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Background and Purpose: Tolerance to the analgesic effects of opioids and resultant dose escalation is associated with worsening of side effects and greater addiction risk. Here, we compare the development of tolerance to the conventional opioid fentanyl with a novel pH-sensitive μ-opioid receptor (MOR) agonist, (±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide (NFEPP) that is active only in acidic inflammatory microenvironments., Experimental Approach: An opioid tolerance model was developed in male C57BL/6 mice, with and without dextran sulphate sodium colitis, using increasing doses of either fentanyl or NFEPP over 5 days. Visceral nociception was assessed in vivo by measuring visceromotor responses (VMRs) to noxious colorectal distensions and in vitro measuring colonic afferent nerve activity of mesenteric nerves and performing patch-clamp recordings from isolated dorsal root ganglia neurons. Somatic thermal nociception was tested using a tail immersion assay. Cardiorespiratory effects were analysed by pulse oximeter experiments., Key Results: VMRs and tail immersion tests demonstrated tolerance to fentanyl, but not to NFEPP in colitis mice. Cross-tolerance also occurred to fentanyl, but not to NFEPP. The MOR agonist DAMGO inhibited colonic afferent nerve activity in colitis mice exposed to chronic NFEPP, but not those from fentanyl-treated mice. Similarly, in patch-clamp recordings from isolated dorsal root ganglia neurons, DAMGO inhibited neurons from NFEPP-, but not fentanyl-treated mice., Conclusion and Implications: NFEPP did not exhibit tolerance in an inflammatory pain model, unlike fentanyl. Consequently, dose escalation to maintain analgesia during an evolving inflammation could be avoided, mitigating the potential risk of side effects., (© 2024 British Pharmacological Society.)

Clostridioides difficile is the leading cause of antibiotic-associated infectious diarrhea. The development of C.difficile infection is tied to perturbations of the bacterial community in the gastrointestinal tract, called the gastrointestinal microbiota. Repairing the gastrointestinal microbiota by introducing lab-designed bacterial communities, or defined microbial communities, has recently shown promise as therapeutics against C.difficile infection, however, the mechanisms of action of defined microbial communities remain unclear. Using an antibiotic- C.difficile mouse model, we report the ability of an 18-member community and a refined 4-member community to protect mice from two ribotypes of C.difficile (CD027, CD078; p < 0.05). Furthermore, bacteria-free supernatant delivered orally to mice from the 4-member community proteolyzed C.difficile toxins in vitro and protected mice from C.difficile infection in vivo (p < 0.05). This study demonstrates that bacteria-free supernatant is sufficient to protect mice from C.difficile; and could be further explored as a therapeutic strategy against C.difficile infection., (© 2024. The Author(s).)

Background & Aims: Abdominal pain is a major symptom of diseases that are associated with microbial dysbiosis, including irritable bowel syndrome and inflammatory bowel disease. Germ-free mice are more prone to abdominal pain than conventionally housed mice, and reconstitution of the microbiota in germ-free mice reduces abdominal pain sensitivity. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons., Methods: In vivo and in vitro assays of visceral sensation were performed on mice treated with the nonabsorbable antibiotic vancomycin (50 μg/mL in drinking water) for 7 days and water-treated control mice. Bacterial dysbiosis was verified by 16s rRNA analysis of stool microbial composition., Results: Treatment of mice with vancomycin led to an increased sensitivity to colonic distension in vivo and in vitro and hyperexcitability of dorsal root ganglion (DRG) neurons in vitro, compared with controls. Interestingly, hyperexcitability of DRG neurons was not restricted to those that innervated the gut, suggesting a widespread effect of gut dysbiosis on peripheral pain circuits. Consistent with this, mice treated with vancomycin were more sensitive than control mice to thermal stimuli applied to hind paws. Incubation of DRG neurons from naive mice in serum from vancomycin-treated mice increased DRG neuron excitability, suggesting that microbial dysbiosis alters circulating mediators that influence nociception. The cysteine protease inhibitor E64 (30 nmol/L) and the protease-activated receptor 2 (PAR-2) antagonist GB-83 (10 μmol/L) each blocked the increase in DRG neuron excitability in response to serum from vancomycin-treated mice, as did the knockout of PAR-2 in NaV1.8-expressing neurons. Stool supernatant, but not colonic supernatant, from mice treated with vancomycin increased DRG neuron excitability via cysteine protease activation of PAR-2., Conclusions: Together, these data suggest that gut microbial dysbiosis alters pain sensitivity and identify cysteine proteases as a potential mediator of this effect., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Targeting the acidified inflammatory microenvironment with pH-sensitive opioids is a novel approach for managing visceral pain while mitigating side effects. The analgesic efficacy of pH-dependent opioids has not been studied during the evolution of inflammation, where fluctuating tissue pH and repeated therapeutic dosing could influence analgesia and side effects. Whether pH-dependent opioids can inhibit human nociceptors during extracellular acidification is unexplored. We studied the analgesic efficacy and side-effect profile of a pH-sensitive fentanyl analog, (±)- N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide (NFEPP), during the evolution of colitis induced in mice with dextran sulphate sodium. Colitis was characterized by granulocyte infiltration, histological damage, and acidification of the mucosa and submucosa at sites of immune cell infiltration. Changes in nociception were determined by measuring visceromotor responses to noxious colorectal distension in conscious mice. Repeated doses of NFEPP inhibited nociception throughout the course of disease, with maximal efficacy at the peak of inflammation. Fentanyl was antinociceptive regardless of the stage of inflammation. Fentanyl inhibited gastrointestinal transit, blocked defaecation, and induced hypoxemia, whereas NFEPP had no such side effects. In proof-of-principle experiments, NFEPP inhibited mechanically provoked activation of human colonic nociceptors under acidic conditions mimicking the inflamed state. Thus, NFEPP provides analgesia throughout the evolution of colitis with maximal activity at peak inflammation. The actions of NFEPP are restricted to acidified layers of the colon, without common side effects in normal tissues. N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide could provide safe and effective analgesia during acute colitis, such as flares of ulcerative colitis., (Copyright © 2023 International Association for the Study of Pain.)

