Mast cell modulation: A novel therapeutic strategy for abdominal pain in irritable bowel syndrome

Summary

Irritable bowel syndrome (IBS) is one of the most prevalent gastrointestinal disorders characterized by recurrent abdominal pain and an altered defecation pattern. Chronic abdominal pain represents the hallmark IBS symptom and is reported to have the most bothersome impact on the patient’s quality of life. Unfortunately, effective therapeutic strategies reducing abdominal pain are lacking, mainly attributed to a limited understanding of the contributing mechanisms. In the past few years, exciting new insights have pointed out that altered communication between gut immune cells and pain-sensing nerves acts as a hallmark driver of IBS-related abdominal pain. In this review, we aim to summarize our current knowledge on altered neuro-immune crosstalk as the main driver of altered pain signaling, with a specific focus on altered mast cell functioning herein, and highlight the relevance of targeting mast cell-mediated mechanisms as a novel therapeutic strategy for chronic abdominal pain in IBS patients.

Graphical abstract

New insights in the pathophysiology of irritable bowel syndrome (IBS) offer the promise toward more effective therapeutic strategies for abdominal pain in patients with IBS. Van Remoortel et al. highlight exciting new mechanistic information on the role of altered neuro-immune crosstalk and its potential for treating chronic abdominal pain in IBS.

Introduction

Irritable bowel syndrome (IBS) presents as a chronic gastrointestinal (GI) disorder characterized by abdominal pain associated with altered bowel habits in the absence of an organic cause. Typically, laboratory testing, radiological, and/or endoscopic investigations fail to identify an underlying cause of these symptoms, and IBS diagnosis largely relies on the fulfillment of the Rome IV diagnostic criteria¹ (Figure 1A). Based on the predominant stool pattern, patients with IBS are classified into four subgroups: IBS with diarrhea (IBS-D), IBS with constipation (IBS-C), IBS with mixed stool pattern (IBS-M), and IBS unclassified (IBS-U). The prevalence of IBS is estimated at 5%–20% of the general population, albeit this is dependent on the diagnostic criteria used.^2,3,4 IBS not only has a considerable negative societal impact for patients but also reduces work productivity and causes a disproportionate use of health care resources. According to the International Foundation for Functional GI Disorders, the economic burden of IBS the United States (US) is estimated at 21 billion US dollars per year, while in Europe the estimated total annual cost amounts up to up to 3,000 euros/year per patient.^5,6 IBS etiopathogenesis is complex, including disturbances of the microbiota-gut-brain axis,^7,8,9 post-infectious reactivity,^10,11 food sensitivity,^12,13 and malabsorption.¹⁴ Currently, IBS treatment is mainly restricted to efforts in correcting altered defecation patterns via the use of antidiarrheal medication or laxatives.¹⁵ Yet, such approach yields disappointing results and often leaves patients desperate, as their most debilitating symptom, abdominal pain, remains untreated.¹⁶ The distress of IBS patients is often faced with incomprehension, even from physicians, and has fueled the false belief that “their problem most likely lies in the mind,” as often cited by patients.^17,18 This highlights an obvious medical need to improve clinical management of IBS, which remains unmet due to the lack of knowledge of the underlying pathophysiological mechanisms of IBS, and in particular abdominal pain.

Figure 1.

Clinical characteristics of IBS

(A) Rome IV criteria for the clinical diagnosis of IBS.

(B) Visceral hypersensitivity is the main driver of abdominal pain experienced by IBS patients, and it presents as an abnormally increased pain sensitivity from the bowel, mainly characterized by allodynia (red) and hyperalgesia (orange). Allodynia refers to the process where stimuli that normally are perceived as non-painful are perceived as painful. Hyperalgesia refers to the process where stimuli that normally are perceived as painful are perceived as even more painful.

The last 50 years have seen a significant evolution in the focus of IBS pathophysiology research. Initially, IBS was considered as a psychosomatic disorder without any gross indicators of disease in the GI tract. With the development of new tools to study the function of the GI tract in the ‘70s and ‘80s, research into IBS pathophysiology shifted toward studying changes in GI motility.¹⁹ Disappointingly, the connection between symptoms and disordered motility remains limited and has not translated into efficacious treatment options for abdominal pain. Over the past two decades, visceral hypersensitivity, rather than dysmotility, has become the main research focus linking IBS pathophysiology and abdominal pain. Visceral hypersensitivity is a term used to describe abnormal pain signaling originating from the viscera, manifesting as a painful response to normal stimuli (allodynia) and/or an exaggerated painful response to painful stimuli (hyperalgesia) (Figure 1B). To date, visceral hypersensitivity is considered a central disease mechanism underlying abdominal pain, and it has become clear that gaining a better understanding of the mechanisms driving visceral hypersensitivity offers the promise of novel treatment options for this patient population.

