This is big news for Irritable Bowel Syndrome (IBS) patients: 11 percent of the world’s population suffers from IBS, but the fight against chronic pain has taken a major step forward with scientists identifying receptors in the nervous system which cause the condition in the hope of developing effective treatments.
Flinders University researchers at SAHMRI have discovered receptors that cause itchy skin also exist in the human gut and activate neurons, which result in IBS patients feeling like they’re experiencing chronic gut pain or a seriously painful ‘gut itch.’
In millions of Americans with IBS, it looks like these ‘itch’ receptors might be more present than in healthy people.
This means that more neurons are activated, causing the feeling of more pain.
NHMRC and Matthew Flinders Research Fellow in Gastrointestinal Neuroscience, Professor Stuart Brierley, says these gut itch receptors could offer a new way of targeting the underlying cause of gut pain, rather than using traditional drugs (like opioids), which don’t fix the problem right now.
“We found receptors which bring about an itchy feeling on skin also do the same in in the gut, so these patients are essentially suffering from a ‘gut itch’.
We’ve translated these results to human tissue tests and now hope to help create a treatment where people can take an oral medication for IBS.”
Professor Stuart Brierley explains research identifying receptors in the nervous system which cause irritable bowel syndrome in the hope of developing effective treatments. Credit: Flinders University
“Patients with IBS suffer from chronic abdominal pain and experience rewiring of their nervous system so they feel pain when they shouldn’t – we decided to ask important questions about how nerves in the gut are activated to cause pain in order to seek out potential solutions.”
Australian researchers identify a link between itchy skin and gut pain, caused by identical receptors signalling the nervous system. Credit: Professor Stuart Brierley, Flinders University
Professor Brierley, also the Director of the Visceral Pain Research Group at SAHMRI, says pain experienced by IBS sufferers takes place when itch receptors are coupled with what’s known as the ‘wasabi receptor’ in the nervous system, which normally helps people react to consuming wasabi- the Japanese condiment.
“If you think about what happens when you eat wasabi, it activates a receptor on the nerves and sends a pain signal – that’s exactly what’s happening within in their gut as they experience an itchy effect or wasabi effect in the gut.”
“Having shown these mechanisms contribute to chronic gut pain, we can now work out ways to block these receptors and thereby stop the ‘gut itch’ signal traveling from the gut to the brain.
This will be a far better solution that the problems currently presented by opioid treatments”.
Irritable bowel syndrome (IBS) is a functional bowel disorder in which recurrent abdominal pain is associated with a change in bowel habit, typically constipation (IBS-C), diarrhea (IBS-D), or a mixed (constipation and diarrhea) bowel habit (IBS-M) (1).
IBS is a common disorder in Western populations, which affects around 11% of the global population (2), with a higher prevalence in women than in men (1).
Although the aetiology of IBS remains unclear, low-grade inflammation has been widely described in this disorder, with several fundamental studies implicating proinflammatory molecules in the pathophysiology of IBS symptoms (3).
We previously showed that the amounts of several polyunsaturated fatty acid (PUFA) metabolites, also defined as bioactive lipids, are statistically significantly altered in biopsy samples from patients with IBS compared to those in samples from healthy controls (4).
This is in agreement with previous studies focused on the prostanoid subtype of PUFA metabolites (5–7).
The functional relationship between PUFAs and pain has been the subject of many studies (8).
Both basic and clinical studies have revealed that a dietary intake of n-3 series PUFAs results in a reduction in pain associated with rheumatoid arthritis (9, 10), dysmenorrhea (11), inflammatory bowel disease (12), and neuropathy (13), whereas n-6 series PUFAs are high in abundance in patients with chronic pain, including IBS patients (4, 14, 15).
The n-3 PUFA metabolites, such as resolvins (Rvs), are analgesic in multiple pain models, an effect attributed to inhibition of certain transient receptor potential (TRP) channels (16). For example, RvE1 specifically inhibits TRPV1 signaling (17), whereas RvD1 attenuates the function of TRPA1 and TRPV4 (18) and RvD2 inhibits TRPV1 and TRPA1 activity (19).
These effects have been observed with other types of n-3 PUFAs, such as maresin 1 (Mar1), which also has inhibitory effects on TRPV1 channel function (20) and reduces pain. The quantification of Rvs in the knee synovia of patients suffering from inflammatory arthritis suggests that synthesis of specialized proresolving mediators (SPMs) at the site of inflammation may be a mechanism of endogenous pain relief in humans. In contrast, n-6 PUFA metabolites are pro-nociceptive by stimulating nerve fibers through the activation of immune cells (21, 22).
Nonetheless, several n-6 PUFA metabolites, such as thromboxane A2 (TXA2), PGE2, leukotriene B4 (LTB4), and PGD2, can directly stimulate sensory nerve fibers (23–26). However, some n-6 PUFA metabolites, such as lipoxins, can inhibit pain (27).