Background: Monosodium glutamate (MSG) has been identified as a trigger of abdominal pain in irritable bowel syndrome (IBS), but the mechanism is unknown. This study examined whether MSG causes visceral hypersensitivity using a water-avoidance stress (WAS) mouse model of visceral pain., Methods: Mice were divided into four groups receiving treatment for 6 days: WAS + MSG gavage, WAS + saline gavage, sham-WAS + MSG gavage, and sham-WAS + saline gavage. The acute effects of intraluminal administration of 10 μM MSG on jejunal extrinsic afferent nerve sensitivity to distension (0-60 mmHg) were examined using ex vivo extracellular recordings. MSG was also applied directly to jejunal afferents from untreated mice. Glutamate concentration was measured in serum, and in the serosal compartment of Ussing chambers following apical administration., Key Results: Acute intraluminal MSG application increased distension responses of jejunal afferent nerves from mice exposed to WAS + MSG. This effect was mediated by wide dynamic range and high-threshold units at both physiologic and noxious pressures (10-60 mmHg, p < 0.05). No effect of MSG was observed in the other groups, or when applied directly to the jejunal afferent nerves. Serum glutamate was increased in mice exposed to WAS + MSG compared to sham-WAS + saline, and serosal glutamate increased using WAS tissue (p = 0.0433)., Conclusions and Inferences: These findings demonstrate that repeated exposure to MSG in mice leads to sensitization of jejunal afferent nerves to acute ex vivo exposure to MSG. This may contribute to visceral hypersensitivity reported in response to MSG in patients with IBS., (© 2023 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

Background: Using data from the Rome Foundation Global Epidemiology Study examining prevalence of disorders of gut-brain interaction (DGBI) in 33 countries, this study explored the prevalence of all 22 disorders in Canada. It examined differences related to geography and sociodemographic factors, health impact and compared these findings to other countries., Methods: The Rome Foundation survey was conducted through the Internet, targeting 2000 Canadian participants. The survey used the validated ROME IV diagnostic questionnaire to identify the 22 DGBI and an in-depth supplemental questionnaire that included quality assurance techniques., Key Results: There were 2029 respondents with a mean age of 48 years and 50% females. Diagnostic criteria for at least one of the 22 DGBI were met by 41.3%, similar to other countries. Functional constipation, functional diarrhea, and unspecified functional bowel disorders were most common. Irritable bowel syndrome prevalence was 4.2% using Rome IV and 10.1% using ROME III criteria. DGBI were associated with poorer quality of life and increased psychological symptom scores. Prevalence of DGBI ranged from 48% in Quebec to 36% in British Columbia. Prescription pain medication was reported by 17% with DGBI compared to 9% without DGBI and correlated negatively with mental health and physical well-being. Quebecers and francophones were in limited number but reported higher DGBI prevalence and symptom severity compared to others., Conclusions & Inferences: A large proportion of Canadians suffer from DGBI which seriously impact their well-being. Findings highlight the need for further research and education, including understanding whether significant regional and cultural differences contribute to DGBI., (© 2023 John Wiley & Sons Ltd.)

Objective: Dietary therapies for irritable bowel syndrome (IBS) have received increasing interest but predicting which patients will benefit remains a challenge due to a lack of mechanistic insight. We recently found evidence of a role for the microbiota in dietary modulation of pain signalling in a humanised mouse model of IBS. This randomised cross-over study aimed to test the hypothesis that pain relief following reduced consumption of fermentable carbohydrates is the result of changes in luminal neuroactive metabolites., Design: IBS (Rome IV) participants underwent four trial periods: two non-intervention periods, followed by a diet low (LFD) and high in fermentable carbohydrates for 3 weeks each. At the end of each period, participants completed questionnaires and provided stool. The effects of faecal supernatants (FS) collected before (IBS FS) and after a LFD (LFD FS) on nociceptive afferent neurons were assessed in mice using patch-clamp and ex vivo colonic afferent nerve recording techniques., Results: Total IBS symptom severity score and abdominal pain were reduced by the LFD (N=25; p<0.01). Excitability of neurons was increased in response to IBS FS, but this effect was reduced (p<0.01) with LFD FS from pain-responders. IBS FS from pain-responders increased mechanosensitivity of nociceptive afferent nerve axons (p<0.001), an effect lost following LFD FS administration (p=NS) or when IBS FS was administered in the presence of antagonists of histamine receptors or protease inhibitors., Conclusions: In a subset of IBS patients with improvement in abdominal pain following a LFD, there is a decrease in pronociceptive signalling from FS, suggesting that changes in luminal mediators may contribute to symptom response., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

While effective in treating abdominal pain, opioids have significant side effects. Recent legalization of cannabis will likely promote use of cannabinoids as an adjunct or alternative to opioids, despite a lack of evidence. We aimed to investigate whether cannabinoids inhibit mouse colonic nociception, alone or in combination with opioids at low doses. Experiments were performed on C57BL/6 male and female mice. Visceral nociception was evaluated by measuring visceromotor responses (VMR), afferent nerve mechanosensitivity in flat-sheet colon preparations, and excitability of isolated DRG neurons. Blood oxygen saturation, locomotion, and defecation were measured to evaluate side effects. An agonist of cannabinoid 1 receptor (CB1R), arachidonyl-2'-chloroethylamide (ACEA), dose-dependently decreased VMR. ACEA and HU-210 (another CB1R agonist) also attenuated colonic afferent nerve mechanosensitivity. Additionally, HU-210 concentration-dependently decreased DRG neuron excitability, which was reversed by the CB1R antagonist AM-251. Conversely, cannabinoid 2 receptor (CB2R) agonists did not attenuate VMR, afferent nerve mechanosensitivity, or DRG neuron excitability. Combination of subanalgesic doses of CB1R and µ-opioid receptor agonists decreased VMR; importantly, this analgesic effect was preserved after 6 d of twice daily treatment. This combination also attenuated afferent nerve mechanosensitivity and DRG neuron excitability, which was inhibited by neuronal nitric oxide synthase and guanylate cyclase inhibitors. This combination avoided side effects (decreased oxygen saturation and colonic transit) caused by analgesic dose of morphine. Activation of CB1R, but not CB2R, decreased colonic nociception both alone and in synergy with µ-opioid receptor. Thus, CB1R agonists may enable opioid dose reduction and avoid opioid-related side effects. SIGNIFICANCE STATEMENT One of the most cited needs for patients with abdominal pain are safe and effective treatment options. The effectiveness of opioids in the management of abdominal pain is undermined by severe adverse side effects. Therefore, strategies to replace opioids or reduce the doses of opioids to suppress abdominal pain is needed. This study in mice demonstrates that cannabinoid 1 receptor (CB1R) agonists inhibit visceral sensation. Furthermore, a combination of subanalgesic doses of µ-opioid receptor agonist and CB1R agonist markedly reduce abdominal pain without causing the side effects of high-dose opioids. Thus, CB1R agonists, alone or in combination with low-dose opioids, may be a novel and safe treatment strategy for abdominal pain., (Copyright © 2022 the authors.)