In this review, we will outline how a unique translational approach starting from the patient to the bench has shown that mast cells, and their increased release of mediators, play a hallmark role in driving abdominal pain in the IBS-affected gut. We will discuss how their altered activity is triggered, how this contributes to increased activation and sensitization of the gut pain signaling axis, and highlight the clinical relevance of targeting mast cells and their mediators as a novel treatment option for the management abdominal pain in IBS patients.

From bedside to bench: Mechanistic insights in abdominal pain in IBS patients

To date, it is well accepted that abnormal pain signaling represents one of the major mechanisms involved in the pathogenesis of IBS. In the early 1970s, Ritchie reported increased pain responses in patients with IBS in rectal barostat studies.²⁰ Barostat experiments evaluate pain responses to the stepwise inflation of a balloon positioned in a patient’s rectum. Notably, patients with IBS experienced higher pain scores compared to healthy controls, which introduced the idea that visceral hypersensitivity might be an important biomarker for IBS.²⁰ Importantly, this finding has been repeatedly confirmed in over 60% of patients with IBS and is strongly associated with the severity of GI symptoms of IBS patients.^{21,22,23,24,25,26} Unsurprisingly, much research has been devoted to understanding the mechanisms contributing to this altered pain signaling. In this section, we will focus on the mechanisms involved in gut pain signaling and we will outline how using a translational approach starting from patient material has allowed to clarify the mechanisms underlying visceral hypersensitivity in IBS patients.

A first look at gut pain signaling

The bowel is innervated by two intricate neuronal networks: an intrinsic innervation, called the enteric nervous system (ENS) or the “second brain,” and an extrinsic innervation, often referred to as the “gut-brain axis.” While the intrinsic ENS represents a local neural circuitry within the gut wall that is able to maintain basic GI functions such as secretion, absorption, and motility,²⁷ the gut-brain axis allows the transmission of visceral sensations, including pain, from the gut to the central nervous system (CNS). Within this gut-brain axis, pain perception is mediated by specialized “pain-sensing” neurons, also referred to as gut nociceptors (Figure 2). The nerve terminals of gut nociceptors reside in the gut wall and send information via their cell bodies located in the dorsal root ganglia (DRGs) to the spinal cord and the central nervous system (Figure 2). As such, gut nociceptors are a class of sensory neurons specialized in detecting and responding to actual or potentially damaging stimuli, such as thermal, mechanical, or chemical stimuli. To allow the detection of such stimuli, gut nociceptor peripheral nerve terminals are equipped with a plethora of receptors and ion channels that are classified as either pro-nociceptive (or excitatory) or anti-nociceptive (or inhibitory).²⁸ Typical examples of pro-nociceptive receptors/ion channels include transient receptor potential (TRP) channels (TRPV1, TRPA1, and TRPV4), acid-sensing ion channels, purinergic and histaminergic receptors (H1-4R), and protease-activated receptors (PAR1-2), while opioid and cannabinoid receptors are typical examples of anti-nociceptive receptors. The balance between pro- and anti-nociceptive input signals gut nociceptors receive at their peripheral endings will determine whether they will transmit pain signals to the spinal cord and to the brain.²⁸ This implies that the abnormally increased pain signaling seen in IBS patients may result both from increased excitatory inputs and from reduced anti-nociceptive inputs to gut nociceptors.

Figure 2.

Neuroanatomy of the colonic nociceptive innervation

Colonic pain sensation is mediated by specialized afferent nerves, the splanchnic and pelvic nerves, of which the pseudo unipolar neuronal cell bodies are in the dorsal root ganglia (DRGs) located near the spinal cord. The peripheral nerve endings of these afferent nerves reside in the different layers of the gut wall (mucosa, submucosa, and muscularis externa), where they act as sentinels that detect potential dangers. As opposed to the enteric nervous system (ENS), comprising a local and intrinsic neuronal signaling circuitry, gut nociceptors are part of an extrinsic gut-to-brain signaling axis that transmits its pain signals via the splanchnic and pelvic nerves to the DRG and spinal cord. In the dorsal horn of the spinal cord, this nociceptive input is integrated by spinal cord neurons and further transmitted toward the brain, resulting in pain perception. ENS, enteric nervous system.

