Cathepsin S is a cysteine protease that has been implicated in inflammatory bowel diseases (IBD) for its ability to promote visceral pain. Given its pro-inflammatory roles, we hypothesized that cathepsin S would drive other symptoms associated with IBD. Using activity-based probes, we investigated cysteine cathepsin activation in human and murine colitis. We observed a significant increase in fecal cathepsin S in patients with ulcerative colitis compared to healthy controls, while cathepsin S in mucosal biopsies was unchanged. Mice with experimental colitis exhibited a modest increase in mucosal activity of both cathepsin S and X compared to naïve mice. Luminal secretion of cathepsin S was dramatically increased upon colitis induction, although differences between mouse colonies were observed. To investigate the contribution of cathepsin S and cathepsin X to colitis, we induced colitis in cathepsin-deficient mice. Cathepsin X-deficient mice exhibited no clear differences in disease indicators compared to wild-type mice. While cathepsin S-deficient mice exhibited less rectal bleeding, less splenomegaly and marginally improved histological scores, weight loss, diarrhea, colon shortening, and myeloperoxidase activity were not significantly different from wild-type mice. To determine whether pharmacologic inhibition of cathepsin S activity would ameliorate symptoms of colitis, a reversible inhibitor LY3000328 was administered to mice at the initiation of colitis. LY3000328 provoked a clear upregulation of cathepsin S and L activity in the mucosa, most likely through a compensatory mechanism. This increase in protease activity was associated with exacerbated histological scores and slight splenomegaly. Collectively, these results suggest that cathepsin S, but not cathepsin X, may contribute to some of the symptoms of experimental colitis. While cathepsin S has potential to be a therapeutic target in colitis, improved strategies to sustain its inhibition are required in future.

Background: Irritable bowel syndrome (IBS) is a chronic abdominal pain disorder that affects women twice as often as men. While luminal mediators of both host and bacterial origin have been implicated in modulating abdominal pain in IBS patients, gonadal hormones have also been shown to influence pain signaling. Estrogen has been identified as a pronociceptive mediator that can modulate central and peripheral neural pathways. Given this, we hypothesized that the estrous cycle modulates sensory neuronal excitability, thereby altering the sensitivity to luminal mediators and this contributes to the female predominance of IBS. Aim: Identify the impact of the estrous cycle on nociceptive signaling and compare the effects of fecal supernatants (FS) from male and female IBS patients on abdominal pain pathways. Methods: Current clamp recordings measured rheobase and voltage clamp measured voltage-gated Na+ current in thoracolumbar dorsal root ganglia (DRG) neurons. FS from male and female IBS patients reporting high levels of abdominal pain were used. FS were perfused through male, female, and ovariectomized murine colonic preparations while performing extracellular colonic afferent nerve recordings to measure changes in action potential frequency during spontaneous firing and in response to colonic distension. Phase of estrous cycle in female mice was determined through analysis of vaginal swabs. Ovariectomies were performed 4 weeks prior to assays. Results: Current clamp recordings revealed an increase in excitability due to a 20% reduction in rheobase in DRG neurons taken from proestrus/estrus female mice compared to males, metestrus/diestrus females and ovariectomized females (p < 0.05). Voltage-gated Na+ current density was increased by 40% in neurons from proestrus/estrus mice compared to metestrus/diestrus female and male mice (p < 0.01). Extracellular afferent nerve recordings revealed that FS from female IBS patients reporting high abdominal pain (N=3) increased afferent nerve discharge (p < 0.05) in proestrus/estrus female mice by 70%. Single unit analysis of nociceptive axons showed that their activation was increased over 50% following FS perfusion. H owever, this excitatory effect was abolished in ovariectomized mice. Interestingly, FS from male IBS patients reporting high abdominal pain (N=4) had no effect in male mice while female IBS patient FS (N = 6) increased afferent nerve discharge (p< 0.05). Conclusion: This work suggests that the estrous cycle impacts abdominal pain signaling, which may contribute to the female predominance of IBS. This work is funded by CIHR and The Weston Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract Background Abdominal pain is a debilitating symptom in patients with inflammatory bowel disease. Previously we have shown that combining sub-analgesic doses of cannabinoid 1 receptor (CB1R), but not cannabinoid 2 receptor (CB2R), and mu-opioid receptor (MOR) agonists synergistically inhibits colonic nociception in healthy mice. However, it is unknown whether this combination has analgesic efficacy in a pre-clinical model of colitis. Aims To determine the effects of combining sub-analgesic doses of CBR and MOR agonists on colonic nociception during acute colitis. Methods Colitis was induced in male and female C57BL/6 mice with 2.5% dextran sulfate sodium in drinking water. Extracellular afferent nerve recordings were obtained from ex vivo flat sheet preparations of mouse distal colon. Mechanosensitivity of single afferent axons was assessed via probing of the colon with a 1g von Frey hair before and after superfusion of agonists of CB1R or CB2R plus MOR. To examine effects in vivo, visceromotor response (VMR) to colorectal distention (volume range 20-80 µL) was measured via electromyography. Mice were injected intraperitoneally with vehicle, or a combination of CB1R or CB2R agonist plus morphine 30-minutes prior to VMR experiment. Data were analyzed using a one or two-way ANOVA with Bonferroni test. N denotes number of mice; n denotes number of single afferent axons. Results In afferent nerve recordings, in contrast to healthy mice, the CB2R agonist HU-308 (1 µM and 3 µM) inhibited colonic mechanosensitivity in mice with colitis (1 µM: p<0.01, N=5, n=12; 3 µM: p<0.01, N=7, n=10); a lower concentration (300 nM) had no effect (p=0.52, N=6, n=10). The CB1R agonist ACEA (10 µM) reduced mechanosensitivity during acute colitis (p<0.05, n=11, N=6), whereas 100 nM (p>0.99, n=7, N=5) and 1 µM (p=0.25, n=10, N=6) had no effect. A combination of sub-analgesic concentrations of ACEA (100 nM) and DAMGO (MOR agonist, 1 nM) inhibited colonic mechanosensitivity in healthy mice (p<0.01, N=4, n=8) and during acute colitis (p<0.05, N=6, n=8). While a combination of sub-analgesic concentrations of HU-308 (300 nM) and DAMGO (1 nM) had no effect in healthy mice (p=0.70, N=4, n=8), it inhibited colonic mechanosensitivity during acute colitis (p<0.01, N=8, n=15). In VMR experiments, a combination of a sub-analgesic dose of ACEA (0.3 mg/kg) with morphine (0.3 mg/kg) reduced VMR (p<0.01, N=7) during acute colitis. Similarly, a combination of a sub-analgesic dose of HU-308 (1 mg/kg) and morphine (0.3 mg/kg) reduced VMR (p<0.01, N=6) during acute colitis. Conclusions A CB2R agonist inhibits colonic nociception during acute colitis, but not in healthy mice. A sub-analgesic combination of CB1R and MOR agonists can inhibit pain in healthy and inflamed mice, while combining sub-analgesic CB2R and MOR agonists is only inhibitory during acute colitis. Funding Agencies NRC

Abstract Background Intestinal microbiota have been implicated in the expression of irritable bowel syndrome (IBS) as patients present with altered gut microbial profiles and microbial metabolic activity. We have previously identified bacterial histamine to strongly influence mast cell accumulation through only IBS activation of the H4 receptor, leading to visceral hyperalgesia in a subset of patients with IBS. We hypothesize that a subset of IBS patients with high histamine-producing microbiota exhibit an aberrant histamine metabolism. Investigating the microbiota-driven pathways involved in histamine metabolism is key to understanding abdominal pain pathophysiology in IBS patients. Aims 1. To study whether variations of histamine levels are due to bacterial metabolism using in vitro and in vivo approaches. 2. To identify the prevalence of high histamine-producing and histamine-degrading bacteria in a clinical cohort via in silico analyses. Methods Using in vitro approaches, stool samples from healthy control (HC) donors and IBS patients were inoculated in minimal media in aerobic/anaerobic conditions, with/without excess histidine or added histamine. Bacterial histamine production and degradation were assessed in culture supernatants by ELISA. After identification through Sanger sequencing, individual colony capacity to degrade and produce histamine was assessed. Host and microbial contributions to histamine metabolism will be identified through analyses of germ-free mice colonized with IBS and HC stool samples. Results IBS patients (n=23) tested were found to consistently produce higher levels of histamine compared to HC (n=3). Among the tested isolated colonies from IBS patients (n=179) 61% produced histamine compared to 