Chemical and molecular factors in irritable bowel syndrome: current knowledge, challenges, and unanswered questions

Abstract

Several chemical and molecular factors in the intestine are reported to be altered and to have a potentially significant role in irritable bowel syndrome (IBS), particularly in IBS with diarrhea. These include bile acids; short-chain fatty acids; mucosal barrier proteins; mast cell products such as histamine, proteases, and tryptase; enteroendocrine cell products; and mucosal mRNAs, proteins, and microRNAs. This article reviews the current knowledge and unanswered questions in the pathobiology of the chemical and molecular factors in IBS. Evidence continues to point to significant roles in pathogenesis of these chemical and molecular mechanisms, which may therefore constitute potential targets for future research and therapy. However, it is still necessary to address the interaction between these factors in the gut and to appraise how they may influence hypervigilance in the central nervous system in patients with IBS.

irritable bowel syndrome (IBS) is traditionally diagnosed on the basis of symptoms: recurrent abdominal pain and alterations in bowel function experienced as diarrhea (IBS-D), constipation (IBS-C), or mixed variant with constipation alternating with diarrhea (IBS-M). A variety of central and peripheral mechanisms initiate perturbation of gastrointestinal motor and sensory functions and lead to IBS symptoms (9, 41). These observations lead to the hypothesis that identification of the peripheral factors, their dysfunctions, and altered tissue expression will lead to a greater understanding of the pathobiology, diagnosis of subgroups, and prevention or reversal of the symptoms of IBS.

The objectives are to review the salient literature and knowledge gaps, focusing on chemical and molecular factors that could help understand the pathobiology of IBS and conceivably serve as potential therapeutic targets in IBS. The microbiota in IBS has been reviewed extensively elsewhere (4, 49) and is not included in this review of endogenous chemical and molecular mediators. Thus we will focus on bile acids (BAs), short-chain fatty acids (SCFAs), mucosal barrier proteins, proteases, mast cell products, and pathobiologically relevant mucosal molecules such as mRNA, proteins, and miRNA. These factors are linked in different ways in the complex mechanisms leading to the symptomatology in IBS (Table 1, Fig. 1).

Table 1.

Chemical and molecular factors in the multifactorial mechanisms implicated in mucosal functions in IBS

	Secretion/Absorption	Barrier	Inflammation	Sensation
Lumen	Bile acids; short-chain fatty acids	Bile acids	Bacterial proteases/toxins
Mucosa	Ion channel expression	Tight junction expression; microRNA expression		MicroRNA expression; neurotransmitters; mesotrypsin
Lamina propria		Serine proteases	Mast cells; serine proteases

Fig. 1.

Endogenous chemical and molecular factors in IBS.

Table 2.