Consistent with the role of TRP channels in the transduction of noxious stimuli, we previously showed a correlation between PUFA metabolites and TRP channel activation, particularly for the TRPV4 agonist 5,6-epoxyeicosatrienoic acid (5,6-EET) and pain intensity in IBS-D patients (4).
Furthermore, PUFA metabolites from colonic biopsies of IBS-C patients induced Ca2+ influx in sensory neurons independently of TRPV4, suggesting that the PUFA metabolites produced in IBS-C and IBS-D are distinct (4). Thus, the aim of this study was to identify algogenic PUFA metabolites specifically produced in IBS-C patients and decipher the mechanism by which they may activate sensory nerves.
Herein, we showed that 5-oxoETE, an n-6 PUFA subtype selectively increased in abundance in colonic tissues from IBS-C patients, induced hypersensitivity in a manner dependent on the G protein–coupled receptor (GPCR) Mrgprd.
Discussion
In this study, we showed that: (i) Concentrations of the PUFA metabolite 5-oxoETE were statistically significantly increased in biopsies from patients with IBS-C compared to biopsies from patients with other IBS subtypes or from HCs; (ii) 5-oxoETE induced somatic, as well as visceral, hyperalgesia, without promoting inflammation; (iii) 5-oxoETE activated both mouse and human sensory neurons; (iv) In mice, 5-oxoETE signaled in a manner dependent on Mrgprd. Together, these data suggest a role for 5-oxoETE and Mrgprd-expressing, IB4-positive sensory neurons in visceral hypersensitivity in IBS-C patients.
Eicosanoids and docosanoids are the most important lipids implicated in inflammatory processes. They derive from the oxidation of twenty and twenty-two carbon PUFAs, respectively (29).
Several PUFA metabolites are increased in abundance in the intestinal mucosa from patients with IBDs (including, TXA2, PGE2, LTB4, and PGD2), which induce visceral afferent fiber activation (23–26).
Here, we showed that PGE2, 5,6-EET, and TXB2 were statistically significantly increased in abundance in the intestinal mucosa of IBS-D patients, whereas no alteration in PUFA metabolism was observed in patients with IBS-M. Furthermore, when lipid extracts from HCs and all IBS patients were compared, we observed a statistically significant decrease in the amounts of 14-HDoHE and 17-HDoHE, which are SPM precursors (30).
Because SPMs possess an analgesic effect (31), the pain associated with IBS could be also the consequence of a decrease in SPM abundance, leading to sensory neuron activation. A complete characterization of the different SPMs produced by the metabolism of EPA, DHA, or DPA will be of interest for the characterization of bioactive lipids potentially linked with pain in IBS patients.
We showed that the concentration of 5-oxoETE increased in only the colonic biopsies of IBS-C patients, highlighting its potential relevance as a new marker of this disease. 5-oxoETE, which derives from arachidonic acid (AA) metabolism, is produced by various inflammatory cells. In addition, it can also be synthesized from 5-HETE by stromal cells, possibly by transcellular biosynthesis (32). 5-oxoETE is formed by the oxidation of 5-HETE by 5-hydroxyeicosanoid dehydrogenase (5-HEDH) (33), a microsomal enzyme that is highly selective for 5S-HETE and requires NADP+ as a cofactor (34).
5-HEDH is found in neutrophils as well as in various other inflammatory and stromal cells, including monocytes (35), dendritic cells (36), and intestinal epithelial cells (37). 5-oxoETE is a potent chemoattractant for human and rat eosinophils and it indirectly promotes the survival of these cells (38).
However, we observed no cellular infiltration either in the paw or intestinal mucosa of mice administered with 5-oxoETE.
This discrepancy may be due to the rapid metabolism of 5-oxoETE in vivo (32) or the absence of other molecules, such as interleukin-5, which act in synergy to attract inflammatory cells during inflammatory processes or allergies (39).
In our experiments, the injection of 5-oxoETE alone, without cofactors, could thus explain the absence of infiltration of tissues by polymorphonuclear cells. The formation of 5-oxoETE requires NADP+ (40).
Accordingly, oxidative stress associated with IBS-C (41) may improve the conversion rate of NADPH into NADP+ in epithelial cells, thereby resulting in the synthesis of greater amounts of 5-oxoETE.
In a previous study, we reported that PUFA metabolites extracted from biopsies of IBS-C and IBS-D patients triggered an increase in [Ca2+]i in primary sensory neurons, whereas those extracted from biopsies of IBS-M patients had no effect (4).
We further identified the PUFA metabolite 5,6-EET as a TRPV4 agonist with algogenic activity, specifically associated with IBS-D sub-group (4). By contrast, no PUFA metabolite with TRP agonist activity was found to be increased in abundance in IBS-C patient biopsies (4).
