which causes defects in apical junctional complex integrity, by increasing the spaces between epithelial cells, and generally contributes to exacerbation of clinical symptoms, such as diarrhea and pain [16].

There is substantial literature providing evidence for the association between the frequency and severity of abdominal pain experienced by IBS patients and the presence of MCs within the gut. More recently, it has been shown that an allergic background worsens IBS symptoms by enhancing the infiltration of MCs into the cecum and rectum mucosa, and promoting the secretion of soluble factors responsible for paracellular permeability. Patients with food allergy are more susceptible to develop symptoms typical for IBS-D, rather than IBS-C, which probably results from a different level of tryptase released by MCs or even other pathways involved in MC activation. This notion warrants further investigation [17].

Apart from immune cells, enterochromaffin (EC) cells, whose serum concentration is increased especially in IBS-D subtype, are responsible for the modulation of nerve activity in the epithelium of lower GI tract by the release of serotonin (5-hydroxytryptamine, 5-HT). Because 5-HT participates in multiple GI functions, including vasodilation, peristalsis, electrolyte secretion and absorption, and in pain perception, it is reasonable to think that the symptoms observed in IBS can stem from alterations in serotonergic signaling. The highest level of 5-HT is found in the amygdala, integral to the emotional responses and visceral stimulation, which indicates that BGA also plays a critical role in signal transmission. The 5-HT reuptake transporter protein (SERT) regulates the action of 5-HT receptor within the GI tract by maintaining transmitter homeostasis and terminating the transmission. Worth noting, in SERT knockout mice, the level of 5-HT augments in an uncontrolled manner which aggravates the inflammatory response [18]. Several studies demonstrate that gene expression of SERT is downregulated in the colon and rectum of IBS patients, which is associated with increased 5-HT mucosal availability, augmented reflex activity and luminal hypersecretion that frequently result in diarrhea-like symptoms [19]. Moreover, the colonic 5-HT release correlates with MC infiltration and thereby can drive abdominal pain, irrespective of IBS subtype. Overall, IBS-D patients have considerably higher number of EC cells in comparison with IBS-C patients; nonetheless, some reports reveal no differences in EC quantity between IBS subtypes.

These data indicate that any disturbances in expression and/or content of 5-HT receptors, as well as changes in SERT activity impact sensorimotor function and thereby affect severity of abdominal pain/discomfort perceived by IBS patients. Additionally, the therapeutic efficacy of drugs affecting 5-HT receptors also supports the contribution of 5-HT in IBS pathophysiology (for more information see: Clinical treatment).

More recently, the upregulation and sensitization of receptors located on the peripheral nerve terminals of nociceptors are thought to imply visceral hypersensitivity. Notably, a high number of mucosal sensory transient receptor potential cation channel subfamily V type 1 (TRPV1) has been identified in the distal colon of IBS patients. The number of TRPV1 fibres was up to threefold higher in colonic biopsies obtained from IBS-C and IBS-D patients, than in control individuals. Moreover, a positive correlation between TRPV1 staining intensity, visceral pain and low-grade inflammatory infiltration was observed [20, 21].

Although IBS is not generally considered as an inflammatory disease, several lines of evidence indicate that immune and inflammatory mechanisms contribute to its pathophysiology. A low-grade inflammatory response, which changes the enteric neuromuscular and sensory nerve function, has been observed in the GI tract in patients with either “conventional”, as well as PI-IBS. Among various factors that can favor IBS symptoms in these patients, an increased number of immunocytes, especially lamina propria T cells, toll-like receptors (TLRs), intraepithelial lymphocytes (IELs), and—as previously mentioned—mucosal MCs, have been outlined.