Summary of published literature on the role of soluble mediators in IBS

Supernatant of Stool or Mucosal Biopsies	Patient Group	Receptor/Mechanism of Action	Biological Effect	Citation (Ref. no.)
CPA from FSN	Sum of two cohorts; 26 IBS-D, 44 IBS-C, 56 HC	CPA levels are increased in IBS-C vs. HC and IBS-D	Level of CPA correlated with pain. Increased visceral hypersensitivity in PAR2-deficient and normal mice infused with FSN from high-CPA IBS-C vs. normal-CPA IBS-C and HC.	Annahazi et al. (2)
		Decreased occludin in mice and T84 cells after repeated infusion of FSN in IBS-C vs. HC and in colonic mucosal biopsies in 8 IBS-C vs. 6 HC.	Increased intestinal permeability in PAR2 knockout mice and T84 cells infused with FSN in high-CPA IBS-C vs. normal-CPA IBS-C and HC.
Serine protease from mucosal biopsies	10 IBS-D, 5 IBS-C, 12 HC	Activation of PAR2 receptors	Increased hyperexcitability in DRG neurons from wild-type mice vs. PAR2-deficient mice in IBS-D vs. IBS-C and HC.	Valdez Morales et al. (52)
CPA from mucosal biopsies		Cysteine protease receptor	Increased hyperexcitability in DRG neurons from mice in 1 IBS-D.
Serine protease (tryptase), histamine and serotonin from mucosal biopsies	7 IBS-D, 4 IBS-C, 5 HC	Serine protease, serotonin and histamine receptors	Activation of submucosal neurons in surgical specimen of human colon, mediated by H1–H3, 5HT3 and serine protease receptors.	Buhner et al. (8)
Mucosal biopsies	17 IBS-D, 19 IBS-C, 15 IBS-A, 14 HC	Incubation with IBS SUP (all subtypes) decreased ZO-1 mRNA expression in Caco-2 cells	Increased intestinal permeability in Caco-2 cells incubated with SUP of 39 IBS patient (15 IBS-C, 14 IBS-D, 14 IBS-A) vs. 14 HC.	Piche et al. (43)
		Decreased ZO-1 expression in colonic biopsies of 21 IBS vs. 12 HC	Increased permeability in colonic biopsies of 12 IBS (3 IBS-C, 4 IBS-D, 5 IBS-A) vs. 5 HC
Serine proteases from FSN	24 IBS-D, 18 IBS-C, 10 IBS-A, 17 UC	Activation of PAR2 receptors	Increased visceral hypersensitivity in wild-type mice vs. PAR2-deficient mice	Gesce et al. (27)
	23 INF, 25 HC	Increased phosphorylation of myosin light chain; internalization of ZO-1	Increased permeability in wild-type mice vs. PAR2-deficient mice
	22 IBS-D, 17 HC	PAR2-mediated increased brain-derived neurotrophic factor expression in Caco-2 cells and mice; p38 MAPK phosphorylation in Caco-2 cells.	Increased visceral hypersensitivity in mice	Wang et al. (56)
Serine proteases from mucosal biopsies	20 IBS-D, 10 IBS-C, 10 IBS-M, 11 HC	PAR2 mediated increased 5,6-EET in DRG neurons from mice and increased stimulation of transient receptor potential vanilloid-4 in mice treated with SUP in IBS-D vs. HC.	Increased visceral hypersensitivity in mice exposed to SUP in IBS-D patients.	Cenac et al. (15)
Histamine	8 IBS, 7 HC	Mouse single-serosal afferents and DRG sensitized by SUP in IBS compared with control.	Effect mediated by HRH1 interacting with TRPV1.	Wouters et al. (58)
	55 IBS patients (44 analyzed)	HRH1 antagonist decreased the potentiating effect of SUP on DRG neurons in IBS-D patients.	Ebastine, HRH1 antagonist, decreased rectal sensation, abdominal pain, and improved quality of life compared with placebo in a group of IBS patients.
Reactive oxygen species from mucosal biopsies	14 PI IBS-D, 12 HC	Activation of PAR2 receptors on mast cells in rats.	Mast cell activation in rats with production of histamine.	Han et al. (29)
Mesotrypsin in colonic mucosal biopsies	10 IBS, 9 HC	Response to inflammatory stimuli and induction of hyperexcitability of mouse DRG neurons in a PAR2-dependent fashion	Intestinal epithelial cells produce and release mesotrypsin in IBS and upon inflammatory stimulation, specifically at their basolateral side.	Fourcade et al. (24); Lopez-Lopez et al. (37)

CPA, cysteine proteases; FSN, fecal supernatant; protease activated receptors, PAR₂; H, histamine; HC, healthy controls; 5-HT, serotonin; ZO-1, zonula occludens-1; EET, epoxyeicosatrienoic acid; MAPK mitogen-activated protein kinase; IBS-A, alternating-pattern irritable bowel syndrome; UC, ulcerative colitis; INF, infectious acute diarrhea; SUP, supernatant; DRG, dorsal root ganglion; HRH1, histamine receptor H1; TRPV1, transient reporter potential channel V1.