Mediators and mechanisms involved in visceral hypersensitivity

A major contributor to our understanding of the mediators and mechanisms involved in visceral hypersensitivity has been the use of patient-derived mucosal biopsy supernatant. To generate biopsy supernatant, small mucosal biopsies, obtained from the patient via colonoscopy, are incubated in a physiological solution or medium, allowing local mediators present in the tissue to diffuse from the biopsy into the medium.²⁹ Studies have shown that IBS biopsy supernatant contains more histamine, proteases, and serotonin compared to healthy supernatant.^30,31,32,33 Histamine is a biogenic amine mainly known for its role in causing allergic reactions and is primarily produced by innate immune cells, specifically mast cells. An increase in proteases, especially serine proteases, like tryptase,³⁰ produced by mast cells, and trypsin-3,³⁴ produced by epithelial cells, has been consistently observed in the gut of IBS patients. Increased serotonin levels were also observed in colonic biopsies of IBS-D patients,^33,35 which likely results from increased numbers of enteroendocrine cells, although increased mast cell activation could also contribute to increased serotonin production. Importantly, all of these mediators are known to activate nociceptors, supporting sustained pro-nociceptive effects on gut nociceptors due to their increased presence in the environment. Indeed, application of IBS supernatant to murine gut nociceptors in vitro or in vivo induces more pronounced neuronal activation compared to healthy biopsy supernatant, an effect that is reversed by blocking histamine or protease signaling.^30,36 Similarly, IBS supernatant induces increased activation of human submucosal neurons compared to healthy supernatant, an effect mediated by histamine, serotonin, and proteases, further indicating that increased presence of these mediators in the IBS microenvironment plays a direct role in altered neuronal activity.^32,37

Increased firing of gut nociceptors has also been linked to the upregulation of pro-nociceptive receptor and ion channel expression at the neuron plasma membrane. With regards to IBS, several studies have suggested an altered expression and activity of pro-nociceptive TRP channels as a mechanism underlying visceral hypersensitivity. TRP channels are a family of ligand-gated ion channels that are typically expressed on nociceptive sensory neurons. TRP channels are highly conserved across species, and their activation is a central component of nociception. Reports indicate elevated TRPV1 expression in biopsies of patients with IBS, correlating with abdominal pain intensity.^38,39,40 Mediators such as histamine and serotonin, but also nerve growth factor (NGF), can increase TRP channel expression and induce the transport of TRP channels to the cell membrane of gut nociceptors.⁴¹ Mechanistically, such upregulated expression of pro-nociceptive TRP channels drives increased nociceptive signaling upon noxious stimulation.

In addition to increased pro-nociceptive TRP channel expression, sensitization of these TRP channels also plays a central role in the pathophysiology of visceral hypersensitivity in IBS patients. Such sensitization is defined as an augmented TRP channel functioning, typically induced by phosphorylation of intracellular residues, resulting in a lowered activation threshold and increased ion fluxes upon noxious stimulation. Especially the sensitization of the TRP channels TRPV1, TRPV4, and TRPA1 has been repeatedly linked to IBS pathophysiology. IBS patients with visceral hypersensitivity that is confirmed by rectal barostat studies report an increased pain perception to rectal application of the TRPV1 agonist capsaicin compared to patients without visceral hypersensitivity.⁴² Strikingly, even though these patients reported more pain to rectal capsaicin, rectal TRPV1 mRNA and protein expression was similar between IBS patients and healthy individuals, indicating that TRPV1 seems to be sensitized rather than expressed at higher levels in this patient cohort.⁴² This was further confirmed using in vitro studies, where murine DRG neurons exposed to biopsy supernatants of IBS patients showed an increased response to capsaicin compared to neurons exposed to supernatant of healthy subjects, indicating TRPV1 sensitization.^36,43,44 Along the same line, TRPV1 responses to capsaicin were potentiated in rectal submucosal neurons of IBS patients but not in healthy subjects.^43,44 Similar findings have also been observed for other TRP channels such as TRPV4 and TRPA1.⁴⁵ Collectively, it has become clear that bioactive mediators are present in the IBS mucosal microenvironment that cause altered TRP channel expression and sensitization, thereby contributing to altered pain signaling in IBS (Figure 3).

Figure 3.

Molecular mechanisms involved in abnormal pain signaling in IBS patients

The IBS microenvironment is characterized by increased levels of pro-nociceptive mediators such as serotonin, histamine, proteases, growth factors, etc. Through the activation of their cognate receptors on gut nociceptive nerve endings, these mediators can directly activate and modulate the functioning of gut nociceptors (1). Increased signaling by pro-nociceptive mediators induces an increased expression and upregulation of pro-nociceptive ion channels, mostly TRP channels (2). Moreover, pro-nociceptive mediators trigger intracellular signaling cascades that activate downstream protein kinases, resulting in TRP channel phosphorylation and sensitization (3). Collectively, these pro-nociceptive mediators and their effects on TRP channel functioning are the hallmark mechanism underlying aberrant nociceptor excitability in IBS patients. GFR, growth factor receptor; 5-HT, serotonin; 5-HT3R, serotonin 3 receptor; H1R, histamine 1 receptor; PAR2, protease-activated receptor 2; MRGPRs, Mas-related G Protein-coupled receptors; TRP, transient receptor potential; PKA, protein kinase A; PKC, protein kinase C.

