Abdominal pain is a debilitating symptom of inflammatory bowel disease (IBD). Despite advances in understanding IBD pathology, the mechanisms underlying pain remain poorly defined. While studies of tissue biopsies from IBD patients and rodent models have highlighted the roles of proinflammatory cytokines and proteases in pain signaling, these approaches predominantly capture host-derived mediators, overlooking the broader luminal environment influenced by the microbiota. Given the compromised barrier in IBD leading to increased mucosal permeability, examining the luminal milieu would characterize a novel source of factors involved in pain modulation in IBD patients with active disease. Fecal supernatants (FS) from healthy volunteers (HV) of either sex had no effect on ex vivo colonic afferent nerve mechanosensitivity or in vitro dorsal root ganglia (DRG) neuron excitability. In contrast, FS from Crohn's disease (CD) and ulcerative colitis (UC) patients of either sex significantly excited colonic afferent nerves and increased mechanosensitivity ex vivo and increased DRG neuronal excitability in vitro. These were blocked by the serine protease inhibitor and a protease-activated receptor 2 (PAR2) antagonist. Proteomic analysis revealed IBD FS contained elevated levels of trypsin-and elastase-like serine proteases compared with HV FS. Proteomics identified CELA3B, ELA2A, and PRSS1 as key proteases enriched in IBD FS, with distinct activity profiles in UC and CD. These findings establish that proteases within FS from IBD patients directly modulate pain-sensing pathways by activation of PAR2 on colonic afferent nerves, offering a unique insight into luminal contributions to pain and identifying potential therapeutic targets for visceral hypersensitivity in IBD.

Background: Inflammatory bowel disease (IBD) involves relapsing inflammation of the gastrointestinal tract and is frequently associated with abdominal pain, yet effective treatments remain limited. Proteolytic activity is elevated in IBD patients, and proteases can activate visceral pain pathways via protease-activated receptors (PARs). Notably, protease activity from the gut bacterium, Bacteroides vulgatus, has recently been correlated with IBD severity, suggesting a potential microbial contribution to IBD-related pain. We hypothesize that proteases produced by B. vulgatus activate colonic afferent nerves involved in nociception. Methods: B. vulgatus cell-free supernatant (ATCC 8482 strain) or brain heart infusion (BHI) media on nociceptive neuronal activity were examined using ex vivo single-unit colonic afferent recordings from C57Bl/6 mice. Cellular mechanisms were probed by Ca 2 + imaging of dissociated DRG neurons exposed to supernatant superfusion. To specifically assess sensitization, we examined whether exposure of DRG neurons to B. vulgatus supernatant enhanced TRPV1-mediated Ca 2 + influx to capsaicin. A protease inhibitor cocktail (PIC) evaluated protease involvement, and Nav1.8-PAR2 knockout mice determined PAR2 dependence. In vivo visceromotor responses (VMR) to colorectal distension (CRD) were assessed after intracolonic administration of supernatant. Colonic permeability was measured ex vivo via FITC-dextran flux in Ussing chambers. PAR2 N-terminal cleavage by B. vulgatus supernatant was assessed using a fluorescent enzymatic assay. Results: Superfusion of DRG neurons with B. vulgatus supernatant, but not BHI, significantly increased Ca 2+ influx in response to the pro-nociceptive mediator, capsaicin (100 nM; p< 0.0001) by 49%. Ex vivo luminal perfusion of B. vulgatus supernatant, but not BHI, significantly increased spontaneous activity by 50% (p< 0.001) and mechanosensitivity of colonic afferent axons by 48% (p< 0.05). Pre-incubation of B. vulgatus supernatant with a PIC blocked the excitatory effect observed in both bioassays. Additionally, this excitation was suppressed using DRG neurons and colons from Nav1.8 PAR2 knockout mice, whereas it was maintained in the Cre controls. In vivo enema administration of B. vulgatus supernatant significantly increased VMR to CRD by 183% (p< 0.05), whereas consecutive BHI enemas had no effect. Exposure to B. vulgatus supernatant significantly increased colonic mucosal permeability, as indicated by elevated FITC-dextran flux across the colonic epithelium compared with BHI application, an effect blocked using the PIC (p = 0.05). Lastly, PAR2 cleavage was detected in the B. vulgatus supernatant, which was blocked using serine and metalloprotease inhibitors. Conclusion: Proteases secreted by B. vulgatus disrupt permeability and sensitize nociceptive visceral afferent nerves and may be a novel target for pain management in IBD. This work highlights the potential for microbiome-derived proteases to contribute directly to abdominal pain and mucosal injury in IBD patients. Funded by CIHR. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