The gut microbiota has been implicated in chronic pain disorders, including irritable bowel syndrome (IBS), yet specific pathophysiological mechanisms remain unclear. We showed that decreasing intake of fermentable carbohydrates improved abdominal pain in patients with IBS, and this was accompanied by changes in the gut microbiota and decreased urinary histamine concentrations. Here, we used germ-free mice colonized with fecal microbiota from patients with IBS to investigate the role of gut bacteria and the neuroactive mediator histamine in visceral hypersensitivity. Germ-free mice colonized with the fecal microbiota of patients with IBS who had high but not low urinary histamine developed visceral hyperalgesia and mast cell activation. When these mice were fed a diet with reduced fermentable carbohydrates, the animals showed a decrease in visceral hypersensitivity and mast cell accumulation in the colon. We observed that the fecal microbiota from patients with IBS with high but not low urinary histamine produced large amounts of histamine in vitro. We identified Klebsiella aerogenes , carrying a histidine decarboxylase gene variant, as a major producer of this histamine. This bacterial strain was highly abundant in the fecal microbiota of three independent cohorts of patients with IBS compared with healthy individuals. Pharmacological blockade of the histamine 4 receptor in vivo inhibited visceral hypersensitivity and decreased mast cell accumulation in the colon of germ-free mice colonized with the high histamine-producing IBS fecal microbiota. These results suggest that therapeutic strategies directed against bacterial histamine could help treat visceral hyperalgesia in a subset of patients with IBS with chronic abdominal pain.

Opioid tolerance (OT) leads to dose escalation and serious side effects, including opioid-induced hyperalgesia (OIH). We sought to better understand the mechanisms underlying this event in the gastrointestinal tract. Chronic in vivo administration of morphine by intraperitoneal injection in male C57BL/6 mice evoked tolerance and evidence of OIH in an assay of colonic afferent nerve mechanosensitivity; this was inhibited by the δ-opioid receptor (DOPr) antagonist naltrindole when intraperitoneally injected in previous morphine administration. Patch-clamp studies of DRG neurons following overnight incubation with high concentrations of morphine, the µ-opioid receptors (MOPr) agonist [D-Ala 2 , N-Me-Phe 4 , Gly 5 -ol]-Enkephalin (DAMGO) or the DOPr agonist [D-Ala 2 , D-Leu 5 ]-Enkephalin evoked hyperexcitability. The pronociceptive actions of these opioids were blocked by the DOPr antagonist SDM25N but not the MOPr antagonist D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH 2 The hyperexcitability induced by DAMGO was reversed after a 1 h washout, but reapplication of low concentrations of DAMGO or [D-Ala 2 , D-Leu 5 ]-Enkephalin restored the hyperexcitability, an effect mediated by protein kinase C. DOPr-dependent DRG neuron hyperexcitability was blocked by the endocytosis inhibitor Pitstop 2, and the weakly internalizing DOPr agonist ARM390 did not cause hyperexcitability. Bioluminescence resonance energy transfer studies in HEK cells showed no evidence of switching of G-protein signaling from G i to a G s pathway in response to either high concentrations or overnight incubation of opioids. Thus, chronic high-dose opioid exposure leads to opioid tolerance and features of OIH in the colon. This action is mediated by DOPr signaling and is dependent on receptor endocytosis and downstream protein kinase C signaling. SIGNIFICANCE STATEMENT Opioids are effective in the treatment of abdominal pain, but escalating doses can lead to opioid tolerance and potentially opioid-induced hyperalgesia. We found that δ-opioid receptor (DOPr) plays a central role in the development of opioid tolerance and opioid-induced hyperalgesia in colonic afferent nociceptors following prolonged exposure to high concentrations of MOPr or DOPr agonists. Furthermore, the role of DOPr was dependent on OPr internalization and activation of a protein kinase C signaling pathway. Thus, targeting DOPr or key components of the downstream signaling pathway could mitigate adverse side effects by opioids., (Copyright © 2022 the authors.)

Objective: The effectiveness of µ-opioid receptor (MOPr) agonists for treatment of visceral pain is compromised by constipation, respiratory depression, sedation and addiction. We investigated whether a fentanyl analogue, (±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide (NFEPP), which preferentially activates MOPr in acidified diseased tissues, would inhibit pain in a preclinical model of inflammatory bowel disease (IBD) without side effects in healthy tissues., Design: Antinociceptive actions of NFEPP and fentanyl were compared in control mice and mice with dextran sodium sulfate colitis by measuring visceromotor responses to colorectal distension. Patch clamp and extracellular recordings were used to assess nociceptor activation. Defecation, respiration and locomotion were assessed. Colonic migrating motor complexes were assessed by spatiotemporal mapping of isolated tissue. NFEPP-induced MOPr signalling and trafficking were studied in human embryonic kidney 293 cells., Results: NFEPP inhibited visceromotor responses to colorectal distension in mice with colitis but not in control mice, consistent with acidification of the inflamed colon. Fentanyl inhibited responses in both groups. NFEPP inhibited the excitability of dorsal root ganglion neurons and suppressed mechanical sensitivity of colonic afferent fibres in acidified but not physiological conditions. Whereas fentanyl decreased defecation and caused respiratory depression and hyperactivity in mice with colitis, NFEPP was devoid of these effects. NFEPP did not affect colonic migrating motor complexes at physiological pH. NFEPP preferentially activated MOPr in acidified extracellular conditions to inhibit cAMP formation, recruit β-arrestins and evoke MOPr endocytosis., Conclusion: In a preclinical IBD model, NFEPP preferentially activates MOPr in acidified microenvironments of inflamed tissues to induce antinociception without causing respiratory depression, constipation and hyperactivity., Competing Interests: Competing interests: NWB is a founding scientist of Endosome Therapeutics Inc., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Up to 20% of people worldwide develop gastrointestinal symptoms following a meal 1 , leading to decreased quality of life, substantial morbidity and high medical costs. Although the interest of both the scientific and lay communities in this issue has increased markedly in recent years, with the worldwide introduction of gluten-free and other diets, the underlying mechanisms of food-induced abdominal complaints remain largely unknown. Here we show that a bacterial infection and bacterial toxins can trigger an immune response that leads to the production of dietary-antigen-specific IgE antibodies in mice, which are limited to the intestine. Following subsequent oral ingestion of the respective dietary antigen, an IgE- and mast-cell-dependent mechanism induced increased visceral pain. This aberrant pain signalling resulted from histamine receptor H 1 -mediated sensitization of visceral afferents. Moreover, injection of food antigens (gluten, wheat, soy and milk) into the rectosigmoid mucosa of patients with irritable bowel syndrome induced local oedema and mast cell activation. Our results identify and characterize a peripheral mechanism that underlies food-induced abdominal pain, thereby creating new possibilities for the treatment of irritable bowel syndrome and related abdominal pain disorders.

Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR 2 ) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR 2 -dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared with matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR 2 in Na V 1.8-positive neurons ( Par 2 Na v 1.8 ), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1. Par 2 Na v 1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR 2 -dependent hyperexcitability of trigeminal neurons from WT female mice. Par 2 deletion, LI-1, and inhibitors of adenylyl cyclase or protein kinase A (PKA) prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N terminus of PAR 2 at Asn 30 ↓Arg 31 , proximal to the canonical trypsin activation site. Lgmn activated PAR 2 by biased mechanisms in HEK293 cells to induce Ca 2+ mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not β-arrestin recruitment or PAR 2 endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR 2 by biased mechanisms that evoke cancer pain. SIGNIFICANCE STATEMENT Oral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR 2 ) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared with matched normal oral tissue. Lgmn evokes pain-like behavior through PAR 2 Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR 2 Inhibitors of adenylyl cyclase and protein kinase A (PKA) prevented the effects of Lgmn. Lgmn activated PAR 2 to induce calcium mobilization, cAMP formation, and activation of protein kinase D (PKD) and PKA, but not β-arrestin recruitment or PAR 2 endocytosis. Thus, Lgmn is a biased agonist of PAR 2 that evokes cancer pain., (Copyright © 2021 the authors.)

Whether G protein-coupled receptors signal from endosomes to control important pathophysiological processes and are therapeutic targets is uncertain. We report that opioids from the inflamed colon activate δ-opioid receptors (DOPr) in endosomes of nociceptors. Biopsy samples of inflamed colonic mucosa from patients and mice with colitis released opioids that activated DOPr on nociceptors to cause a sustained decrease in excitability. DOPr agonists inhibited mechanically sensitive colonic nociceptors. DOPr endocytosis and endosomal signaling by protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways mediated the sustained inhibitory actions of endogenous opioids and DOPr agonists. DOPr agonists stimulated the recruitment of Gα i/o and β-arrestin1/2 to endosomes. Analysis of compartmentalized signaling revealed a requirement of DOPr endocytosis for activation of PKC at the plasma membrane and in the cytosol and ERK in the nucleus. We explored a nanoparticle delivery strategy to evaluate whether endosomal DOPr might be a therapeutic target for pain. The DOPr agonist DADLE was coupled to a liposome shell for targeting DOPr-positive nociceptors and incorporated into a mesoporous silica core for release in the acidic and reducing endosomal environment. Nanoparticles activated DOPr at the plasma membrane, were preferentially endocytosed by DOPr-expressing cells, and were delivered to DOPr-positive early endosomes. Nanoparticles caused a long-lasting activation of DOPr in endosomes, which provided sustained inhibition of nociceptor excitability and relief from inflammatory pain. Conversely, nanoparticles containing a DOPr antagonist abolished the sustained inhibitory effects of DADLE. Thus, DOPr in endosomes is an endogenous mechanism and a therapeutic target for relief from chronic inflammatory pain., Competing Interests: Competing interest statement: N.W.B. is a founding scientist of Endosome Therapeutics Inc. Research in the laboratories of N.A.V., N.W.B., and D.P.P. is funded in part by Takeda Pharmaceuticals International.

The pathophysiology of irritable bowel syndrome and the detection of biomarkers of specific mechanisms and/or predictors of therapeutic response remain elusive. This roadblock reflects, in large part, the complexity and heterogeneity of the disorder. Recently, there has been growing evidence of a dietary and/or microbiome interaction with the host that may trigger symptoms in a subset of patients. While a number of techniques are available to examine these potential interactions, "omic" approaches such as metabolomics are becoming more widely used. Metabolomics measures hundreds and potentially thousands of known and unknown small molecule chemicals (metabolites) to provide a unique look into mechanisms that underlie symptom generation and potential predictors of therapeutic response. In this issue of the journal, Lee et al use nuclear magnetic resonance (NMR) to demonstrate the value of this approach to study IBS. This review examines the use of metabolomics to better understand IBS, focusing on what has been learned to date, practical and technical considerations, its potential for future research and how the study by Lee et al have contributed to these concepts., (© 2020 John Wiley & Sons Ltd.)

Background: The low FODMAP (fermentable oligo-, di-, monosaccharides, and polyols) diet reduces functional gastrointestinal symptoms (FGID) when implemented by dietitian-delivered education in clinical trials, but it is unknown how well the diet is followed in routine clinical care and if differences exist when implemented by physician or dietitian. This study aimed to evaluate the real-world experience of patients recommended the diet., Methods: This case-series interviewed FGID patients attending a gastroenterology clinic with previous recommendation to trial the low FODMAP diet, examining who recommended the diet and what their percentage improvement was. To evaluate implementation of the diet's 3 phases, questions were constructed based on current literature and clinical guidelines regarding length of initial restriction and food knowledge (Phase-1), number of foods re-challenged (Phase-2) and food re-introduction as tolerated (Phase-3). The comprehensive nutrition assessment questionnaire provided daily FODMAP intake. Data were analyzed using chi-squared tests., Key Results: In 80 patients (21 male), the diet was recommended by the gastroenterologist in 53%, general practitioner 22%, dietitian 9% and other 15%. 30% saw a dietitian for guidance. 55% reported a ≥50% symptom improvement. The diet was followed appropriately during Phase-1 by 78% (with vs without a dietitian, 96% vs 71%; P = .02), Phase-2 by 48% (70% vs 39%; P = .02) and Phase-3 by 40% (65% vs 29%; P < .01). A FODMAP intake of <12 g/d (considered therapeutic) was achieved by 44% (72% vs 31%; P < .01)., Conclusions & Inferences: Symptom improvement was reported in half of patients, but many did not reach the therapeutic FODMAP intake target, especially without dietitian education. Compliance was poor in Phase-2 and Phase-3 but improved with dietitian guidance., (© 2019 John Wiley & Sons Ltd.)

Background: A low fermentable carbohydrate (FODMAP) diet is used in quiescent inflammatory bowel disease when irritable bowel syndrome-like symptoms occur. There is concern that the diet could exacerbate inflammation by modifying microbiota and short-chain fatty acid (SCFA) production. We examined the effect of altering dietary FODMAP content on inflammation in preclinical inflammatory models., Methods: C57BL/6 mice were given 3% dextran sodium sulfate (DSS) in drinking water for 5 days and recovered for 3 weeks (postinflammatory, n = 12), or 5 days (positive-control, n = 12). Following recovery, DSS-treated or control mice (negative-control, n = 12) were randomized to 2-week low- (0.51 g/100 g total FODMAP) or high-FODMAP (4.10 g) diets. Diets mimicked human consumption containing fructose, sorbitol, galacto-oligosaccharide, and fructan. Colons were assessed for myeloperoxidase (MPO) activity and histological damage. Supernatants were generated for perforated patch-clamp recordings and cytokine measurement. Cecum contents were analyzed for microbiota, SCFA, and branched-chain fatty acids (BCFA). Data were analyzed by two-way ANOVA with Bonferroni., Key Results: Inflammatory markers were higher in the positive-control compared with negative-control and postinflammatory groups, but no differences occurred between the two diets within each treatment (MPO P > .99, histological scores P > .99, cytokines P > .05), or the perforated patch-clamp recordings (P > .05). Microbiota clustered mainly based on DSS exposure. No difference in SCFA content occurred. Higher total BCFA occurred with the low-FODMAP diet in positive-control (P < .01) and postinflammatory groups (P < .01)., Conclusions and Inferences: In this preclinical study, reducing dietary FODMAPs did not exacerbate nor mitigate inflammation. Microbiota profile changes were largely driven by inflammation rather than diet. Low FODMAP intake caused a shift toward proteolytic fermentation following inflammation., (© 2019 John Wiley & Sons Ltd.)