Among these bioactive mediators, histamine represents one of the main candidates involved in TRP channel sensitization in IBS. As described earlier, increased histamine levels have repeatedly been reported in mucosal biopsies of IBS patients. In gut nociceptors, histamine typically signals through the histamine 1 receptor (H1R), initiating intracellular G protein-mediated signaling cascades driving downstream TRP channel phosphorylation and sensitization. Indeed, exposure of murine DRG neurons to histamine directly sensitizes TRPV1, TRPV4, and TRPA1 channels, an effect reversed by histamine 1 receptor antagonism or receptor knockout.^43,45 Likewise, the sensitizing effects of IBS supernatant on TRPV1, TRPV4, and TRPA1 are directly reversed by H1R antagonism, further indicating a direct role for histamine in TRP channel sensitization. Proteases have also been involved in pain sensitization in IBS.⁴⁶ Among the different classes of proteases, serine proteases like trypsin-3 and tryptase have been found to be increased in the IBS microenvironment.^30,34 These proteases can signal through the activation of their cognate protease-activated receptor (PAR2) expressed throughout the body, including by gut nociceptive innervation. PAR2 is activated by protease-mediated cleavage of the receptor’s extracellular N terminus, which in turn acts as a ligand inducing intracellular G protein-mediated signaling and downstream sensitization of ion channels, such as TRP channels. Evidence from preclinical studies has indeed indicated that proteases can promote DRG neuron hyperexcitability in vitro and visceral hypersensitivity in vivo,^30,47,48 likely via PAR2-mediated sensitization of TRP channels.^49,50,51

More recently, the Mas-related G protein-coupled receptors (Mrgprs) members Mrgpra3 and Mrgprc11 were found to play a role in the sensitization of gut afferents in mice via TRPA1, yet their putative ligands and exact role in IBS pathophysiology remain to be studied.⁵² Finally, mediators such as NGF and brain-derived neurotrophic factor are known to increase TRP channel expression and sensitization, but, although increased levels of these growth factors have been found in IBS patients, their exact role in visceral hypersensitivity remains elusive.^53,54

In conclusion, the increased presence of pro-nociceptive mediators in the IBS micromilieu, together with their downstream effects on TRP channel functioning in gut nociceptors, plays a crucial role in the abnormally increased pain signaling in IBS patients. Interestingly, how this increased pain signaling links to the altered bowel habits seen in IBS remains to be unraveled further. Of course, the pro-nociceptive mediators that are increased in the IBS micromilieu, such as histamine and serotonin found in IBS supernatant, not only increase nociceptor excitability but also affect the activity of human enteric neurons that control absorption/secretion and motility.^32,37 Alternatively, more recent preclinical evidence suggests that increased activity of gut nociceptors could also affect gut motility through a direct spinal reflex pathway that affects the activity of enteric neurons.⁵⁵

Mast cells strike a nerve: Role of altered neuro-immune crosstalk in IBS

An important question that rises is where do the mediators causing abnormal pain signaling come from? The answer to this question lies within the gut’s immune system. Although obvious macroscopic or microscopic inflammation is not observed in IBS patients, cumulative evidence from the past decades suggests an important role of mucosal immune activation in IBS patients. Already in the early 2000s, a long-lasting increase in T cell and mononuclear cell numbers was observed in the mucosa of patients who had developed IBS after an episode of acute infectious gastroenteritis,^56,57 a finding that was recently confirmed by a meta-analysis.⁵⁸ Data comparing cytokine gene expression or cytokine release by mucosal biopsies between IBS and healthy individuals are rather inconsistent, as are data on serum cytokine levels in IBS patients, often due to small patient populations.⁵⁹ However, more recent studies comprising larger study groups have provided evidence for increased serum levels and mucosal gene expression of inflammatory markers in subgroups of IBS patients.^60,61 Likewise, an unbiased gene expression array approach revealed a subset of patients (33%) who displayed an upregulation of interleukin-1β (3-fold), prostaglandin synthase PTGS2 (2.1-fold), and the mast cell-specific Mas-Related G protein-Coupled Receptor X2 (MRGPRX2) (10.7-fold), yet these expression changes were unrelated to clinical IBS symptoms.⁶² Nevertheless, research findings from the past two decades clearly highlight that immune activation could play an important role in the pathophysiology of IBS.