Bile Acids

Patients with IBS-D may have increased total fecal bile acid excretion, and the percentage of primary fecal bile acids is positively correlated with Bristol stool score and stool frequency (20). There is a significant overall relationship between BAs in serum and stool in IBS. IBS-D is associated with increased primary and reduced secondary BAs in feces relative to healthy controls, whereas IBS-C is associated with marginally reduced primary and increased secondary BAs in feces relative to healthy controls (19). Novel observations of BA malabsorption in patients with IBS-D are reduced levels of FGF-19, variations in klotho-β and FGFR4 genes in association with looser consistency and accelerated colonic transit (10, 57), and identification of an effector G protein-coupled BA receptor (TGR5) on motor, secretory, and enteroendocrine cells in the intestine (1, 45).

Unanswered questions or gaps in knowledge.

While there have been advances in understanding the prevalence and mechanisms of BA diarrhea, further studies of the interaction between microbial flora and BAs are needed, as bacteria in the cecum deconjugate primary BAs and can thereby alter their signaling. In addition, the possible secretory and motor-stimulating effect of primary BAs and the potential role of rapid colonic transit in decreasing the conversion of primary to secondary BAs need further clarification. Finally, there is evidence of abnormal levels of total BAs (48, 57) in patients with IBS-C or chronic constipation, but these studies have involved relatively small numbers of patients and require further replication.

Short-Chain Fatty Acids

An estimated 2–20% of complex carbohydrates escapes absorption in small intestine and reaches the colon (35) in healthy volunteers. Approximately 90% of the starches entering the colon are fermented by gut bacteria into SCFAs and absorbed or used by colonocytes. A major proportion of SCFAs in the mammalian colon is comprised of butyric, acetic, and propionic acids (46). Mucosal butyrate enhances net sodium and chloride ion absorption in isolated stripped mucosa from distal rat colon to a greater extent than propionate and acetate (7). The enhanced absorption of water is attributed, at least in part, to the increased expression of sodium-hydrogen exchanger channels (40).

Abnormalities of SCFAs in stool have been reported in IBS patients. There is evidence of increased colonic fermentation of SCFAs (and gas production) with lower stool mass and slower colonic transit in patients with IBS-C (47) or IBS (39). In a third study, there were higher levels of acetate, propionate, and total organic acids and higher counts of Veillonella and Lactobacillus in IBS patients compared with controls, and those with higher organic acid levels presented with worse gastrointestinal symptoms, quality of life, and negative emotions (50).

On the other hand, some effects of the SCFAs that may be associated with diarrhea include acceleration of colonic transit by stimulation release of serotonin (5-HT) (26) and the secretory effects of SCFAs such as propionate-induced ion and fluid secretion in guinea pig distal colon in vitro, and increased expression of FFA2 receptor (the SCFA receptor) (32). In a study of 63 consecutive IBS patients who received a 4-wk FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) restricted diet, there was marked modulation of fecal fermentation: saccharolytic fermentation (e.g., acetic and n-butyric acids) decreased, while proteolytic fermentation (e.g., i-butyric and i-valeric acids) increased, though there was no relationship between fecal SCFA levels and IBS symptoms (53).

Unanswered questions or gaps in knowledge.

SCFA abnormalities have been observed in patients with IBS-D and IBS-C; however, it is not clear whether this is a cause-effect relationship. It is important to know whether altered composition or concentration of SCFAs is responsible for the transit abnormalities seen in IBS or whether the changes in SCFAs reflect the abnormality in colonic transit similar to the relationship between colonic transit and bile acid composition. The relative proportions of SCFAs may also be influenced by different microbial flora, as has been demonstrated in people of different ethnicity (30). Future studies need to further elucidate the interactions between microbiome, SCFAs, and bile acids, with standardization of diet, longitudinal or repeat studies, and studies conducted in diverse populations who ingest different diets, as well as the effects of FODMAP restriction.