Neutrophil elastase is a serine protease that has been implicated in the pathogenesis of inflammatory bowel disease. Due to post-translational control of its activation and high expression of its inhibitors in the gut, measurements of total expression poorly reflect the pool of active, functional neutrophil elastase. Fluorogenic substrate probes have been used to measure neutrophil elastase activity, though these tools lack specificity and traceability. PK105 is a recently described fluorescent activity-based probe, which binds to neutrophil elastase in an activity-dependent manner. The irreversible nature of this probe allows for accurate identification of its targets in complex protein mixtures. We describe the reactivity profile of PK105b, a new analogue of PK105, against recombinant serine proteases and in tissue extracts from healthy mice and from models of inflammation induced by oral cancer and Legionella pneumophila infection. We apply PK105b to measure neutrophil elastase activation in an acute model of experimental colitis. Neutrophil elastase activity is detected in inflamed, but not healthy, colons. We corroborate this finding in mucosal biopsies from patients with ulcerative colitis. Thus, PK105b facilitates detection of neutrophil elastase activity in tissue lysates, and we have applied it to demonstrate that this protease is unequivocally activated during colitis.

Proteases sustain hyperexcitability and pain by cleaving protease-activated receptor-2 (PAR 2 ) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR 2 coupling to Gα q , Gα s , and β-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR 2 at distinct sites and activate it by biased mechanisms that induce coupling to Gα s , but not to Gα q or β-arrestins. Because proteases activate PAR 2 by irreversible cleavage, and activated PAR 2 is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR 2 from the Golgi apparatus or de novo synthesis of new receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR 2 -dependent activation of protein kinase D (PKD) in the Golgi of HEK293 cells, in which PKD regulates protein trafficking. The proteases stimulated translocation of the PKD activator Gβγ to the Golgi, coinciding with PAR 2 mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR 2 -Kaede fusion protein from the Golgi to the plasma membrane of KNRK cells. After incubation of HEK293 cells and dorsal root ganglia neurons with CS, NE, or trypsin, PAR 2 responsiveness initially declined, consistent with PAR 2 cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gβγ, and protein translation inhibited recovery of PAR 2 responsiveness. PKD and Gβγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR 2 by canonical and biased mechanisms stimulate PKD in the Golgi; PAR 2 mobilization and de novo synthesis repopulate the cell surface with intact receptors and sustain nociceptive signaling by extracellular proteases., (© 2019 Zhao et al.)

Background: Gastrointestinal (GI) motility and functional disorders comprise over two-third of referrals to GI specialists yet training programs are disproportionately focused on endoscopy, inflammatory bowel disease and liver disease. Trainees at many centres receive minimal or no formal training in motility disorders and have little or no exposure to motility testing. Our purpose was to develop an educational intervention to address this learning need., Methods: We designed a formal training program comprised of didactic sessions, workshops and hands-on motility sessions with live demonstrations designed to be held over the course of a weekend. Faculty for the course were experienced GI motility experts from across Canada. Resident trainees from all Canadian GI fellowship programs were invited to attend. Pre- and post-tests were administered to measure the baseline learning needs and the impact of the program. Course evaluations were completed by attendees., Results: Three annual courses were offered over the past 3 years. Both adult and paediatric gastroenterology trainees attended the programs. The majority of training programs from Canada were represented. Baseline testing of attendees revealed a fundamental lack of understanding of GI motility concepts and their clinical implications. Postcourse test scores demonstrated a significant improvement in motility knowledge. Course evaluations of the content and faculty presentations received uniformly positive reviews., Conclusions: There is a pervasive lack of clinical knowledge of GI motility among Canadian GI subspecialty trainees. A focused weekend intensive course is one step in addressing this learning need., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Canadian Association of Gastroenterology.)

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS), but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight, we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin β7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore, phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFα secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFα production, while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms, but not markers of immune activation, decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation, but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms.

Purpose: Examine the association between bulky DNA adduct levels in colon mucosa and colorectal adenoma prevalence, and explore the correlation between adduct levels in leukocytes and colon tissue., Methods: Bulky DNA adduct levels were measured using 32 P-postlabelling in biopsies of normal-appearing colon tissue and blood donated by 202 patients. Multivariable logistic regression was used to examine associations between DNA adducts, and interactions of DNA adduct-DNA repair polymorphisms, with the prevalence of colorectal adenomas. Correlation between blood and tissue levels of DNA adducts was evaluated using Spearman's correlation coefficient., Results: An interaction between bulky DNA adduct levels and XPA rs1800975 on prevalence of colorectal adenoma was observed. Among individuals with lower DNA repair activity, increased DNA adduct levels were associated with increased colorectal adenoma prevalence (OR = 1.41 per SD increase, 95%CI: 0.92-2.18). Conversely, among individuals with normal DNA activity, an inverse association was observed (OR = 0.60 per SD increase, 95%CI: 0.34-1.07). Blood and colon DNA adduct levels were inversely correlated (ρ = -0.20)., Conclusions: Among genetically susceptible individuals, higher bulky DNA adducts in the colon was associated with the prevalence of colorectal adenomas. The inverse correlation between blood and colon tissue measures demonstrates the importance of quantifying biomarkers in target tissues.

Telomere length has been associated with risk of several cancers. However, studies of the relationship between telomere length and colorectal cancer risk have been inconsistent. This study examined the relationship between telomere length in normal colon tissue and the prevalence of colorectal adenoma, a precursor to colorectal cancer. This nested case-control study consisted of 85 patients aged 40 to 65 undergoing a screening colonoscopy: 40 cases with adenoma(s) detected at colonoscopy and 45 controls with normal colonoscopy. During the colonoscopy, two pinch biopsies of healthy, normal appearing mucosa were obtained from the descending colon. Relative telomere length (rTL) was quantified in DNA extracted from colon mucosa using quantitative real-time PCR. Logistic regression was used to assess the relationship between telomere length and adenoma prevalence and estimate odds ratios and 95% confidence intervals. rTL was significantly longer in colon tissue of individuals with adenomas compared to healthy individuals (p = 0.008). When rTL was categorized into quartiles according to the distribution of rTL among controls, individuals with the longest telomeres had increased odds of adenoma when compared to individuals with shortest telomeres (OR = 4.58, 95% CI: 1.19, 17.7). This study suggests that long telomeres in normal colon tissue are associated with increased colorectal cancer risk., Competing Interests: The authors have declared that no competing interests exist.

Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR 2 ), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR 2 -dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR 2 , which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR 2 , which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR 2 -dependent hyperexcitability of nociceptors, and PAR 2 association with β-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR 2 A cholestanol-conjugated PAR 2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR 2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR 2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases., Competing Interests: Conflict of interest statement: P.M. and G.A.H. work for Takeda Pharmaceuticals, Inc. Research in N.W.B.’s laboratory is supported in part by Takeda Pharmaceuticals, Inc. N.W.B. and G.A.H. are founding scientists of Endosome Therapeutics, Inc.

Background: Functional gastrointestinal symptoms in irritable bowel syndrome (IBS) and quiescent inflammatory bowel disease (IBD) cause significant morbidity and a reduction in quality of life. Multiple dietary therapies are now available to treat these symptoms, but supporting evidence for many is limited. In addition to a further need for studies demonstrating efficacy and mechanism of action of dietary therapies, the risk of nutritional inadequacy, alterations to the microbiome and changes in quality of life are key concerns requiring elucidation. Identifying predictors of response to dietary therapy is an important goal as management could be tailored to the individual to target specific dietary components, and thereby reduce the level of dietary restriction necessary., Purpose: This review discusses the available dietary therapies to treat symptoms in patients with IBS and patients with quiescent IBD suffering from IBS symptoms, with the aim to understand where current dietary evidence lies and how to move forward in dietary research in this field., (© 2017 John Wiley & Sons Ltd.)

Aims and Background: Psychological stress accompanies chronic inflammatory diseases such as IBD, and stress hormones can exacerbate pain signalling. In contrast, the endogenous opioid system has an important analgesic action during chronic inflammation. This study examined the interaction of these pathways., Methods: Mouse nociceptive dorsal root ganglia (DRG) neurons were incubated with supernatants from segments of inflamed colon collected from patients with chronic UC and mice with dextran sodium sulfate (cDSS)-induced chronic colitis. Stress effects were studied by adding stress hormones (epinephrine and corticosterone) to dissociated neurons or by exposing cDSS mice to water avoidance stress. Changes in excitability of colonic DRG nociceptors were measured using patch clamp and Ca 2+ imaging techniques., Results: Supernatants from patients with chronic UC and from colons of mice with chronic colitis caused a naloxone-sensitive inhibition of neuronal excitability and capsaicin-evoked Ca 2+ responses. Stress hormones decreased signalling induced by human and mouse supernatants. This effect resulted from stress hormones signalling directly to DRG neurons and indirectly through signalling to the immune system, leading to decreased opioid levels and increased acute inflammation. The net effect of stress was a change endogenous opioid signalling in DRG neurons from an inhibitory to an excitatory effect. This switch was associated with a change in G protein-coupled receptor excitatory signalling to a pathway sensitive to inhibitors of protein kinase A-protein, phospholipase C-protein and G protein βϒ subunits., Conclusions: Stress hormones block the inhibitory actions of endogenous opioids and can change the effect of opioid signalling in DRG neurons to excitation. Targeting these pathways may prevent heavy opioid use in IBD., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 μm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K + currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain. SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human donor signal to DRG neurons. Their secretory products contain serine proteases that suppress excitability via activation of protease-activated receptor-4. Moreover, from this community of commensal microbes, Faecalibacterium prausnitzii strain 16-6-I 40 fastidious anaerobe agar had the greatest effect. Our study suggests that therapies that induce or correct microbial dysbiosis may affect the excitability of primary afferent neurons, many of which are nociceptive. Furthermore, identification of the bacterial strains capable of suppressing sensory neuron excitability, and their mechanisms of action, may allow therapeutic relief for patients with gastrointestinal diseases associated with pain., (Copyright © 2017 the authors 0270-6474/17/3711758-11$15.00/0.)

Background: Changes to the structure and function of the innervation of the gut contribute to symptom generation in inflammatory bowel diseases (IBD). However, delineation of the mechanisms of these effects has proven difficult. Previous work on sympathetic neurons identified interleukin (IL)-17A as a novel neurotrophic cytokine. Since IL-17A is involved in IBD pathogenesis, we tested the hypothesis that IL-17A contributes to neuroanatomical remodeling during IBD., Methods: Immunohistochemistry for tyrosine hydroxylase was used to identify sympathetic axons in mice with dextran sulphate sodium (DSS)-induced colitis and controls. Axon outgrowth from sympathetic neurons in response to incubation in cytokines or endoscopic patient biopsy supernatants was quantified., Key Results: DSS-induced colitis led to an increase in tyrosine hydroxylase immunoreactivity in the inflamed colon but not the spleen. Colonic supernatants from mice with colitis and biopsy supernatants from Crohn's disease patients increased axon outgrowth from mouse sympathetic neurons compared to supernatants from uninflamed controls. An antibody that neutralized IL-17A blocked the ability of DSS-induced colitis and Crohn's disease supernatants to induce axon extension., Conclusions and Inferences: These findings identify IL-17A as a potential mediator of neuroanatomical remodeling of the gut innervation during IBD., (© 2017 John Wiley & Sons Ltd.)

Introduction: Meat consumption is a risk factor for colorectal cancer. This research investigated the relationship between meat-derived carcinogen exposure and bulky DNA adduct levels, a biomarker of DNA damage, in colon mucosa., Methods: Least squares regression was used to examine the relationship between meat-derived carcinogen exposure (PhIP and meat mutagenicity) and bulky DNA adduct levels in normal-appearing colon tissue measured using 32 P-postlabelling among 202 patients undergoing a screening colonoscopy. Gene-diet interactions between carcinogen exposure and genetic factors relevant to biotransformation and DNA repair were also examined. Genotyping was conducting using the MassARRAY ® iPLEX ® Gold SNP Genotyping assay., Results: PhIP and higher meat mutagenicity exposures were not associated with levels of bulky DNA adducts in colon mucosa. The XPC polymorphism (rs2228001) was found to associate with bulky DNA adduct levels, whereby genotypes conferring lower DNA repair activity were associated with higher DNA adduct levels than the normal activity genotype. Among individuals with genotypes associated with lower DNA repair (XPD, rs13181 and rs1799179) or detoxification activity (GSTP1, rs1695), higher PhIP or meat mutagenicity exposures were associated with higher DNA adduct levels. Significant interactions between the XPC polymorphism (rs2228000) and both dietary PhIP and meat mutagenicity on DNA adduct levels was observed, but associations were inconsistent with the a priori hypothesized direction of effect., Conclusion: Exposure to meat-derived carcinogens may be associated with increased DNA damage occurring directly in the colon among genetically susceptible individuals., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Irritable bowel syndrome (IBS) patients increasingly seek out acupuncture therapy to alleviate symptoms, but it is unclear whether the benefit is due to a treatment-specific effect or a placebo response. This study examined whether true acupuncture is superior to sham acupuncture in relieving IBS symptoms and whether benefits were linked to purported acupuncture mechanisms., Methods: A double blind sham controlled acupuncture study was conducted with Rome I IBS patients receiving twice weekly true acupuncture for 4 weeks (n=43) or sham acupuncture (n=36). Patients returned at 12 weeks for a follow-up review. The primary endpoint of success as determined by whether patients met or exceeded their established goal for percentage symptom improvement. Questionnaires were completed for symptom severity scores, SF-36 and IBS-36 QOL tools, McGill pain score, and Pittsburg Sleep Quality Index. A subset of patients underwent barostat measurements of rectal sensation at baseline and 4 weeks., Key Results: A total of 53% in the true acupuncture group met their criteria for a successful treatment intervention, but this did not differ significantly from the sham group (42%). IBS symptom scores similarly improved in both groups. Scores also improved in the IBS-36, SF-36, and the Pittsburg Sleep Quality Index, but did not differ between groups. Rectal sensory thresholds were increased in both groups following treatment and pain scores decreased; however, these changes were similar between groups., Conclusions & Inferences: The lack of differences in symptom outcomes between sham and true treatment acupuncture suggests that acupuncture does not have a specific treatment effect in IBS., (© 2017 John Wiley & Sons Ltd.)

