Underlying molecular and cellular mechanisms in childhood irritable bowel syndrome

Visceral sensation mechanisms and pathways have been extensively reviewed by others [11]. Communication of sensory nerves responsive to noxious stimuli (chemical, mechanical, or inflammatory) with the central nervous system occurs via vagal, pelvic, and splanchnic nerve pathways [11, 12]. Splanchnic and pelvic nerve signals are transmitted via spinal visceral afferents to the spinal dorsal horn where they subsequently communicate to the brain via several ascending pathways including the spinothalamic, spinoreticular, and spinomesencephalic tracts. Descending inhibitory pathways influence visceral sensation by modulating (gating) the ascending visceral afferent pathways [12]. Visceral sensation processing is further modulated by mediators and receptors including neurotransmitter receptors, cannabinoid receptors, opioid receptors, gamma-aminobutyric acid receptors, glutamate receptors, glucocorticoid receptors, inflammatory receptors, and ion channel receptors [11]. Additional psychological factors such as attention/distraction, expectations of pain, emotion, stress, and coping strategies also play a role (Fig. 1).

Visceral hypersensitivity may be present in a subgroup of children with IBS. Through the use of rectal barostat studies, which inflate a balloon in the rectum while measuring pressure and volume, some children with IBS have been found to have increased visceral sensitivity [13]. In a small rectal barostat study in children with IBS (n = 10), Iovino et al. identified that emotional instability (anxiety, depression, discontent, impulsiveness, and anger) modulated visceral perception [14]. Supporting this are data suggesting children with pain-related FGIDs who had more anxiety and somatization also had increased pain frequency and severity; however, the study did not measure visceral sensitivity [15].

Abnormalities within the descending pain modulatory system are increasingly considered to be important in the development of the pro-nociceptive state encountered with visceral hypersensitivity [12]. Williams et al. recently found, using a diffuse noxious inhibitory control study design, that girls with IBS (in comparison to healthy girls) had impaired pain inhibition [16]. Hypnotherapy (hypnosis with therapeutic intent) appears, in part, to use descending inhibitory pathways for control of pain [17] while also reducing psychological factors such as stress [18]; it has demonstrated efficacy in several randomized controlled trials for childhood IBS [19].

While the majority of work in new pharmacological visceral pain therapies is occurring within animal models, there are new emerging clinical therapies. For example, efforts have focused on inflammatory receptors known as tachykinin receptors, of which there are three types [20]. Within a phase II randomized, placebo-controlled, parallel group study, Ibodutant^® (MEN 15596), a neurokinin 2 antagonist, demonstrated outcome efficacy (overall IBS symptom relief for 3 of 4 weeks) in a post hoc analysis of adults with IBS-D with pain [20]. However, it should be noted that in the overall IBS population studied, Ibodutant^® was not superior to placebo. In infants with colic, an abstract has reported that Nepadutant^® (MEN-11420), a neurokinin 2 antagonist, had efficacy in a phase II, randomized, double blind, placebo-controlled trial [21]. Though infants with colic may go on to manifest childhood FGIDs [22], there are currently no studies using neurokinin 2 antagonists in childhood IBS. We anticipate further studies with new pharmacologic therapies directed at modulating visceral pain.

In addition to drug development, alternative therapies are now being investigated for their ability to modulate visceral pain. Menthol, an active ingredient of peppermint oil, may reduce visceral pain through ion channels such as transient receptor potential ion channel melastatin subtype 8 [23]. A recent meta-analysis of peppermint oil identified an improvement in global assessment and IBS scores in adults with IBS [24]. Given a previous successful small, short-term study in children with IBS [25] and a recent small pharmacodynamic study in children with IBS [26], we anticipate further studies of peppermint oil in children with IBS.

Genetic factors in IBS recently have been reviewed in detail and are beyond the scope of this review [27]. Therefore, we provide a brief overview with more focus on genetics as they relate to serotonin signaling.

Relatives with a family history of IBS have a two- to threefold increased risk for IBS [27]. Twin studies have estimated the genetic heritability of IBS to range between 22 and 57 % [28] with higher prevalence seen amongst monozygotic in comparison to dizygotic twins [29]. The potential role of environmental contributions is still being investigated.