To date, the link between immune activation and abdominal pain in IBS lies in mast cell activation. Mast cells are members of the innate immune system, typically found at mucosal surfaces where they exert numerous physiological functions, such as blood flow regulation, coagulation and wound healing, tissue homeostasis, antimicrobial defense, and pain modulation.⁶³ Mast cells are classically activated via crosslinking of high-affinity immunoglobulin E (IgE) receptors (FcεRI) found at their surface by antigen-IgE complexes.⁶⁴ Increasing evidence also highlights the role of IgE-independent mechanisms, such as the MRGPRX2 pathway, in mast cell activation.⁶⁵ The human intestine harbors distinct mast cell subtypes identified based on their protease expression.⁶⁶ Most mast cells (98%) in the mucosa contain high levels of tryptase but little to no chymase. This mast cell subset is often referred to as mucosal mast cells (MMCs). In the submucosa, a deeper layer of connective tissue that harbors a network of enteric neurons, blood vessels, and connective tissue, only a minority of mast cells have an MMC phenotype, while up to 77% of mast cells contain tryptase, chymase, and carboxypeptidase. This is reminiscent of connective tissue mast cells (CTMCs) also found in the skin. Mast cells produce and release a plethora of bioactive mediators, such as histamine, proteases, serotonin, cytokines, and chemokines, endowing them with an important role in tissue physiology.⁶³ As described earlier, the gut nociceptive innervation is equipped with receptors for these mediators; hence mast cells are likely candidates to convey information of potential harm or danger.

The link between mast cells and the pathophysiology of IBS was first introduced by Weston et al., who published a landmark paper reporting increased MMC numbers in the terminal ileum of IBS patients.⁶⁷ Along the same line, Barbara et al. reported increased mast cell numbers in rectal biopsies of IBS patients.³¹ However, looking at the number of mast cells as such has proven to be a poorly reliable biomarker, as some groups failed to detect any differences in mast cell numbers between healthy and IBS patients or even reported lower mast cell counts in biopsies of IBS patients.^58,68,69,70 Importantly, rather than increases in the absolute number of mast cells, it has become clear that changes in the functioning of mast cells are a hallmark of IBS pathophysiology, characterized by their increased activity and mediator release.^71,72 Indeed, increased levels of mast cell mediators, such as histamine and proteases, are repeatedly found to be increased in the mucosa of IBS patients.^{30,31,32,33,37} Moreover, degranulated mast cells are found in close proximity to mucosal nerves in IBS patients, a finding that correlates with the frequency and severity of abdominal pain reported by these patients.^31,36 More direct evidence supporting a major role for upregulated mast cell functioning stems from functional studies using IBS biopsy supernatant. As described before, in vitro exposure of murine gut nociceptors or human submucosal neurons to IBS supernatant induces neuronal hyperexcitability and TRP channel sensitization, effects that can be blocked by targeting mast cell mediators using histamine antagonists or protease inhibitors. In vivo, mast cell stabilizers, histamine antagonists, or protease inhibitors effectively reduce visceral hypersensitivity in murine models, directly confirming the causal role of mast cells in the induction and maintenance of visceral hypersensitivity.^30,36,73 These preclinical findings highlight the crucial role of mast cell-derived mediators as a central driver of neuronal sensitization, ultimately leading to the development of visceral hypersensitivity and chronic abdominal pain in IBS patients.

New insights into abdominal pain triggers in IBS

Effective therapies for abdominal pain in IBS patients are lacking, mainly attributed to a poor understanding of the pathobiological drivers underlying this disorder. As discussed in the previous sections, increased mast cell activity and mediator release contribute to nociceptor sensitization and abdominal pain, yet the mechanisms initiating this pathway have remained under debate. Although several triggers have been proposed, such as food, stress, gut microbiota, and disturbed intestinal permeability or so called “leaky gut,” their link with abdominal pain has remained associative and often lacks convincing experimental evidence. Of interest, new patient-focused translational studies have highlighted the role of food as a major trigger of mast cell activation.

You are what you eat: Food as a trigger of abdominal pain

Food is a major trigger of symptoms in IBS patients. The majority of patients report a rapid onset or exacerbation of their GI symptoms following the consumption of certain foods, usually within 3 h after consumption.^16,74,75 Interestingly, up to 50% of patients report symptom onset within 1 h after consumption and show an overlapping postprandial syndrome (e.g., functional dyspepsia). These observations imply that not only the large intestine but also the small intestine could be involved in early symptom generation toward foods.¹³ Among these foods, wheat, gluten, milk/dairy products, caffeine, fatty or spicy foods, fermentable oligosaccharides/disaccharides/monosaccharides and polyols (FODMAPs), or foods rich in biogenic amines (fermented food) are most frequently reported as triggers for their symptoms.^75,76 The role of FODMAPs in symptom generation is particularly well established in IBS patients. FODMAPs are short-chain carbohydrates that are poorly absorbed by the small bowel and which cause luminal distension, a major trigger for visceral pain in a sensitized gut, as a result of their osmotic effects and their fermentation by colonic bacteria in the colon result.⁷⁷