33% of HC (n=54), and 20% degraded histamine compared to 11% of HC. Of these colonies, 13% of only IBS isolates demonstrated the capacity to both degrade and produce histamine. Facultative anaerobes were found to possess both higher production and degradation capacity. Both pH and histamine concentration determine bacterial ability to produce or degrade histamine. Conclusions Based on these findings, both healthy and IBS individuals exhibit varying levels of histamine production/degradation, most prominently through facultative anaerobes. The intestinal environment and bacterial community composition are major regulators of bacterial histamine metabolism. The observed in vitro capacity to produce/degrade histamine, and its biological implications, will be confirmed with gnotobiotic humanized mice, ultimately aiding in designing microbiota-directed therapies for the management of visceral hypersensitivity. Funding Agencies CIHRFarncombe Innovation Fund

Abstract Background An impairment of vagally-mediated satiety signalling has been implicated in the caloric imbalance that leads to weight gain during obesity. Previous studies have suggested that a reduction in the excitability of vagal afferent neurons with cell bodies in nodose ganglia (NG) was responsible, but the cellular mechanisms are unclear. Host and bacterially derived mediators present in the small intestine and stool provide a physiologically relevant model to help elucidate the role luminal mediators play in modulating vagal afferent neuronal excitability. Aims We hypothesize that the microbiota of obese individuals and mice produce mediators that impair NG neuron excitability and satiety in mice. Methods Perforated patch clamp was used to measure the excitability of NG neurons following exposure to human and mouse fecal supernatants (FS), mouse jejunal supernatants (JS), and mice serum samples. Human FS were from ampersand:003E 5 healthy human donors or FS from ampersand:003E 5 obese donors. Mice FS, JS and serum samples were collected from ampersand:003E 5 obese mice fed a high-fat diet and ampersand:003E 5 control mice fed a normal diet. Results NG neurons incubated in FS from obese participants were significantly less excitable (rheobase was 30% higher and action potential discharge at 2x rheobase was 50% lower) than NG neurons exposed to FS from non-obese participants. NG neurons incubated in FS or JS from obese mice were also significantly less excitable (rheobase was 65% higher and action potential discharge at 2x rheobase was 50% lower) than NG neurons incubated with FS or JS from control mice. Lastly, NG neurons incubated with obese mouse serum were significantly less excitable (rheobase was 50% higher and action potential discharge at 2x rheobase was 80% lower) than NG neurons incubated with serum from control mice. We then attempted to identify mediators that may account for this inhibitory effect by using receptor antagonists that block GABA, ghrelin, and orexin signalling. Ghrelin and GABA receptor antagonists did not block the inhibitory effect of obese patients’ FS on NG neurons but the orexin receptor 1 antagonist (SB-334867;10µM) did. Following this, we incubated the orexin receptor 1 antagonist (SB-334867;10µM) on NG neurons incubated with mouse FS and JS and observed a similar blocking of inhibitory effects back to control values. Conclusions These findings suggest that the gut luminal contents of obese mice and humans contain an orexin receptor agonist that inhibits satiety and may contribute to over-eating. Funding Agencies CIHRNSERC

Abstract Background Irritable bowel syndrome (IBS) is more than twice as common in women and female patients report more severe abdominal pain. This suggests that sex-specific mechanisms may contribute to the pathophysiology of IBS. Many IBS patients have altered gut microbiota and luminal meditators, implicating the gut microbiota in abdominal pain. These luminal mediators can alter excitability of visceral nociceptors, thus potentially contributing to abdominal pain in IBS. Furthermore, in a subset of IBS patients a low FODMAP diet (LFD) reduces the effect of luminal mediators on pain sensing neurons. The LFD may improve abdominal pain at greater rates in women, and numerous putative mechanisms contributing to abdominal pain in IBS are susceptible to sex-specific mediators. However, it is unknown whether luminal mediators have similar effects on visceral pain signaling in both males and females. We hypothesize that luminal mediators will cause greater differences in neuronal excitability and pain signaling in female mice due to sex-specific factors. Purpose To determine