Animal studies have led to significant advances in our understanding of pain mechanisms in the intestine that could lead to altered signaling in disorders such as irritable bowel syndrome. However, how these translate to the human afferent nervous system is unclear. Recent studies have demonstrated that it is possible to use a variety of techniques, including electrophysiological recordings, to begin to examine these concepts in humans. This mini-review examines these studies to explore how well animal studies translate to humans suffering from irritable bowel syndrome, highlights some of the advantages and technical limitations of these approaches, and identifies some priorities for future studies using human tissues., (Copyright © 2017 the American Physiological Society.)

Agonist-evoked endocytosis of G protein-coupled receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled receptors from intracellular stores are unexplored. Protease-activated receptor-2 (PAR2) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2-stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca(2+) signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: A relationship between stress and the symptoms of irritable bowel syndrome (IBS) has been well established but the cellular mechanisms are poorly understood. Therefore, we investigated effects of stress and stress hormones on colonic descending inhibition and transit in mouse models and human tissues., Methods: Stress was applied using water avoidance stress (WAS) in the animal model or mimicked using stress hormones, adrenaline (5 nM), and corticosterone (1 μM). Intracellular recordings were obtained from colonic circular smooth muscle cells in isolated smooth muscle/myenteric plexus preparations and the inhibitory junction potential (IJP) was elicited by nerve stimulation or balloon distension oral to the site of recording., Key Results: Water avoidance stress increased the number of fecal pellets compared to control (p < 0.05). WAS also caused a significant increase in IJP amplitude following balloon distension. Stress hormones also increased the IJP amplitude following nerve stimulation and balloon distension (p < 0.05) in control mice but had no effect in colons from stressed mice. No differences were observed with application of ATP between stress and control tissues, suggesting the actions of stress hormones were presynaptic. Stress hormones had a large effect in the nerve stimulated IJP in human colon (increased >50%). Immunohistochemical studies identified alpha and beta adrenergic receptor immunoreactivity on myenteric neurons in human colon., Conclusions & Inferences: These studies suggest that WAS and stress hormones can signal via myenteric neurons to increase inhibitory neuromuscular transmission. This could lead to greater descending relaxation, decreased transit time, and subsequent diarrhea., (© 2016 John Wiley & Sons Ltd.)

Background: Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis., Methods: We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader., Key Results: NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p < 0.05 at 200 min and p < 0.01 at 220-240 min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p < 0.05 at 140 min, p < 0.01 at 180 min, p < 0.001 at 200-240 min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r = 0.77, p = 0.017)., Conclusions & Inferences: Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders., (© 2015 John Wiley & Sons Ltd.)

Proteases that cleave protease-activated receptor-2 (PAR(2)) at Arg(36)↓Ser(37) reveal a tethered ligand that binds to the cleaved receptor. PAR(2) activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR(2) at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR(2)-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR(2), causes inflammation and pain by activating PAR2 and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR(2) at Ala(66)↓Ser(67) and Ser(67)↓Val(68). Elastase stimulated PAR(2)-dependent cAMP accumulation and ERK1/2 activation, but not Ca(2+) mobilization, in KNRK cells. Elastase induced PAR(2) coupling to Gαs but not Gαq in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK(2)) or β-arrestin to PAR(2), consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR(2)-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR(2)-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR(2)- and TRPV4-mediated influx of extracellular Ca(2+) in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR(2)- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR(2) causes Gαs-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR(2) as a mediator of protease-driven inflammation and pain., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: The development of postinfectious-irritable bowel syndrome is associated with psychological stress but this relationship is poorly understood. The mouse Citrobacter rodentium model enhances the postinfectious excitability of colonic nociceptors, which can be further amplified by water-avoidance stress (WAS). This study tested whether concurrent infectious colitis and chronic stress enhance and sustain nociceptor excitability more than stress after resolution of infection., Methods: Male C57 mice were gavaged with C. rodentium. WAS (1 h/day) was performed at different time-points relative to the infection. After the final session of WAS, T9-T13 colonic-projecting DRG neurons were isolated, cultured overnight and patch-clamped to assess excitability. To investigate potential mechanisms, histological damage scores and colonic cytokine production were assessed., Key Results: WAS more than 30 days after C. rodentium infection produced no greater DRG excitability than WAS in uninfected mice. However, when overlapped with chronic stress (3 sessions of WAS; 7 days before, 9 days during and 9 days after C. rodentium or sham gavage), C. rodentium significantly enhanced DRG excitability vs saline-gavaged chronically stressed mice. Bodyweights and colonic damage scores were unchanged. Both WAS and C. rodentium gavage were found to significantly alter colonic cytokines at postinfection day 30., Conclusions & Inferences: Chronic stress and infectious colitis combine in an additive manner to heighten and prolong the sensitivity of visceral nociceptors. The effect relies on temporal coincidence of stress and infection, does not involve substantial exacerbation of inflammation, and may involve combined direct stress hormone and immune signaling on DRG neurons., (© 2014 John Wiley & Sons Ltd.)