Several genetic disturbances related to IBS have been identified [27]. Perhaps the most extensive investigations to date have related to 5-hydroxytryptamine (5-HT, serotonin). Serotonin is a bioamine neurotransmitter primarily produced in the gut which is important for modulating intestinal motility, inflammation, and secretion [30]. Serotonin is synthesized by tryptophan hydroxylase within enteroendocrine cells and enteric neurons [31]. Under normal circumstances, the serotonin transporter (SERT) from gene SLC6A4 terminates 5-HT action via uptake into enterocytes and serotonergic neurons. However, genotypes which would alter SERT expression have been identified. These include homozygous genotypes of the promoter length polymorphism 5-HT transporter-linked polymorphic region (5-HTTLPR) which is upstream of SLC6A4. Short allele polymorphisms affect SLC6A4 transporter expression and have been associated with IBS-D and IBS-C [27]. 5-HT levels have been found to be markedly increased in rectal biopsies of adults with IBS-D who were homozygous for the short allele for 5-HTTLPR [32]. However, the field is still being actively investigated as a recent meta-analysis identified that the long/long genotype of 5-HTTLPR is associated with IBS-C but did not find associations of 5-HTTLPR mutations with IBS-D [33].

Beyond the gut alone, the short allele of 5-HTTLPR also is associated with several other factors including depression, anxiety, neuroticism, anxiety, increased sympathetic tone, decreased parasympathetic tone, post-infectious IBS, and higher cortisol levels [27]. Whether this and/or other serotonin-related mutations are found in children with IBS is currently unknown.

Intestinal permeability is regulated, in part, by inter-epithelial tight junctions composed of protein complexes. Within these protein complexes, three proteins have received particular attention within IBS: occludin, claudin-1, and zonula occludens-1 [34]. Though these protein complexes were not specifically evaluated, increased intestinal permeability has been identified in children with IBS [35]. In adults with IBS, increased intestinal permeability correlates with increased visceral hypersensitivity and abdominal pain symptoms [36].

Intestinal permeability recently has been found to be regulated by a class of endogenously expressed noncoding RNAs (21–23 nucleotides in length) known as microRNAs. MicroRNAs regulate gene expression, including gene expression of tight junction proteins. Specifically, in adults with IBS-D, by downregulating nuclear factor-ĸβ-repressing factor and claudin-1 mRNA expression, microRNA 29 increases intestinal barrier permeability [37]. Further studies to understand the unique role of microRNA 29 in those with IBS-D as opposed to other subtypes are needed. In addition, it is not currently known whether these molecules may serve as biomarkers in children with IBS.

Nevertheless, within the field of gastroenterology, there are increasing efforts to address pathology by improving intestinal barrier function. In children with IBS, a randomized controlled trial demonstrated that Lactobacillus GG improved both abdominal pain and intestinal permeability [38]. In adults with IBS, glutamine increased claudin-1 expression in patients with IBS-D [34]. In adults with celiac disease, Leffler et al. have studied the effect of larazotide (an inhibitor of Vibrio cholera zonula occludens toxin) which promotes tight junction assembly [39]. Though there were mixed results, a low dose of larazotide in adults with celiac disease on a gluten-free diet improved persistent symptoms [39].

We anticipate there will be further efforts in children with IBS to address increased intestinal permeability. In addition to specific therapies directed at tight junction proteins, there is increasing knowledge of other factors that alter intestinal permeability such as stress and immune activation (Fig. 1) [40].

Carbohydrate malabsorption as a pathway toward generation of symptoms in IBS has received a lot of recent attention, particularly in light of the efficacy of a low fermentable, oligosaccharide, disaccharide, monosaccharide, and polyols (FODMAP) diet in treating abdominal pain in IBS [58]. FODMAP carbohydrates include lactose, fructose, fructans, galactans, and polyols such as sorbitol. When ingested, they have both an intraluminal fermentation (gas-producing) and osmotic effect [59]. It is these physiologic changes which are believed to exacerbate IBS symptoms.