Molecular Species Involved in Mucosal Barrier

The structural components of intercellular tight junctions can be classified as integral membrane proteins (occludin, claudins, and junctional adhesion molecules), junctional complex proteins [e.g., zonula occludens (ZO) proteins, ZO-1, ZO-2, ZO-3], and cell cytoskeleton structures (microtubules, intermediate filaments, and microfilaments) (14). Abnormalities in the mucosal barrier provide access of luminal factors to immune cells in the lamina propria, leading to the immune activation or inflammation in patients with IBS. In turn, inflammatory cytokines may induce changes in mucosal barrier function.

Thus several chemical and molecular mechanisms lead to initiation and maintenance of the increased mucosal permeability, including alterations in expression, localization, and regulation of tight junction proteins; dysbiosis of the microbial flora resulting in the lack of signals to maintain or increase in signals that break the mucosal barrier; active inflammation and increased proinflammatory cytokines and oxidative species; and increased density of epithelial gaps caused by increased cell shedding (14).

Conceptually, the alteration in barrier function may be related to different molecules that are expressed in the lumen, the mucosa and lamina propria, in addition to the barrier itself (Table 1) in patients with IBS. These include

• Dihydroxyl BAs (16) and miRNA-29 increase intestinal permeability (60, 61).

• Background stress, female sex, and immune activation can influence intestinal mucus barrier (18), gut permeability, visceral pain sensitivity, and gut dysfunction (42, 44).

• Increased permeability and immune activation may be induced by chemotactic relatively small tripeptides produced by bacteria (e.g., formyl met-leu-phe) (23).

• mRNA expression of barrier proteins is generally reduced in IBS-D (e.g., claudin-1 and ribonucleoprotein-1) and increased (e.g., ZO-1 and occludin) in IBS-C (11).

• Proteases (such as the serine proteases and tryptase) produced by luminal bacteria or by mast cells in the lamina propria increase intestinal mucosal permeability through stimulation of type 2 protease-activated receptors (PAR-2) triggering phosphorylation of myosin light chains with subsequent changes in tight junction permeability (22).

Unanswered questions or gaps in knowledge.

In vivo methods that have been used in previous studies to assess intestinal and colonic permeability relied on the urinary measurement of absorbable and poorly metabolized sugars and sugar alcohols, such as lactulose and mannitol. Results from such studies have been difficult to interpret (13) due to limitations that include variation in the timing of urine collection and the possible contamination of urine samples by dietary and other sources of the measured molecule. As a result of these methodological challenges, the proportion of IBS patients with increased intestinal or colonic permeability is still unclear. The methods for in vivo measurement of intestinal permeability are evolving, and Grover et al. (28) have recently validated the use of the stable isotope-labeled pentose [¹³C]mannitol as a probe molecule. Such potentially more accurate methods are yet to be validated in IBS. Pilot studies suggest a beneficial role of probiotics and glutamine (6) in decreasing permeability in the gastrointestinal tract and thus ameliorating symptoms in IBS (5). Larger studies attempting to reverse increased intestinal permeability in patients with IBS and to study the effect of this reversal on various pathophysiological mechanisms such as immune activation and visceral hypersensitivity are needed. Better understanding of those mechanisms and validated in vivo permeability testing may then lead to specific therapies targeting a subset of patients with IBS who have increased mucosal permeability.

Enteroendocrine Cell Products

The roles of peptides and amines in IBS are extensively reviewed elsewhere (10). The best examples are serotonin and the possible role of serotonin-selective uptake transporter (36, 38), the increased plasma postprandial 5-HT in IBS-D, and reduced postprandial levels in IBS-C (3). Serotonin's effects on fluid secretion and stimulation of colonic motility and tone (54) are illustrated by the protean manifestations of carcinoid diarrhea that results predominantly from the effects of serotonin. There is some evidence that serotonin may be involved in control of small intestinal permeability in IBS (33), but confirmation is required.