BACKGROUND:Proteases and histamine, co-secreted by mast cells and bacteria, sensitise colonic nociceptors and contribute to irritable bowel syndrome (IBS) pain. OBJECTIVE:To determine whether irreversible proteolytic cleavage of protease-activated receptor-2 (PAR2) and its continued activity in endosomes amplify and sustain otherwise transient pronociceptive actions of histamine receptors to cause recurrent pain, the defining symptom of IBS. DESIGN:We investigated PAR2 and H1R coexpression in nociceptors using RNAscope and assessed the consequences of coactivation using electrophysiological assays of nociceptor sensitisation and biophysical measurements of receptor and effector activity. RESULTS:PAR2 and H1R were co-expressed by human and mouse dorsal root ganglion nociceptors. Intracolonic infusion of faecal supernatants from patients with IBS with elevated histamine and proteolytic activity enhanced mechanosensitivity of colonic nociceptors in mice. Antagonists of PAR2 or H1R abolished this response. Combined administration of subthreshold concentrations of trypsin and histamine replicated the effects of faecal supernatant and caused hyperexcitability of isolated nociceptors. Pre-activation with trypsin sensitised histamine-induced hyperexcitability in nociceptors from wild-type but not Par2 -/- mice. Endocytosis inhibitors prevented this hypersensitivity, consistent with sustained endosomal signalling of PAR2 and persistent nociceptor hyperexcitability. Trypsin amplified histamine-induced activation of H1R and β-arrestin2 and Gαq effectors at the plasmalemma and in endosomes. Conversely, histamine did not sensitise trypsin-induced hyperexcitability of neurons, in line with the inability of histamine to induce sustained nociceptor hypersensitivity. CONCLUSIONS:By amplifying and maintaining the otherwise transient actions of H1R and possibly other pain receptors, persistent PAR2 endosomal signalling makes a dominant contribution to IBS-related colonic pain.

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.

Protease-activated receptor 2 (PAR2) is a central regulator of intestinal barrier function, inflammation, and pain. Upregulated intestinal proteolysis and PAR2 signaling are implicated in inflammatory bowel diseases (IBDs) and irritable bowel syndrome (IBS), conditions often associated with gut microbiome alterations. To identify potential bacterial regulators of PAR2 activity, we developed a functional assay for PAR2 processing to screen a library of diverse gut microbes. We identify multiple bacteria that secrete proteases capable of cleaving host PAR2. Using chemoproteomic profiling with a covalent irreversible inhibitor, we uncovered a previously uncharacterized Bacteroides fragilis serine protease 1 (Bfp1) and show that it cleaves and activates PAR2 in multicellular and murine models. PAR2 cleavage by Bfp1 disrupts the intestinal barrier, sensitizes nociceptors, and triggers colonic inflammation and abdominal pain. Collectively, our findings uncover Bfp1-mediated PAR2 processing as an axis of host-commensal interaction in the gut that has the potential to be targeted for therapeutic intervention in IBD or IBS.

Protease-activated receptor 2 (PAR2) is a central regulator of intestinal barrier function, inflammation and pain. Upregulated intestinal proteolysis and PAR2-signaling are implicated in inflammatory bowel diseases (IBDs) and irritable bowel syndrome (IBS). To identify potential bacterial regulators of PAR2 activity, we developed a functional assay for PAR2 processing and used it to screen conditioned media from a library of diverse gut commensal microbes. We found that multiple bacteria secrete proteases that cleave host PAR2. Using chemoproteomic profiling with a covalent irreversible inhibitor, we identified a previously uncharacterized Bacteroides fragilis serine protease Bfp1, and showed that it cleaves and activates PAR2 in multicellular and murine models. PAR2 cleavage by Bfp1 disrupts the intestinal barrier, sensitizes nociceptors, and triggers colonic inflammation and abdominal pain. Collectively, our findings uncover Bfp1-mediated PAR2-processing as a new axis of host-commensal-interaction in the gut that has the potential to be targeted for therapeutic intervention in IBD or IBS.