Exposure to heterocyclic aromatic amines (HAAs), carcinogens produced when meat is cooked at high temperatures, is an emerging risk factor for colorectal cancer (CRC). In a cross-sectional study of 342 patients undergoing a screening colonoscopy, the role of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), the three most abundant HAAs found in cooked meats, and total mutagenic activity in cooked meats were examined in relation to colorectal adenoma risk. Given that genetic differences in the ability to biotransform HAAs and repair DNA are postulated to modify the HAA-CRC relationship, gene-diet interactions were also examined. Among the total study population, no relationships were observed between dietary HAAs or meat mutagenicity, and colorectal adenoma risk; however, in males, positive associations between dietary HAAs/meat mutagenicity exposures and adenoma risk were suggestive of a relationship. In a separate analysis, polymorphisms in CYP1B1 were found to be associated with colorectal adenoma risk. Additionally, gene-diet interactions were observed for dietary PhIP and polymorphisms in CYP1B1 and XPD, dietary DiMeIQx and XPD polymorphisms, and meat mutagenicity exposure and CYP1B1 polymorphisms. Overall, increased colorectal adenoma risk was observed with higher HAA/meat mutagenicity exposures among those with polymorphisms which confer greater activity to biotransform HAAs and/or lower ability to repair DNA. This research supports the link between dietary HAAs and genetic susceptibility in colorectal adenoma etiology. The vast majority of CRCs arise from colorectal adenomas; thus, the results of this study suggest that changes in meat preparation practices limiting the production of HAAs may be beneficial for CRC prevention.

Inflammation involving the helper T cell 17 (Th17) subset of lymphocytes has been implicated in a number of diseases that affect the nervous system. As the canonical cytokine of Th17 cells, interleukin 17A (IL-17A) is thought to contribute to these neuroimmune interactions. The main receptor for IL-17A is expressed in many neural tissues. IL-17A has direct effects on neurons but can also impact neural function via signaling to satellite cells and immune cells. In the central nervous system, IL-17A has been associated with neuropathology in multiple sclerosis, epilepsy syndromes and ischemic brain injury. Effects of IL-17A at the level of dorsal root ganglia and the spinal cord may contribute to enhanced nociception during neuropathic and inflammatory pain. Finally, IL-17A plays a role in sympathetic axon growth and regeneration of damaged axons that innervate the cornea. Given the widespread effects of IL-17A on neural tissues, it will be important to determine whether selectively mitigating the damaging effects of this cytokine while augmenting its beneficial effects is a possible strategy to treat inflammatory damage to the nervous system., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR2) at R(36)↓S(37) and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR2 at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR2 at E(56)↓T(57), which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR2 coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca(2+), activate ERK1/2, recruit β-arrestins, or induce PAR2 endocytosis. Cat-S caused PAR2-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR2 or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR2 antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR2 in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR2 that causes PAR2- and TRPV4-dependent inflammation and pain. They expand the role of PAR2 as a mediator of protease-driven inflammatory pain., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: The methylation of DNA is recognized as a key epigenetic mechanism and evidence for its role in the development of several malignancies is accumulating. We evaluated the relationship between global methylation in DNA derived from normal appearing colon mucosal tissue and blood leukocytes, and colorectal adenoma risk., Methods: Patients, aged 40 to 65, scheduled for a screening colonoscopy were recruited. During the colonoscopy, two pinch biopsies of healthy, normal appearing mucosa were obtained from the descending colon. A fasting blood sample was also collected. The methylation status of LINE-1 (long interspersed nuclear element-1) repetitive sequences, as a surrogate measure of global methylation, was quantified in DNA extracted from normal colon mucosa and blood leukocytes. Statistical analysis of the relationship between global DNA methylation and adenoma risk was conducted on 317 participants, 108 subjects with at least one pathologically confirmed adenoma and 209 subjects with a normal colonoscopy., Results: A statistically significant inverse relationship was observed between LINE-1 methylation in colon tissue DNA and adenoma risk for males and for both sexes combined for the lowest methylation quartile compared to the highest (adjusted ORs = 2.94 and 2.26 respectively). For blood, although the overall pattern of odds ratio estimates was towards an increase in risk for lower methylation quartiles compared to the highest methylation quartile, there were no statistically significant relationships observed. A moderate correlation was found between LINE-1 methylation levels measured in tissue and blood (Pearson correlation 0.36)., Conclusions: We observed that lower levels of LINE-1 DNA methylation in normal appearing background colon mucosa were associated with increased adenoma risk for males, and for both sexes combined. Though these findings provide some support for a relationship between LINE-1 DNA methylation in colon mucosal tissue and adenoma risk, large prospective cohort studies are needed to confirm results. Until such investigations are done, the clinical usefulness of LINE-1 methylation as a biomarker of increased adenoma risk is uncertain. Regardless, this study contributes to a better understanding of the role of global DNA methylation as an early event in CR carcinogenesis with implications for future etiologic research.

There is increasing interest in clarifying the role of global DNA methylation levels in colorectal cancer (CRC) etiology. Most commonly, in epidemiologic studies, methylation is measured in DNA derived from blood leukocytes as a proxy measure of methylation changes in colon tissue. However, little is known about the correlations between global methylation levels in DNA derived from colon tissue and more accessible tissues such as blood or buccal cells. This cross-sectional study utilized DNA samples from a screening colonoscopy population to determine to what extent LINE-1 methylation levels (as a proxy for genome-wide methylation) in non-target tissue (e.g., blood, buccal cells) reflected methylation patterns of colon mucosal tissue directly at risk of developing CRC. The strongest Pearson correlation was observed between LINE-1 methylation levels in buccal and blood leukocyte DNA (r = 0.50; N = 67), with weaker correlations for comparisons between blood and colon tissue (r = 0.36; N = 280), and buccal and colon tissue (r = 0.27; N = 72). These findings of weak/moderate correlations have important implications for interpreting and planning future investigations of epigenetic markers and CRC risk.

Sensitization of dorsal root ganglia (DRG) neurons is an important mechanism underlying the expression of chronic abdominal pain caused by intestinal inflammation. Most studies have focused on changes in the peripheral terminals of DRG neurons in the inflamed intestine but recent evidence suggests that the sprouting of central nerve terminals in the dorsal horn is also important. Therefore, we examine the time course and reversibility of changes in the distribution of immunoreactivity for substance P (SP), a marker of the central terminals of DRG neurons, in the spinal cord during and following dextran sulphate sodium (DSS)-induced colitis in mice. Acute and chronic treatment with DSS significantly increased SP immunoreactivity in thoracic and lumbosacral spinal cord segments. This increase developed over several weeks and was evident in both the superficial laminae of the dorsal horn and in lamina X. These increases persisted for 5 weeks following cessation of both the acute and chronic models. The increase in SP immunoreactivity was not observed in segments of the cervical spinal cord, which were not innervated by the axons of colonic afferent neurons. DRG neurons dissociated following acute DSS-colitis exhibited increased neurite sprouting compared with neurons dissociated from control mice. These data suggest significant colitis-induced enhancements in neuropeptide expression in DRG neuron central terminals. Such neurotransmitter plasticity persists beyond the period of active inflammation and might contribute to a sustained increase in nociceptive signaling following the resolution of inflammation.