Densities of rectal enteroendocrine cells in IBS have been investigated, and the density of peptide YY cells was significantly lower and somatostatin cell density higher in IBS patients than in the controls (21). However, there were generally no differences between IBS-D and IBS-C.

Enteroendocrine cells are stimulated to secrete their products by molecular entities in the intestinal or colonic lumen, such as SCFA and bile acids (1). For example, Alemi et al. (1) showed that BAs stimulated release of the peristaltic transmitters 5-HT and calcitonin gene-related peptide (CGRP); antagonists of these transmitters suppressed BA-induced peristalsis, consistent with localization of TGR5 (BA receptor) to enterochromaffin cells and intrinsic primary afferent neurons. In their elegant studies, TGR5-knockout mice did not show peristalsis or transmitter release in response to BAs (1).

Unanswered questions or gaps in knowledge.

It is unclear whether the enteroendocrine cell products influence intestinal permeability or whether they are influenced by the microbiome. A very recent metagenomic study observed chromogranin A (CgA), a protein secreted by enteroendocrine cells, was exclusively associated with 61 microbial species whose abundance collectively accounted for 53% of microbial composition. Low CgA concentrations were seen in individuals with a more diverse microbiome (59). In addition, studies using organoids show increased stimulation of enteroendocrine cell 5-HT and serotonergic receptors by the SCFA acetate (51).

Proteases

There is abundant literature documenting increased mast cells in the lamina propria (reviewed in Ref. 13), proximity of mast cells to intestinal nerves, and the effects of fecal supernatants or mucosal extracts from patients with IBS on immune activation and afferent stimulation including transient receptor potential cation channel, subfamily V, member 4 (TRPV4) channel activation in animal models (15) (Table 3). In another study (58), supernatants from biopsies of patients with IBS were shown to induce nociception in a murine model and these effects mediate via histamine receptor H1 (HRH1) sensitization of TRPV1 receptors. These interesting interactions were confirmed in humans with IBS, in whom ebastine, a nonsedating HRH1 antagonist, was associated with a decrease in pain in a placebo-controlled clinical trial. A third fecal supernatant from IBS-D patients also increased production of brain-derived neurotrophic factor (BDNF) in colonic epithelial cells and induced visceral hypersensitivity in mice (55). The increased BDNF acts on enteroglial cells in the colon of IBS patients, and the enteroglial cells that localize in proximity to colonic nerves express substance P and correlate with abnormal pain scores in IBS patients (56). Recent data also suggest that mucosal biopsies from IBS patients have increased expression of mesotrypsin (24, 37), and effects of mesotrypsin appear to be mediated by PAR-2 receptors (34, 37).

Table 3.

Mechanisms of action and possible therapeutic roles of different microRNAs involved in IBS

