De rol van mestcellen in het prikkelbaredarmsyndroom

Irritable bowel syndrome (IBS) is one of the most prevalent disorders of the gut-brain axis, affecting on average 4% of the world population and 3% of Belgians (1). IBS is characterised by recurrent abdominal pain that is associated with a change in stool frequency and/or form (2). Patients are categorised based on their defecation pattern in an attempt to stratify the highly heterogeneous group and aid in treatment selection and scientific research. These subgroups include diarrhoea-predominant (IBS-D) patients, constipation-predominant (IBS-C) patients, and patients with alternating periods of diarrhoea and constipation (IBS-M). Patients who cannot be classified based on their defecation pattern are referred to as IBS-U. Abdominal pain is the hallmark symptom of IBS and is often caused by increased pain perception in the gut, also called visceral hypersensitivity (VHS) (3). Current clinical management of IBS consists of dietary and lifestyle advice and drugs that normalise defecation or reduce cramping (4). Treatment options targeting abdominal pain are limited, exhibit modest efficacy or are prone to cause more severe side effects. A more complete understanding of IBS pathophysiology would greatly improve the development of effective treatments. Mast cell (MC) activation in the gastrointestinal tract is known to play a key role in IBS pathophysiology, mainly characterised by a greater number of MCs close to enteric and extrinsic nerve terminals and an increase in MC activation (5). This results in the release of MC mediators such as histamine, with sensitisation of visceral afferents and increased pain signalling. In a pilot study, we previously reported clinical improvement by treatment with the histamine receptor H1 (HRH1) antagonist ebastine (6). In chapter 3, we report the results of a multicentre trial in which we compared the treatment effect of ebastine to placebo in non-constipated IBS patients. We show that a 12-week treatment of ebastine (20 mg daily) significantly increased the proportion of patients with a clinical response, especially in the last 6 weeks of treatment. Ebastine also had a significant treatment effect on the weekly abdominal pain scores compared to placebo, but not on stool consistency and quality of life. This would indicate that HRH1-antagonism can reduce VHS and represents a novel therapeutic option for managing IBS-related abdominal pain. Our group previously provided evidence that MCs in the intestinal mucosa of IBS patients are sensitised with dietary antigen-specific immunoglobulin E (IgE) and therefore more prone to degranulation upon food antigen stimulation (7). However, MCs can also undergo degranulation via an IgE-independent pathway, for example via the Mas-related G-protein-coupled receptor X2 (MRGPRX2). In chapter 4, we report the presence of MCs expressing MRGPRX2 in the human colon. Activation of the receptor in situ leads to MC degranulation, and subsequent sensitisation of transient receptor potential vanilloid 1 (TRPV1), indicating MRGPRX2 is functional in the human gut and might contribute to VHS. While expression levels of the receptor in rectal biopsies were similar between IBS patients and healthy subjects, we did observe increased levels of MRGPRX2 agonistic activity in rectal biopsy supernatant when comparing calcium responses before and after incubation with an MRGPRX2 antagonist in MRGPRX2-expressing Chinese Hamster Ovary cells. Additionally, we show evidence of increased levels of substance P in IBS patient samples, which is a known MRGPRX2 agonist and possibly involved in stress-induced abdominal pain. Our results suggest a potential role for MRGPRX2-mediated MC activation in IBS, implying that MRGPRX2-antagonists may represent a novel therapeutic approach to treat IBS patients. As we know from our results in chapter 3, a significant proportion of patients do not improve with ebastine. Therefore, other mediators such as proteases might be involved in the development of VHS. From research in mouse models of VHS, we know that histamine and tryptase, two MC mediators, can sensitise neurons and induce VHS via sensitisation of the TRP channels TRPV1 and TRPV4, and TRPA1 (6,8-11). While proteases in the gut are primarily known for their role in food digestion, they can also mediate processes that depend on cellular communication (12). Trypsin and tryptase are suggested to play a role in this pathway based on increased expression levels in the intestinal mucosa and increased activity levels measured with substrate-based kinetics (13-17). In chapter 5, we confirm these previous findings of increased trypsin-like activity levels and show no significant increase in chymotrypsin- and elastase-like activity levels, the two other subclasses of serine proteases. Additionally, our data strongly suggest that the increased trypsin-like activity in IBS patient samples is more likely to originate from proteases with trypsin- and tryptase-like substrate preferences, rather than thrombin-like proteases. Using an activity-based probe to unambiguously identify active proteases in the complex proteome of rectal biopsy supernatant, preliminary results suggest increased levels of trypsin-2 and chymotrypsin-like elastase 3A in a subgroup of IBS patients displaying high trypsin-like activity levels. These findings support the involvement of proteases in IBS and guide the development of protease inhibitors as potential new treatment options for IBS management. While it is clear that activation of the protease-activated receptor 2 (PAR2) is necessary for TRPV1, TRPV4, or TRPA1 sensitisation in murine models, it is uncertain how proteases mediate sensitisation of TRP channels in humans. Especially as it appears that proteases in colonic biopsy supernatant from IBS patients could only activate human enteric neurons via PAR1, and not PAR2 (18-20). As human dorsal root ganglion neurons are hard to obtain, and experiments with enteric neurons and stem cells are highly time-intensive, we decided to modify the Human Embryonic Kidney cell line, which has neuronal characteristics (21). In chapter 6, we describe in detail the stable incorporation of human TRPV1, TRPV4, and TRPA1 with a C-terminal hemagglutinin-tag under an inducible promotor and the calcium indicator GCaMP6m-Xc under a constitutive promotor, determining optimal TRP expression and confirming functionality of the new model, which enables functional assessment of protease-mediated TRP sensitisation. In summary, the results of this thesis provide new insights into the mechanisms underlying abdominal pain in IBS. We show that HRH1 antagonism should be considered as a novel treatment option for IBS patients. Additionally, our results indicate two novel therapeutic strategies for managing IBS-related abdominal pain, specifically antagonism of MRGPRX2 and inhibition of overactive proteases, highlighting promising new avenues for targeted treatment approaches. References: 1. Sperber, A. D. et al. Worldwide Prevalence and Burden of Functional Gastrointestinal Disorders, Results of Rome Foundation Global Study. 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