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The quest for appropriate diagnostic tools and therapies for IBS runs in parallel with the different pathogenic mechanisms investigated. One of these mechanisms is visceral hypersensitivity, resulting from a disturbed neuronal signalling at the peripheral and/or central levels.1 ,2 Rolland-Fourcade et al 3 show, in this issue of Gut, that intestinal epithelial cells (Caco-2 cells) release a trypsin-like activity at the basolateral side of the cells after stimulation with a classical inflammatory stimulus (lipopolysaccharide) or with a stress-related stimulus (epinephrine), and they identified it as trypsin-3. They confirmed the clinical relevance of trypsin-3 by proving its presence in colonic tissue from patients with IBS (all subgroups). Besides, trypsin-3 increased epithelial permeability in the Caco-2 monolayers and increased the excitability of mouse dorsal root ganglia neurons in vitro. Moreover, it induced Ca+2 transients in human submucosal neurons in vitro and caused visceral hyperalgesia in response to colorectal distension in two rat models of visceral hypersensitivity.3 The novelty of this manuscript lies within the identification of a specific protease, namely trypsin-3 and its source, the intestinal epithelium, providing additional evidence for serine proteases and protease-activated receptors (PAR).
The importance of visceral hypersensitivity as a key contributor to the generation of symptoms in patients with IBS and thus an interesting target in the therapeutic counselling was recently endorsed by Simrén et al.4 Mast cells and their mediators, including histamine and proteases, are generally accepted to play a role in visceral hypersensitivity with histamine1 receptor antagonists representing a good example from ‘bench to bedside’ research.5 ,6 Proteases can exert their activity via PARs, with PAR1 and PAR4 activation showing anti-nociceptive and PAR2 activation showing pronociceptive effects.6 Protease inhibition has been suggested as a therapeutic option in both IBS and IBD since an increased trypsin-like activity and enhanced tryptase contents have been shown in tissues from patients with IBS. In preclinical studies, PAR2-antagonists and (serine) protease inhibitors indeed have shown beneficial effects on inflammation, intestinal permeability and recently on visceral hypersensitivity (for reviews, see refs 2 ,7 ,8). One has to be aware that besides mast cells releasing different serine proteases, such as tryptase and chymase, and inflammatory cells, the pancreas and the microbiome encompass an important source of proteases.
Based on the existing data, one might definitely conclude that proteases are interesting molecules both as diagnostic biomarkers and/or as therapeutic targets in IBS. However, some important issues need to be solved and studied in more detail.
The first issue relates to the origin of the proteases, the interactions between different proteases and their pathogenic potential in IBS. Proteases are present in large amounts and in different compartments both in the GI lumen (from pancreatic or microbial origin) and the GI wall (classically assumed derived from mast cells and inflammatory cells). In Gut, an additional source and its mechanisms are postulated regarding a specific protease, trypsin-3, released by intestinal epithelial cells and able to influence visceral sensitivity and the intestinal barrier function as stated above.3 In general, we need to unravel in more detail whether specific proteases are ubiquitously present or rather cell-specific as suggested here for trypsin-3. Next, it will be interesting to unravel how the epithelium-specific trypsin-3 relates to or interacts with, for instance, the mast cell-derived tryptase as both are postulated as important mediators in visceral hypersensitivity. Regarding the role of the different sources, there is evidence that the increased faecal serine protease activity detected in diarrhoea-predominant patients with IBS is from human and most probably from pancreatic origin.9 This does however not exclude a role for microbial and other luminal sources of proteases. We generally assume that these luminal proteases derived from the microbiome, pancreas or even food substances will remain in the GI lumen without crossing the mucosal barrier. However, we do not know exactly what happens in circumstances of impaired barrier dysfunction—as evidenced in many GI diseases—taking into account that proteases themselves are able to alter the intestinal permeability.2 ,8
A second important issue that needs attention is the mechanism of action of the proteases considering the presence and activity of endogenous protease inhibitors as well. Specific protease inhibitors are needed in order to develop safe drugs that do not interfere with all the physiological roles of the different proteases and endogenous protease inhibitors, but on the other hand it is plausible that a combination of proteases needs to be targeted in order to obtain a sustained effect as well.6 ,7 ,9 Regarding the mechanism of action, a PAR2-mediated pathway has been suggested for several proteases. In their present study, Rolland-Fourcade et al seem to exclude the involvement of PAR1 and PAR4 as the main receptor targets of trypsin-3.2 However, we cannot exclude a role for different PAR receptors when other (serine) proteases are involved. Moreover, the signalling cascade needs to be investigated in more detail, such as the involvement of transient receptor potential (TRP) channels downstream of the PAR receptors. The TRP vanilloid receptors TRPV1 and TRPV4 as well as the TRP ankyrin receptor TRPA1 have been linked to PAR2 activation and TRP channels also seem to be crucial for histaminergic signaling related to visceral hypersensitivity.5 ,6
Finally, the methodologies used to measure protease activities need further development. One has to be aware that the substrates used to determine the activity of trypsin in this and other papers are cleaved by trypsin and by tryptase, and thus this non-specificity of the substrates used only provides information on a more broad trypsin-like activity. Recently, activity-based probes have been developed that bind the activity site of two serine proteases in an activity-dependent manner, namely trypsin-like and elastin-like activities, with proven efficacy in vitro but not yet in vivo. 10
In summary, the role of serine proteases in IBS becomes more and more intriguing with evidence for the presence of different sources including the microbiome, the mast cells and as shown in this issue also the intestinal epithelium. Their effects on intestinal permeability and visceral sensitivity offer potential as diagnostic markers and/or therapeutic targets, keeping in mind the pitfalls and questions remaining.
Competing interests None declared.
Provenance and peer review Commissioned; internally peer reviewed.
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