whether FS from IBS patients (IBS FS) affects nociceptors from female mice more than nociceptors from male mice. Method Neurons from dorsal root ganglia from male and female mice were incubated overnight in media containing fecal supernatant (FS) from IBS patients (N=2 females) before and after the LFD, or healthy controls (HC, N=1 female and 1 male). Ratiometric Ca2+ imaging with FURA-2-AM was employed to quantify TRPV1 channel sensitization following application of capsaicin (100nM for 1 minute) as a measure of neuronal excitability. Data was analyzed using chi-squared test as well as two-way and mixed-effects model ANOVA as appropriate, followed by Sidak’s multiple comparisons test. Result(s) IBS FS caused a 177% larger Ca2+ influx in response to capsaicin compared to HC FS in female mice (p=0.0148, N=6-7 mice, neurons=43-49). In male mice, IBS FS increased Ca2+ influx by only 13% compared to HC FS (p=0.79, N=5 mice, neurons=28-35). In female mice, 117% more neurons responded to capsaicin after incubation with IBS FS versus HC FS (p=0.0004), while in male mice, only 17% more neurons responded following incubation with IBS FS (p=0.46). Finally, FS from the same IBS patients following a LFD reduced neuronal Ca2+ influx by 39% compared to IBS FS in female mice (p=0.0434, N=4-6 mice, neurons=18-49). In male mice, LFD FS reduced Ca2+ influx by 11% versus IBS FS (p=0.98, N=5 mice, neurons=28-35). Conclusion(s) Nociceptive neurons from female mice are more sensitive to the pro-nociceptive effects of FS from IBS patients, as well as a reduction of these excitatory effects following the LFD. This suggests a potential role of sex hormones in pain signaling in IBS. Disclosure of Interest None Declared

Abstract Background Irritable bowel syndrome (IBS) is a chronic abdominal pain disorder that affects women twice as often as men. The gut microbiota has been implicated as a key player in the modulation of abdominal pain in IBS. Given this, we hypothesised that the production of pro-nociceptive mediators within the gut lumen are increased in females, and this contributes to the female predominance of IBS. Purpose Compare the effects of FS from male and female IBS patients on abdominal pain pathways and identify the impact of female mouse estrous cycle on abdominal pain. Method Fecal supernatants (FS) were perfused through murine colonic preparations while performing extracellular colonic afferent nerve recordings to measure changes in action potential frequency in response to colonic distension. Phase of estrous cycle in female mice was determined through vaginal swabs. FS from male and female IBS patients reporting low, moderate, and high levels of abdominal pain were used. Result(s) FS from female IBS patients (N=6) increased afferent nerve discharge (p < 0.05) whereas FS from male IBS patients has no effect (N=4). However, single unit analysis of nociceptive axons revealed that male IBS FS increased nociceptor activity in female mice taken during the proestrus/estrus stage (p < 0.05), but not female mice taken during the metestrus/diestrus stage or male mice. Further investigation found that IBS FS from female patients with high abdominal pain (N=6), but not patients with moderate (N=5) or low pain (N=3), increased visceral afferent nerve discharge by 70%. Single unit analysis of nociceptive axons showed that their activation was increased by almost 50% following FS perfusion from high abdominal pain patients only (p < 0.05). Histamine concentrations and proteolytic activity are increased in FS from female IBS patients with high abdominal pain compared to male IBS patients. Conclusion(s) This work suggests that luminal mediators that impact abdominal pain are increased in female IBS patients compared to male IBS patients, and females appear to be more sensitive to their pro-nociceptive effects. Together, these sex differences may contribute to the female predominance of IBS. Disclosure of Interest None Declared