Diets which eliminate one substrate (e.g., lactose) at a time have not demonstrated efficacy in randomized controlled trials for childhood FGIDs [60]. However, elimination of a more comprehensive group appears to be more successful. A low FODMAP diet has demonstrated efficacy in ameliorating GI symptoms in children and adults with IBS in double blind, randomized controlled trials [61, 62]. In addition, low FODMAP diets alter the gut microbiome composition. One study found that a 4-week low FODMAP diet decreased luminal Bifidobacteria versus a habitual diet [63]. Another study in adults with IBS during a randomized, crossover, double blind trial found decreases in total bacterial abundance and diversity on the low FODMAP diet versus a typical Australian diet [62]. The role of these changes in the gut microbiome in affecting IBS symptoms while on the low FODMAP diet remains to be elucidated.

Lactose (one of the FODMAP carbohydrates) has received attention for many decades as a potential culprit for inducing gastrointestinal symptoms. Lactose malabsorption is not more prevalent in adults with IBS. Kumar et al. evaluated the prevalence of known lactase polymorphisms in those with IBS in comparison to healthy controls and found a similar frequency between groups [64]. However, underlying IBS pathological factors may play a role in lactose malabsorption symptom expression. Yang et al. evaluated lactose malabsorption in adults with IBS-D and healthy adults via a randomized blinded crossover trial; both groups had the lactase non-persistence polymorphism (C/C-13910). Subjects received doses of 10, 20, and 40 g of lactose [65]. In addition, they underwent hydrogen breath testing to identify lactose malabsorption, rectal barostat testing to measure visceral hypersensitivity, and ileocolonic biopsies to assess neuroimmune activation. Yang et al. found that those with an increase in gastrointestinal symptoms in response to lactose malabsorption had both increased visceral hypersensitivity and increased ileocolonic mast cells compared to both healthy controls and those with IBS who had no increase in GI symptoms following lactose malabsorption [65].

Dietary fiber supplementation, acting as a bulking agent, has long been proposed as a therapy for IBS. Based on meta-analyses and systematic reviews, the efficacy of fiber treatment in IBS is mixed; conclusions vary based on the different types of fiber used and, in part, on the different review methods employed [66‐71]. Although not conclusive, data suggest that psyllium, a soluble fiber, reduces abdominal pain and/or improves stooling symptoms in adults with IBS [70‐72]. A preliminary report suggests efficacy for psyllium fiber supplementation in children with IBS [73].

The role of bile acids in gut physiology and potential role in FGIDs has recently been reviewed [74, 75]. Briefly, bile acids are steroid-derived detergent molecules produced within the liver and secreted into the intestinal lumen that aid in fat digestion. Primary bile acids (produced by the liver) include both chenodeoxycholic acid (CDCA) and cholic acid. In normal physiologic conditions ~95 % of secreted bile acids are absorbed in the ileum via the ileal bile acid transporter and become part of the enterohepatic circulation. Within the ileal enterocyte bile acids induce expression of nuclear farnesoid X receptor (FXR); this in turn leads to expression of fibroblast growth factor 19 (FGF-19). FGF-19 is secreted and activates fibroblast growth factor receptor 4 on the hepatocyte membrane; this is followed by downregulation of both cholesterol 7 alpha-hydroxylase and subsequent bile acid synthesis. Therefore, bile acids absorbed in the ileum normally have negative feedback on hepatic bile acid production.

Primary bile acids that reach the colon such as CDCA may have both potent colonic secretory and motor stimulatory effects [76]. However, the majority of bile acids that are not absorbed in the ileum are believed to undergo deconjugation and dihydroxylation by colonic microbiota, leading to the formation of secondary bile acids: deoxycholic acid and lithocholic acid. As a result, the potential effects of primary bile acids may be altered. For example, cholic acid does not have colonic secretory effects, whereas following its conversion to deoxycholic acid by colonic microbiota, it does [77].

