Protease-Induced Excitation of Dorsal Root Ganglion Neurons in Response to Acute Perturbation of the Gut Microbiota Is Associated With Visceral and Somatic Hypersensitivity.

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.

Changes in signalling from faecal neuroactive metabolites following dietary modulation of IBS pain.

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.

Synthetic Cannabinoid Agonist WIN 55212-2 Targets Proliferation, Angiogenesis, and Apoptosis via MAPK/AKT Signaling in Human Endometriotic Cell Lines and a Murine Model of Endometriosis.

Endometriosis (EM) is characterized by the growth of endometrium-like tissue outside the uterus, leading to chronic inflammation and pelvic pain. Lesion proliferation, vascularization, and associated inflammation are the hallmark features of EM lesions. The legalization of recreational cannabinoids has garnered interest in the patient community and is contributing to a greater incidence of self medication; however, it remains unknown if cannabinoids possess marked disease-modifying properties. In this study, we assess the effects of synthetic cannabinoid, WIN 55212-2 (WIN 55), in EM-representative in vitro and in vivo syngeneic mouse models. WIN 55 reduced proliferation and angiogenesis in vitro, via MAPK/Akt-mediated apoptosis. These findings were corroborated in a mouse model of EM, where we found reduced TRPV1 expression in the dorsal root ganglia of the EM mouse model exposed to WIN 55, suggesting reduced signaling of pain stimuli. Ultimately, these pieces of evidence support the use of cannabinoid receptor agonists as a potential therapeutic intervention for EM associated pain and inflammation.