Abstract Background Irritable bowel syndrome (IBS) is characterized by chronic abdominal pain. Previous work has shown IBS fecal supernatants (FS) increase the excitability of nociceptive neurons compared to healthy volunteer FS. Altered production of intestinal mediators, including elevated histamine content and proteolytic activity, have been implicated in these effects. We hypothesized that the effects of histamine and proteases on neuronal excitability may be synergistic. Purpose 1) Determine whether the excitatory effect of IBS patient FS can be abolished by histamine receptor antagonists and protease inhibitors. 2) Investigate whether the excitatory effects of histamine and proteases potentiate each other’s effects on visceral nociception Method Extracellular recordings were performed, measuring action potentials from mechanosensitive extrinsic afferent nerves innervating the mouse colon during colonic distensions to 60 mmHg. Colons were perfused (20 minutes) with histamine receptor antagonists (1 µM pyrilamine, 10 µM ranitidine, 30 nM clobenpropit, 1 µM JNJ7777120) or protease inhibitors (10 µM EDTA, 0.2 µM bestatin, 0.03 µM Pepstatin A, 0.03 µM E-64) prior to treatment with the IBS patient FS. Perforated patch clamp experiments were used to measure neuronal excitability from mouse dorsal root ganglion neurons by recording rheobase (minimum current required to elicit an action potential fire). This technique was used to examine the effect of subthreshold concentrations of histamine (1µM) and trypsin (5nM), as well as their simultaneous administration on the neurons. Result(s) Luminal administration of histamine antagonists or protease inhibitors blocked the excitatory effect of the IBS patient FS (two-way ANOVA P=0.44 for protease antagonists, two-way ANOVA P=0.37 for histamine antagonists). Patch clamp experiments revealed that a subthreshold concentration of either histamine (1µM) or trypsin (5nM) had no effect on neuronal rheobase, whereas the combination significantly decreased neuronal rheobase (one-way ANOVA with Tukey’s test, P=0.006). Image Conclusion(s) These findings demonstrate that either histamine receptor antagonists or protease inhibitors inhibit the excitatory effect of IBS patient FS and suggest that a potentiating effect exists between the actions of histamine and proteases on nociceptive neurons. Disclosure of Interest None Declared

Biopsies have important value in assessing for nonerosive reflux disease.

Abstract Background Abdominal pain is a debilitating symptom of Crohn’s disease (CD). Despite the current treatment options for this disease, abdominal pain is an unresolved problem that commonly persists in the absence of active inflammation. This suggests that something other than inflammation is driving the pain during the quiescent phase. We have previously reported that microbial proteases can directly modulate the excitability of dorsal root ganglia (DRG) neurons, many of which are pain-sensing. We hypothesize that luminal proteases of CD patients are contributing to their abdominal pain. Purpose Determine whether luminal mediators in CD fecal samples induce changes in pain signalling. Method The effects of patient (active CD [n = 3] and healthy volunteer (HV) [n = 3]) fecal supernatant (FS) samples on pain-sensing neurons were assessed using ex-vivo single unit afferent nerve recordings from mouse colons. Each sample was tested in colonic preparations from a least 5 mice. To further examine cellular mechanisms, DRG neurons were isolated and incubated overnight in media containing CD FS or HV FS media. Changes in neuronal excitability were recorded by determining the rheobase (lower rheobase=increased excitability) using patch clamp recordings (n ≥ 9 DRG neurons/group). Protease inhibitors were applied in both bioassays to determine whether these inhibited the excitatory effect of FS. Lastly, total proteolytic activity in the CD and HV fecal samples was calculated using a casein colorimetric protease detection assay. Result(s) FS from HV had no effect on afferent nerve excitability (p = 0.8920). FS from active CD patients increased action potential discharge from colonic afferent nerves by 85% (p<0.0001) and selectively increased the activation of high-threshold units, which are putative nociceptors, by 44% (p=0.0074). A protease inhibitor cocktail (1:1000) and protease-activated receptor (PAR)-2 antagonist GB83 (10µM) both blocked the excitatory effects of CD FS (p<0.05). Overnight incubation with CD FS also had an excitatory effect on DRG neurons compared to HV FS (rheobase decreased by 46%, p<0.05). The effect of CD FS was blocked by GB83 (10µM) (p<0.001) and a serine protease inhibitor (FUT175; 100µM) (p<0.05) independently, but the activity was not blocked by E64 (30nM) a cysteine protease inhibitor. A 200-fold increase (p<0.0001) in total proteolytic activity was found in CD FS compared to HV FS. Conclusion(s) Luminal serine proteases, but not cysteine proteases, appear to be driving nociceptive signalling in CD patients. This provides insight into the generation of pain in CD patients and may be a potential target to mitigate this action. Further research is required to elucidate whether these pro-nociceptive proteases are of bacterial or host origin and their effects in the quiescent phase. Disclosure of Interest None Declared