Recent studies have provided exciting evidence that certain foods can also trigger immune-mediated reactions in the GI tract of IBS patients. Bischoff and colleagues injected food extracts in the cecal mucosa of patients with chronic abdominal symptoms and suspected food allergy.⁷⁸ Up to 77% of patients but none of the healthy controls showed a reaction to at least one injection, characterized by a local weal and flare reaction. Strikingly, the injected antigens eliciting a mucosal reaction matched with the foods inducing complaints in patients. Moreover, mucosal reactions to injected antigens were associated with mast cell activation at the injection site, but not with increased total or antigen-specific IgE concentrations in the serum or skin reactions to the tested antigens, excluding food allergy.⁷⁸ The concept that food could trigger local immune responses in the gut wall of IBS patients wall was further explored by studies using confocal laser endoscopy, an advanced endoscopic imaging technology that, in conjunction with topical or intravenous fluorescent dyes, allows for an “optical biopsy” for real-time diagnosis. Application of food extracts (wheat, yeast, gluten, cow’s milk) to the duodenal mucosa elicited a positive reaction, with increases in intraepithelial lymphocytes, formation of interepithelial gaps, and cell shedding, in two-thirds of patients in two cohorts of patients with IBS without any clear indication of food allergy.^79,80 Of interest, although the total number of mast cells and eosinophils were similar between IBS patients and controls, a positive reaction to food antigens was associated with release of eosinophil-cationic protein, suggesting increased eosinophil activation in the IBS mucosa, yet this requires further study.⁸⁰ Along the same line, our group recently showed that injection of food antigens in the rectosigmoid colonic mucosa of 12 IBS patients and 8 healthy controls elicited a mucosal reaction in IBS patients but not in healthy controls.³⁶ Interestingly, in most IBS patients, those food antigens causing mucosal responses upon injection were also reported by the patient to trigger food-induced symptoms.³⁶ Overall, these recent findings have provided important experimental evidence that food triggers a local mucosal reaction in IBS patients.

Linking food to altered mast cell function

The observation that IBS patients exhibit mucosal responses to food antigens suggests a break of oral tolerance to food. At steady state, the mucosal immune system is primed toward tolerating innocuous antigens, such as food-related antigens, a concept termed oral tolerance.⁸¹ Failure to induce oral tolerance or its loss will elicit an immune response upon antigen exposure, which is responsible for diseases such as food allergy and celiac disease.^82,83 In food allergy, loss of oral tolerance results in the production of food antigen-specific IgE antibodies, leading to IgE-dependent mast cell activation and mast cell degranulation upon subsequent ingestion of the sensitizing food antigen.⁸⁴ Food antigen-specific IgE antibodies are found systemically in food-allergic patients and can be detected in the serum or via skin prick testing. Strikingly, despite showing mucosal responses to food antigens, patients with IBS do not respond to food antigens via skin prick testing and do not exhibit increased food-specific IgE antibody titers in serum.⁸⁵

Recently, compelling evidence has emerged proposing a role for a local IgE response toward food antigens in activating mast cells and triggering abdominal pain in patients with IBS. One of the main clinical risk factors for the development of IBS is infectious gastroenteritis, an event which we hypothesized could disrupt oral tolerance toward food antigens, as similarly reported in celiac disease.⁸² Mice were fed with the dietary antigen ovalbumin (OVA) during an infectious enteritis caused by Citrobacter rodentium. After clearance of the infection, mice were re-exposed to OVA. This resulted in IgE-dependent mast cell activation and histamine-mediated TRPV1 sensitization of gut nociceptors leading to visceral hypersensitivity development in these mice.³⁶ Treatment with IgE-blocking antibodies or using IgE-deficient mice prevented the development of visceral hypersensitivity after OVA re-exposure. Strikingly, contrary to food allergy models, OVA-specific IgE antibodies were only detectable in colonic tissue and not found in the serum.³⁶ These observations suggest that, while food allergy results from a systemic loss of oral tolerance to food antigens, food-induced abdominal pain in IBS rather results from a local loss of tolerance to food antigens. Of note, the mucosa of IBS patients exhibited increased numbers of IgE-sensitized mast cells located in close proximity to nerve endings, an observation that correlated with abdominal pain severity in these patients.³⁶ Collectively, these findings provide a paradigm shift in IBS pathophysiology and support an immunological basis of abdominal pain development in IBS, with a particular role for mast cells.