Type 1 T-helper (Th)-1 and Th-2 cells are able to restrain one another and produce cytokines to maintain a balanced immune response. Dependent on the segment of the intestine and concomitant infectious disease, different expression level of Th-1- and Th-2-derived cytokines and various quantity of immunocytes occur in IBS patients. The peripheral blood obtained from IBS-D patients is abundant in Th-1 derived cytokines, such as proinflammatory IFN-γ, IL-2 and IL-12, and lower number of Th-2—derived cytokines, e.g. IL-4. Higher levels of TNF-α, IL-1β, IL-6 and LPS-induced IL-6 were also observed mostly in the subset of IBS-D. Similarly, in the intestinal mucosa of PI-IBS patients, where the ratio of Th-1 to Th-2 is shifted towards Th-1-derived cytokines, an augmented level of IFN-γ and IL-4 and significantly decreased level of antiinflammatory IL-10 was documented [22]. The imbalance between Th-1 and Th-2 is possibly driven by the infection, which changes the epithelial permeability and prompts subsequent response against microbiotic agents. Although some discrepancies exist in the number of cytokines secreted either by Th-1 or Th-2 cells, and detected in various studies of different IBS subtypes, the single nucleotide polymorphisms (SNP) in the major proinflammatory TNF-α, secreted by Th-1, and impaired production of antiinflammatory IL-10 have been detected, and implied in the mechanism of low-grade inflammation in IBS [23].

TNF-α is a particularly important cytokine produced primarily by macrophages and monocytes, which participates in the pathogenesis of many inflammatory diseases e.g. IBD. TNF-α circulates within the body where it activates MCs and neutrophils, modulates the action of vascular endothelial cells, exhibits tumoricidal activity and orchestrates the cytokine cascade in various immune states. TNF-α, along with IL-6, also control the hypothalamic-pituitary-adrenal (HPA) axis via corticotrophin-releasing hormone. Hence, many studies quantified the level of this prominent inflammatory mediator in patients with IBS.

A significant proportion of IBS patients have an elevated TNF-α in the serum/plasma and stool samples, when compared to healthy individuals [24, 25]; however, while analyzing the IBS subgroups, elevated TNF-α is most prevalent in IBS-D [26]. The level of TNF-α was also increased in peripheral-blood cells among IBS patients, what may influence the symptom perception in the gut mucosa via activation of sensory nerve pathways. Interestingly, changes in the serum level of TNF-α and other cytokines e.g. IL-5, Il-13 are associated with higher anxiety and/or depression [1, 27] (for more information please see Visceral hypersensitivity). The majority of studies highlighted the importance of TNF-α in the pathogenesis of IBS; nonetheless, there are also several studies in which the level of TNF-α was similar as in the healthy individuals. The variability of findings may result from the small sample size, variations in laboratory assays used in the study or heterogeneity of subject data—as up to now IBS is generally symptom-based.

White adipose tissue (WAT), besides its ability to respond to afferent signals from the CNS and hormones, also expresses and secretes both pro- and antiinflammatory adipokines, such as cytokines, chemokines, and hormone—like factors, which participate in a variety of physiological or pathophysiological processes (Fig. 1.3). The crosstalk between the inflamed intestine and the surrounding mesenteric adipose tissue is currently investigated in view of the etiopathology of IBS [28]. Consequently, resistin, leptin, TNF-α, plasminogen activator-1 (PAI-1), IL-6 and angiotensinogen are proinflammatory mediators, whereas adiponectin exerts antinflammatory activity. An overexpression of adipokines, particularly adiponectin, leptin and resistin were documented in mesenteric adipose tissue, while in IBS-D subjects the level of resistin and leptin were increased but adiponectin decreased [29].

The antiinflammatory properties of adiponectin encompass the inhibition of proinflammatory IL–6 and simultaneously induction of the antiinflammatory cytokines IL–10 and IL–1 [30]. Moreover, adiponectin can modulate macrophage phenotype by switching it from the proinflammatory classically activated macrophage (M1) to an antiinflammatory alternatively activated macrophage (M2) [31]. Accordingly, adiponectin knockout mice have elevated expression of M1 markers, including TNF–α and IL–6 in macrophages and stromal vascular fraction (SVFs), as compared with wild type (WT) mice. Administration of adiponectin to WT mice contributes to higher expression of M2-related genes and IL-10 [31]. Nevertheless, not all studies confirm the antinflammatory effect of adiponectin. It was observed that an increased adiponectin level occurs in inflamed, rather than non-inflamed mesenteric adipose tissue, which indicates that adiponectin exerts proinflammatory effects on colonic epithelial cells. In chronic autoimmune or inflammatory diseases the level of adiponectin seems to be elevated. The inconsistency in the reports presenting adiponectin as either proinflammatory or antinflammatory adipokine, may depend on the excess of adipose tissue during inflammation. It is hypothesized that in chronic and autoimmune diseases, such as rheumatoid arthritis, type 1 diabetes or IBD, the increase in adiponectin may be responsible for inflammation-induced catabolic responses. However, an unambiguous determination of what kind of process adiponectin is responsible for is still not possible.