Altered bile acid secretion/absorption and/or metabolism may affect gastrointestinal physiology in those with FGIDs. In a recent meta-analysis, bile salt malabsorption (measured via 23-seleno-25-homotaurocholic acid testing) was found in 28.1 % of adults with IBS-D [78]. Shin et al. demonstrated that adults with IBS-D placed on a high-fat diet (versus those with IBS-C) have higher amounts of both fecal fat and fecal unconjugated secretory bile acids [79]. Though not yet studied in children with IBS one study in children with functional constipation identified that a small subset had excessive sulfation of CDCA [80]. Given the secretory capability of CDCA and its loss following sulfation the authors hypothesized that this may contribute to constipation [80]. A small open-label study of the low FODMAP diet in children with IBS found those who had marked improvement (versus those who did not) while on the diet had higher amounts (amongst other metabolites) of fecal dehydroxylithocholate [9]. Future studies regarding bile acids and their subsequent physiologic effects in children with IBS are needed.

In open-label studies in adults with bile acid malabsorption, bile acid sequestrants such as colestipol have shown efficacy [81]. In addition, obeticholic acid (an FXR agonist) used in a small study of adults with IBS-D with bile acid diarrhea demonstrated both an increase in FGF19 and improvements in several clinical parameters including median stool frequency, stool form, and diarrhea [82]. Studies with these interventions have not been conducted in children with IBS to date.

CDCA has been found to have several beneficial effects in adults with IBS-C including acceleration of colonic transit, increased stool frequency, and loosening of stool consistency [83]. Using an ileal bile acid transporter inhibitor (thereby increasing bile acids in the colon) within a phase II trial for adults with chronic idiopathic constipation, Chey et al. demonstrated an improvement in both stool frequency and constipation-related symptoms [84]. We anticipate further studies attempting to increase colonic secretory bile acid effects in subjects (both adults and children) with IBS-C in the future.

The gut microbiome (collection of microbiota and their genetic material) and its role in health and disease are being increasingly recognized. The gut microbiome composition of adults and children with IBS (versus healthy controls) has been found to be different [85, 86]. Using both 16s rRNA pyrosequencing and PhyloChip technology on fecal samples from children with IBS (versus healthy controls), Saulnier et al. found a significantly greater percentage of the class Gammaproteobacteria (such as H. parainfluenzae) as well as a novel Ruminococcus-like microbe [86]. In addition, Saulnier et al. found that in children with IBS, microbiome composition correlated with both abdominal pain severity and frequency; moreover, microbiome composition was also able to differentiate those with an IBS-U versus IBS-C subtype. Gut microbiome composition was potentially found to be important as a biomarker for improvement on a low FODMAP diet; children who markedly improved had a different microbiome composition as compared to those who did not [9, 61].

The gut microbiome has several functions in health including fermentation/degradation of undigested proteins and carbohydrates, hydrogen disposal, and bile acid transformation [87]. When performing these functions, the microbiome produces several metabolites including but not limited to short-chain fatty acids; gases, such as hydrogen sulfide; and secondary bile acids derived from primary bile acids. Through their presence and metabolic function, gut microbiota may play a role in several of the pathological factors covered so far in this review including dysregulated intestinal immune function, chronic low-grade mucosal inflammation, increased gut permeability, psychosocial distress, and visceral hypersensitivity [41, 87, 88]. Further work to elucidate the mechanisms of action of the gut microbiome and/or their metabolites within the biopsychosocial model of IBS (Fig. 1) is needed.

Therapies which are directed toward the microbiome such as probiotics have demonstrated efficacy in children with IBS. A recent meta-analysis of Lactobacillus rhamnosus GG in children with IBS found a significantly higher rate of treatment responders (no pain or improvement in pain) in those receiving the probiotic versus placebo [89]. In a multicenter, randomized, placebo-controlled, double blind, crossover study in children with IBS, VSL#3 significantly ameliorated gastrointestinal symptoms [90]. The mechanisms involved with these improvements through the use of probiotics remain largely to be elucidated.

Children with IBS may have several interacting factors which play a role in the pathogenesis of their symptoms. These factors are being actively investigated and, given their interrelated nature, are better understood when placed within the context of the biopsychosocial model (Fig. 1). Underpinning these factors are cellular and molecular mechanisms. Future investigations of these mechanisms can be challenging given the likely heterogeneous etiologies driving the symptoms of IBS. Ideally, mechanisms should not be investigated in isolation; rather, interrelationships can be investigated/accounted for within the biopsychosocial model. These mechanisms, when elucidated further, may lead to identification of biomarkers and personalized therapies for children with IBS.