Beyond IgE-mediated activation, IgE-independent mechanisms are also able to activate mast cells, raising the possibility of their contribution to aberrant mast cell functioning in IBS. Cationic substances called basic secretagogues, such as the neuropeptide substance P, can activate mast cells in an IgE-independent manner.^86,87 The receptor involved in these responses, Mas-related G protein-coupled receptor X2 (MRGPRX2), was only recently discovered.⁸⁶ Of note, MRGPRX2-mediated activation of mast cells results in spatiotemporally distinct mediator release compared to IgE-mediated activation.⁸⁸ MRGPRX2 activation results preferentially in the release of tryptase and serotonin instead of histamine, and activation of its mouse counterpart Mrgprb2 has been found to trigger pain hypersensitivity and itch in the skin.^89,90 As previously mentioned, we found a 10.7-fold upregulation of mRNA encoding MRGPRX2 in colonic mucosal biopsies of a subset of patients with IBS,⁶² suggesting that MRGPRX2-mediated activation of mast cells in these patients might contribute to the development of abdominal pain, but this remains a topic of future studies.

From bench to bedside: Targeting mast cells as a novel treatment option for IBS

The current clinical management strategy for IBS patients mainly focusses on correcting altered defecation patterns using antidiarrheal medication for IBS-D patients or laxatives for IBS-C patients. Unfortunately, effective therapies targeting abdominal pain remain very limited. However, the emerging role of mast cells in IBS pathophysiology has paved the way for new studies trialing mast cell-targeted therapies as a novel treatment strategy for abdominal pain in IBS patients (Figure 4).

Figure 4.

Mast cell-targeted therapies as novel treatment strategy for IBS

(A) An effective therapeutic strategy is to target the increased degranulation of gut mast cells. This can be achieved using mast cell stabilizing drugs (cromolyn sodium, ketotifen), which block exocytosis and thereby prevent the release of mediators by activated mast cells. An alternative approach is to prevent IgE-dependent or IgE-independent activation of mast cells. For IgE-dependent signaling, this can be achieved by monoclonal anti-IgE antibodies (omalizumab) that target IgE antibodies and thereby prevent the binding of IgE antibodies to FcεRI receptors on gut mast cells. For IgE-independent signaling, this could be achieved by MRGPRX2 receptor antagonists. Alternatively, Siglec-8 is an inhibitory receptor expressed on mast cells which, upon activation by monoclonal anti-Siglec-8 antibodies, initiates intracellular signaling cascades that prevent that IgE-mediated and non-IgE-mediated activation resulting in mast cell degranulation.

(B) Targeting mast cell mediator signaling represents a very promising therapeutic strategy in IBS. This therapeutic approach relies on blocking the receptors for pro-nociceptive mast cell mediators such as serotonin (alosetron, ramosetron) or histamine (ebastine), thereby effectively preventing pro-nociceptive signaling on the gut pain innervation. IgE, immunoglobulin E; FceR1, Immunoglobulin E receptor; 5-HT, serotonin; 5-HT3R, serotonin 3 receptor; H1R, histamine 1 receptor.

Targeting mast cell degranulation

One possible mast cell-targeted therapy is the use of mast cell-stabilizing drugs. Mast cell stabilizers such as cromolyn sodium and ketotifen prevent calcium ion influx in activated mast cells, thereby inhibiting calcium-induced mast cell degranulation and mediator release, making it an interesting strategy to treat patients with IBS. The mast cell stabilizer cromolyn sodium is effectively used to reduce symptoms and severity of allergic airway disorders such as asthma and allergic rhinitis, as well as to reduce diarrhea and abdominal pain in mastocytosis patients.⁹¹ In a small pilot study conducted with IBS-D patients, patients treated with cromolyn sodium reported significantly reduced abdominal pain and improved stool consistency, an effect that was associated with reduced signs of mast cell activation.⁹² Likewise, we previously showed that treatment with ketotifen, another mast cell-stabilizing drug, decreased visceral hypersensitivity and improved symptoms, further confirming the therapeutic potential of mast cell stabilizers in IBS.⁹³ An alternative to general mast cell stabilization could be to specifically target mast cell activation mechanisms, such as the recently discovered role for local IgE-mediated mast cell activation in food-induced abdominal pain.³⁶ In this context, omalizumab is a humanized monoclonal anti-IgE antibody approved for the treatment of severe allergic asthma and recently also for food anaphylaxis.⁹⁴ This drug inhibits allergic responses by binding to serum IgE antibodies, thus preventing interaction with cellular IgE receptors expressed on mast cells. Interestingly, recent case reports describe clinical improvement of IBS symptoms in severe asthma patients who received omalizumab,⁹⁵ suggesting it could also be used as a therapy to locally interfere with IgE-mediated mast cell activation in IBS patients. Moreover, targeting MRGPRX2-, IgE-independent signaling has advanced significantly as a novel treatment strategy in type 2 inflammatory skin conditions such atopic dermatitis.^96,97 Although MRGPRX2-mediated signaling in IBS needs to be further studied, it could also offer a novel treatment strategy for subsets of IBS patients. Alternatively, targeting membrane receptors that directly inhibit mast cell activation could also offer new therapeutic possibilities. Among these, the sialic-acid-binding immunoglobulin-like lectin (Siglec)-8 (Siglec-8) is an inhibitory receptor expressed on mast cells that, upon targeting by a monoclonal antibody, has been shown to directly inhibit both IgE-mediated and non-IgE-mediated mast cell activation and allergic inflammation,⁹⁸ making it a possible therapeutic target for aberrant mast cell activity in IBS patients.⁹⁹