Unlike adiponectin, resistin exerts proinflammatory effect by inducing expression of vascular endothelial adhesion molecules, what leads to infiltration of leukocytes into the site of immune reaction. Exogenous treatment of mice with recombinant human resistin regulates the release of proinflammatory cytokines from macrophages and adipocytes, including TNF-α, IL-6 and IL-1, which confirms its role in the process of inflammation.

Among a wide range of actions of leptin, e.g. control of the feeding behavior, modulation of the satiety, thermogenesis and lipid and glucose metabolism, the activation and modulation of various cytokines seem particularly important in the pathogenesis of immune and inflammatory disorders. This adipokine stimulates proliferation of naïve T-helper lymphocytes, controls the expression of IL-1, IL-2, IL-6 and TNF-α by T-cells [32, 33], promotes generation of NO and proinflammatory cytokines by macrophages, and contributes to the release of reactive oxygen species (ROS) from neutrophils. The concentration of leptin is higher in IBS-D versus control subjects, indicating its presumable involvement in IBS pathophysiology [29].

Along with a fair number of inflammatory cytokines which participate in the development of low-grade inflammation, proinflammatory adipokines may also indirectly affect the function of sensory innervations of the gut, and thus promote visceral pain.

Of interest, higher endotoxin levels have been observed in a subset of IBS patients, in comparison to healthy control. Gram-negative bacteria-derived endotoxins stimulate various proinflammatory mediators such as TNF-α, and acute phase reactants, including C-reactive protein (CRP), which prompt intestinal mucosal damage and further augment the extent of intestinal permeability and inflammation [34].

Collectively, it seems that increased permeability of the intestinal barrier, low-grade inflammatory response and altered 5-HT signaling may account for local sensitization and enhanced visceral pain observed in IBS patients (Fig. 1.1).

The observation of familial aggregation of IBS has pointed to the idea that genetic background contributes to IBS development. It has been demonstrated that relatives of IBS sufferers are at increased risk of IBS. Twin studies presented consistent results, showing higher concordance rate for IBS between monozygotic than dizygotic twins. Although environmental factors could be responsible for the familial aggregation of IBS, the fact that no association was found between spouses indicates the role of genes. Several genetic factors that seem to be associated with IBS have been recognized [35].

Polymorphisms related to serotonergic system have been found, in agreement with the known function of 5-HT in the pathogenesis of IBS. In particular, a polymorphism in the regulatory region of the SERT gene results in a long (L) or short (S) allele. Homozygous S/S genotype was found to be related to IBS-C and IBS-D in many studies, although some other did not confirm these results. This genotype may result in decreased expression of the gene and, consequently, reduced 5-HT reuptake [35, 36]. In another line of research, single nucleotide polymorphisms (SNPs) associated with IBS-D were found in HTR3 genes encoding serotonin type 3 receptors [37].

Genetic variants connected with neuronal function influence visceral sensitivity. Namely, IBS-associated polymorphisms in genes encoding voltage-gated sodium channel, neurexophilin 1, adrenergic receptors, members of the opioid and cannabinoid receptors, catechol-O-methyltransferase, brain-derived neurotrophic factor and fatty acid amide hydrolase have been reported [35].

Impairment of the immune system function is among the pathophysiological factors of IBS (for more information see: Low-grade inflammation). SNPs associated with IBS were recognized for TNF ligand superfamily member 15 gene (TNFSF15), suggesting it may have a role in immune modulation in IBS. TNFSF15 gene product, TNF-like ligand 1A (TL1A) is expressed in immune cells and participates in the regulation of the inflammatory response, particularly in interactions with pathogens and commensal bacteria in the gut. [38] Polymorphisms in genes associated with intestinal barrier function have also been reported [35].