Micro RNA	Target	Population studied	Results	Suggested mechanism	Potential clinical applicability	Citation (ref. #)
miRNA-24	Serotonin reuptake transporter (SERT)	10 IBS patients and 10 healthy patients; IBS mouse model	miRNA-24 upregulated in IBS patients and intestinal mucosa of mice	miRNA-24 inhibits SERT expression and aggravates IBS	miRNA-24 inhibitor	Liao et al. (36)
miRNA-29	NFKB- repressing factor(NKRF) and claudin-1 genes	183 IBS and 36 HC	Increased levels of miRNA-29 and reduced levels of NKRF and claudin-1 in patients with IBS-D	miRNA-29 targets and reduces claudins and NKRF, which increases intestinal permeability	miRNA-29 inhibitors for IBS with increased permeability	Zhou et al. (60)
		Mir 29 knock-out mice	Decreased intestinal hyperpermeability in Mir-29 knockout mice
	Glutamine synthetase gene GLUL	19 IBS-D and 10 HC	Increased miRNA-29a in IBS-D patients with increased intestinal permeability (42% of IBS-D patients)	miRNA-29a increases membrane permeability by decreasing GLUL gene expression and glutamine level	miRNA-29 inhibitor and glutamine for IBS with increased intestinal permeability	Zhou et al. (61)
miRNA-150 and miRNA-342-3p	Multiple targets including telomerase-related proteins dyskerin, and prosurvival protein kinase AKT2	5 IBS-D, 5 IBS-C, 2 IBS-M, and 31 HC	Increased level of miRNA-150 and miRNA-342-3p in IBS	Inflammatory, pain, and motility pathways	Possible role as biomarkers	Fourie et al. (25)
miRNA-199	Transient receptor potential vanilloid type 1 (TRPV1) signaling	45 IBS-D and 40 HC	Decreased colonic miRNA-199 correlates with visceral pain in patients with IBS-D	miRNA-199a decreases visceral pain via inhibition of TRPV1 signaling	miRNA-199 precursors for pain in IBS	Zhou et al. (63)
		Visceral hypersensitivity rat models	Reduced miRNA-199a in rat DRG and colon tissue associated with visceral hypersensitivity
miRNA-510	5-HT3 receptor type 3 subunit gene HTR3E	98 IBS-D, 99 IBS-C and 100 HC (UK); 119 IBS-D and 195 HC (Germany)	HTR3E variant c. *76G>A, associated with female IBS-D	Variant of HTR3E reduces binding and inhibitory effect of miRNA-510, thus increasing expression of 5-HT3E protein in IBS-D	Unclear clinical impact of miRNA-510 in IBS	Kapeller et al., (31)

HC, healthy controls; NFKB, Nuclear factor-κB; NKRF, NFKB- repressing factor; DRG, Dorsal root ganglia.

Unanswered questions or gaps in knowledge.

The specific mediators in fecal supernatants that result in their effects need to be identified and classified based on the different IBS subtypes. Therapeutic potentials of mast cell modification and protease inhibition have not been adequately appraised with selective pharmacological agents to explore their potential mechanistic role in IBS. For example, the promising effects of ketotifen in a randomized controlled trial may be associated with its effects on mast cell stabilization or the sedating antihistaminic effects of the drug.

Similarly, the potential role of Bowman-Birk protease inhibitors and their variants, which are naturally found in legumes such as soybean, pea, lentil, and chickpea (17), would appear to be worthy of further study, if patients with increased expression of such proteases could be identified in research and, eventually, in clinical studies, thereby appraising potential health-promoting properties of these inhibitors within the gastrointestinal tract.

Altered Expression of Mucosal Molecules in IBS Pathogenesis

Ribonucleic acid (RNA) sequencing showed transcriptomic changes (secretory and barrier) in rectosigmoid mucosa in IBS-D (12), and the findings were confirmed by using RT-PCR relative to healthy controls and IBS-C patients (11).

miRNAs are short RNA molecules that do not code for protein production, but they are capable of posttranslational regulation of expression of several target mRNAs. They are released from their producing cells in protected forms and therefore are stable for long periods in biological fluids. Table 3 summarizes the role of several miRNAs that have been reported to have actions pertinent to IBS (25, 31, 36, 60, 61, 63).

Unanswered questions or gaps in knowledge.

These interesting observations require replication in larger numbers of patients. The safety and the challenging method of delivery of miRNA inhibitors and/or precursors require future dedicated trials (62).

Conclusion

Several molecular and chemical factors may contribute to the development of IBS. The interrelationships between these factors, as well as the role of the central nervous system hypervigilance and “top-down” modulation of these factors, require further exploration. However, the evidence continues to point to significant role in pathogenesis of these intraluminal and mucosal mechanisms, which constitute potential targets for further research and future specific therapy.

GRANTS

M. Camilleri is funded by grant R01-DK92179 from the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

M.C. prepared figures; M.C., I.O., and H.H. drafted manuscript; M.C., I.O., and H.H. edited and revised manuscript; M.C., I.O., and H.H. approved final version of manuscript.