Targeting mast cell mediators and their signaling

Since the activation and/or sensitization of gut nociceptors by mast cell-derived mediators plays a major role in visceral hypersensitivity, an alternative strategy to treat abdominal pain is to block the nociceptive effects of these mediators. Blockade of serotonin signaling, an important mast cell mediator known to activate and sensitize gut nociceptors, has been used to treat abdominal pain in IBS patients. Twenty years ago, encouraging results were obtained with the 5-HT3 receptor antagonist alosetron showing adequate pain relief, a decrease in urgency and stool frequency, and increased stool firmness.¹⁰⁰ However, due to side effects such as ischemic colitis, the use of this compound is currently restricted in the US while it is not available in Europe.¹⁰¹ More recent studies in Japan show promising results with ramosetron, another 5-HT3 receptor antagonist, in IBS-D patients with significantly more global improvement, increased stool consistency, and reduction in abdominal pain and discomfort,¹⁰² further confirming the therapeutic potential of 5-HT3 receptor antagonism. Targeting the activity of proteases via the use of protease inhibitors has proven its efficacy as a treatment for visceral hypersensitivity in preclinical models, yet clinical studies are currently lacking.⁴⁶

Recently, the use of antihistaminic drugs targeting histamine and its signaling pathway on nociceptors has gained interest as a potential treatment option for IBS-D patients. Second-generation anti-histaminergic drugs such as ebastine prevent histamine-mediated signaling by selectively blocking the histamine receptor H1 and are the first-line treatment option for IgE-mediated mast cell disorders such as allergic rhinitis and chronic urticaria.¹⁰³ Based on the observations that blocking the histamine receptor H1 prevented the neuronal excitation by supernatant from IBS biopsies and effectively reduced visceral hypersensitivity in preclinical models, a pilot study evaluating the effect of the H1R antagonist ebastine in IBS patients was designed. After a 12-week treatment period, up to 46% of patients receiving ebastine reported significantly improved symptom relief and reduced abdominal pain, compared to 13% in the placebo-treated group.⁴³ In line with these clinical data, rectal barostat studies confirmed a significant reduction in visceral hypersensitivity symptoms in IBS patients treated with ebastine.⁴³ Recently, a randomized, double-blinded, placebo-controlled phase 2 study further confirmed the potential of ebastine in improving global relief of symptoms and abdominal pain intensity in IBS-D patients.¹⁰⁴

Concluding remarks

Over the past years, it has become clear that an altered neuro-immune crosstalk underlies aberrant pain signaling in IBS patients. Specifically, mast cell mediators generate a pro-nociceptive microenvironment driving increased pain signaling and visceral hypersensitivity. The discovery that a GI infection can break oral tolerance, leading to local production of food antigen-specific IgE antibodies and mast cell activation, is certainly a breakthrough in our understanding of IBS, yet the upstream immunological mechanisms leading to such local allergic responses in IBS patients remain unknown. Undoubtedly, many other factors such as stress, microbiota, and disturbed gut permeability can potentially lead to immune activation and visceral hypersensitivity and remain exciting areas of future research. In this respect, the advent of single-cell omics could offer an unbiased view on the distinct transcriptional or functional states of immune cells in the gut wall of IBS patients, as well as their interactions with other cell types, which will only further help our mechanistic understanding of abdominal pain and visceral hypersensitivity in IBS. Importantly, our basic research findings have been effectively translated into novel and effective treatment options for IBS patients. The use of existing anti-histaminergic drugs to prevent the pro-nociceptive actions of mast cell mediators on gut nociceptors has proven to be effective and holds great therapeutic value. In this respect, an important future challenge will be to clarify the mechanisms driving abdominal pain in IBS patients who fail to respond to H1R antagonism.

In conclusion, a translational bench-to-bedside approach has offered new insights in the mechanisms underlying aberrant pain signaling in IBS patients, and continuing efforts hold promise toward the discovery of novel effective treatment options for this patient population.

Acknowledgments

S.V.R. is supported by an FWO senior postdoctoral fellowship (1289225N). G.B. was funded by KU Leuven internal grants (GOA14-011, C14/18/086) and FWO grant (G0A9516N). All figures were created with Biorender.com.

Author contributions

All authors contributed to researching data for the article, discussion of its content, and the writing, review, and editing of the manuscript.

Declaration of interests

The authors declare no competing interests.