Because we found that only 5-oxoETE was increased in biopsies of IBS-C patients, we hypothesized that this PUFA metabolite might be responsible for the activation of sensory neurons and hypersensitivity-associated with IBS-C. As previously reported in humans, 5-oxoETE may interact with the oxoeicosanoid receptor 1 (OXER1). However, there is no homologous OXE receptor in rodents (40)
. Because the observed 5-oxoETE-–nduced increase in [Ca2+]i in mouse sensory neurons was inhibited by both a PLC inhibitor and PTX, we hypothesized that 5-oxoETE led to the activation of Gαi/o/Gαq-coupled GPCRs. Given that 5-oxoETE acted selectively on IB4+ sensory neurons, the targeted receptor should be specifically expressed on this neuronal subclass. Accordingly, we investigated the role of Mrgprd, a GPCR specifically expressed on IB4+ sensory neurons, which may be coupled to Gαq proteins and to PTX–sensitive Gαi/o proteins (42), and was previously reported as a key player in mechanical hypersensitivity (43–45).
Stimulation of Mrgprd-positive neurons with β-alanine, the prototypical agonist of Mrgprd, increases [Ca2+]i (46), as was observed here after 5-oxoETE treatment. Moreover, in a FLIPR (Fluorescent Imaging Plate Reader) assay developed for the simultaneous identification of Mrgprd agonists and antagonists, a PLC inhibitor completely blocked the FLIPR response to β-alanine, whereas PTX treatment resulted in 50% reduction in [Ca2+]i (47).
Again, similar results were obtained here in experiments with PTX or a PLC inhibitor to inhibit 5-oxoETE–induced activation of primary mouse sensory neurons. Using tissue from adult MrgprdEGFP mice stained with antibodies to GFP, a previous study showed that Mrgprd is expressed in non-peptidergic neurons that innervate the epidermis; however, Mrgprd-positive fibres were not observed in any other visceral organs, including both the small and large intestine (28).
By contrast, numerous studies using different retrograde tracers have identified a minor population (20 to 26%) of IB4+ sensory neurons that innervate the colon (48–50). Indeed, a study also identified Mrgprd mRNA in colonic sensory neurons by single cell RNA-sequencing (51).
To confirm the presence of both Mrgprd mRNA and Mrgprd protein in colonic sensory neurons in the present study, we applied a similar retrograde neurotracing approach using single-cell RT-qPCR analysis and anti-GFP immunostaining in MrgprdEGFP mice. We observed Mrgprd mRNA and Mrgprd protein expression in sensory DRG neurons projecting to the colon at a similar frequency to that observed in previous studies (51), thereby not only confirming the presence of a Mrgprd-positive colonic neuronal subtype, but also reinforcing Mrgprd as a potential target of 5-oxoETE.
The activity of 5-oxoETE towards Mrgprd was attested to by its ability to induce an increase in [Ca2+]i in IB4-positive sensory neurons, but not in mouse neurons in which Mrgprd was knocked down by shRNA or in neurons from Mrgprd-deficient mice. Conversely, whereas Ca2+ transients were stimulated by 5-oxoETE in CHO cells transfected with a plasmid expressing Mrgprd, CHO cells transfected with a control plasmid were not responsive to 5-oxoETE.
Activation of Mrgprd inhibits a fraction of the total M-current, carried primarily by the KCNQ2/3 K+ channel, contributing to an increase in the excitability of DRG neurons (52). Thus, Mrgprd activation by 5-oxoETE might promote the excitability of primary nociceptive afferents by KCNQ inhibition. Several groups have demonstrated that retigabine, a KCNQ2–5 opener, is effective in reducing neuropathic (53) and inflammatory pain (54).
At the visceral level, retigabine reduces capsaicin-induced visceral pain and can inhibit noxious chemosensitivity in human tissue, suggesting that KCNQ channels play an inhibitory role in the transmission of visceral nociception (55, 56).
Given that human sensory DRG neurons express Mrgprd and are activated by 5-oxoETE, we can speculate that 5-oxoETE modulates KCNQ channels through Mrgprd activation, leading to neuronal activation that contributes to the pain symptoms associated with IBS-C. Nevertheless, because OXER1 is expressed in human tissue, we cannot exclude the possibility that this receptor is activated by 5-oxoETE in human tissue.
Together, our current findings build on our previous studies to suggest a pivotal role for PUFA metabolites in the visceral pain associated with IBS (4). Specifically, our study identifies 5-oxoETE with pro-nociceptive activity, as a hallmark of the IBS-C subtype.
More information: Joel Castro et al, Activation of pruritogenic TGR5, MRGPRA3, and MRGPRC11 on colon-innervating afferents induces visceral hypersensitivity, JCI Insight (2019). DOI: 10.1172/jci.insight.131712
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