Abdominal pain poses a significant challenge for individuals with inflammatory bowel disease (IBD). Despite current treatments that target inflammation, IBD-associated abdominal pain often persists even in the absence of inflammation, negatively impacting patients’ quality of life. This persistence suggests that factors other than inflammation may be contributing to the pathology of IBD. Our previous research suggests that bacterial proteases can directly influence the excitability of dorsal root ganglia neurons, many of which are pain-sensing. Building on this, we hypothesize that proteases, both of host and bacterial origin, play a role in pain modulation during the active and remission phases of IBD. The effects of fecal supernatants (FS) from patients with active or remissive IBD and healthy volunteers, on pain-sensing neurons were assessed using ex-vivo single-unit afferent nerve recordings from mouse colons. A protease inhibitor cocktail (PIC; 1:1000) and a protease-activated receptor (PAR)-2 antagonist (GB83; 10µM) were independently applied in the bioassay to determine whether these inhibited the excitatory effect of the FS. In addition, the participant FS were tested for proteolytic cleavage of the N-terminal domain of protease-activated receptor PAR2 using a novel enzymatic assay. FS from healthy volunteers [N=5] had no effect on afferent nerve excitability (p >0.05). FS from active IBD patients [N=15] 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< 0.01). A protease inhibitor cocktail and PAR2 antagonist both independently inhibited the excitatory effects of IBD FS (p >0.05) on afferent nerve activity. In contrast, FS from IBD patients in remission [N=15], did not excite colonic afferent nerves (p >0.05). Interestingly, these findings were found to be consistent when IBD was split into disease subtypes: Crohn’s disease and ulcerative colitis. Furthermore, when normalized to total protein content, active disease yielded significantly greater PAR2 cleavage activity (p< 0.05), while the remission samples were not significantly different than the healthy participants (p >0.05). This PAR2 cleavage activity was found to be correlated to neuronal excitation (R2=0.4721, p< 0.001). Our findings suggest that active IBD leads to the generation of luminal mediators, including proteases acting on PAR2, that activate visceral nociceptive neurons. These luminal mediators are less abundant when inflammation is in remission. These data suggest that targeting proteases could offer a promising therapeutic approach for pain management in IBD.

Background & Aims We recently showed that a bacterial infection can break oral tolerance to food and lead to immunoglobulin E (IgE)-dependent mast cell activation and food-induced abdominal pain, which could constitute an important pathogenic mechanism in postinfectious irritable bowel syndrome (IBS). Here, we investigated whether similar immune mechanisms in response to psychological stress lead to food-evoked pain signaling, and thus potentially explain the pathophysiology in a larger group of patients with IBS. Methods Mice were exposed to ovalbumin (OVA) during water avoidance stress (WAS) and re-exposed to OVA 5 weeks later. Nociception was evaluated by visceromotor responses and afferent nerve recordings to intestinal distension, and patch-clamp recordings of sensory neurons incubated with intestinal supernatants. The role of IgE and type 2 immunity was evaluated using pharmacologic and genetic approaches. Results Re-exposure to OVA increased pain signaling in the colon and small intestine only in mice exposed to OVA during WAS, in the absence of systemic allergy. OVA-induced increases in pain responses depended on mast cells, IgE, and signal transducer and activator of transcription 6 signaling. Notably, incubation of sensory neurons with ileum and colon supernatants from WAS/OVA+OVA mice lowered their threshold of excitability. Finally, treatment with histamine receptor H1 antagonist pyrilamine blocked the increased sensory neuron excitability, and reduced ileal afferent nerve firing to distension in WAS/OVA+OVA mice. Conclusions Psychological stress induces a type 2 immune response to food antigens, with IgE-mediated mast cell activation and increased pain signaling in the small intestine and colon in response to food. These findings may explain the potential role of psychological stress in food-induced symptoms in IBS.

There is an urgent need for analgesics to treat pain that lacks the serious side effects of existing drugs, such as conventional opioids and nonsteroidal anti-inflammatory drugs. Most side effects arise from the non-selective actions of these drugs at sites where the pain is not generated because of the ubiquitous expression of the drug targets in the body regardless of the underlying disease. In this narrative review, we explore 2 mechanistic approaches focusing on visceral nociceptive neurons that have the potential to limit side effects while preserving efficacy. Strategy 1 demonstrates how mechanistic pain studies underlying a specific disorder, such as irritable bowel syndrome, can identify targets specifically upregulated in that condition. We discuss recent findings regarding 2 neuroactive mediators, histamine and proteases, including novel intestinal sources, signalling pathways, and intracellular synergistic actions that could serve as potential therapeutic targets. Strategy 2 examines how acidic microenvironments unique to the sites of inflammation where pain is generated, such as in inflammatory bowel disease, can be exploited. pH-sensitive analgesics have been developed that inhibit μ-opioid receptors at sites of inflammation where tissue pH is low, ie, 6.5, while showing no activity at other sites where tissue pH is normal, ie, 7.4. Collectively, these studies highlight the value of investigating the mechanisms underlying specific disorders, which can lead to novel biomarkers and therapeutic strategies that can enhance the specificity of the new therapies.

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.

Inflammatory bowel disease (IBD) induces hyperexcitability in dorsal root ganglion (DRG) nociceptors, which contributes to abdominal pain. Proteases modulate the excitability of nociceptive neurons via activation of cell-surface protease-activated receptors. Recent findings indicate that serine proteases from Faecalibacterium prausnitzii, a bacterial strain found in the colonic microbiota of healthy individuals, can directly signal to the DRG neurons and reduce excitability, which may suppress abdominal pain. Herein we identify an anti-nociceptive serine protease from F. prausnitzii by heterologous expression and purification of multiple putative anti-nociceptive serine proteases followed by clamp electrophysiology experiments. A single serine protease, Clp-fp, from F. prausnitzii reduced DRG neuronal excitability via activation of protease-activated receptor-4, thereby recapitulating the effect of F. prausnitzii culture supernatants. The identification and characterization of a potentially analgesic serine protease from a commensal gut bacterium makes possible the further exploration of new treatments for IBD-related abdominal pain.

Abdominal pain is a major symptom of diseases associated with microbial dysbiosis. Disruption of the gut microbiota with antibiotics increases visceral pain, and germ‐free mice are more prone to pain than conventionally‐raised mice. 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. Patch clamp electrophysiological recordings of dorsal root ganglion (DRG) neuron excitability were obtained from control mice and mice treated with the non‐absorbable antibiotic vancomycin (50 µg/ml in drinking water) for one week. Ten days prior to recording visceromotor response (VMR) telemetric transmitters were placed into the abdominal cavity of the mice and allowed to recover. VMR was measured by insertion of balloon catheter into the rectum under light anesthetization in both control and vancomycin treated mice, then distended to 80 mmHg and VMR recorded. Bacterial dysbiosis was verified by metagenomic analysis of stool microbial composition. Mice treated with vancomycin were more sensitive to colorectal distension in vivo (VMR increased by 70% at 80 mmHg compared to control), and DRG neurons from vancomycin‐treated mice were hyperexcitable in vitro compared to water‐treated controls (rheobase decreased by 30% relative to control). Interestingly, hyperexcitability of DRG neurons was not restricted to gut projecting neurons, suggesting a widespread effect of gut dysbiosis on pain pathways. Incubation of DRG neurons from naïve mice in serum from vancomycin‐treated mice increased neuron excitability (rheobase decreased by 30% relative to control), suggesting that microbial dysbiosis alters circulating mediators that influence nociception. Multiplex ELISA measurements did not detect any significant changes in serum cytokines or chemokines between vancomycin‐treated and control mice. The cysteine protease inhibitor E64 (30 nM) and the protease‐activated receptor 2 (PAR2) antagonist GB‐83 (10 µM) each blocked the increase in DRG neuron excitability in response to serum from vancomycin‐treated mice. Naïve DRG neurons incubated with fecal supernatants from vancomycin‐treated mice also exhibited increased excitability (rheobase decreased by 40% relative to control), but supernatants derived from colonic tissue failed to cause hyperexcitability. Overall, this data suggests that microbial dysbiosis within the gut alters pain sensitivity. This effect is not caused by inflammation or host derived factors, rather bacterially‐derived cysteine proteases activating PAR2 on DRG neurons.